Figure 4 ( D ) Western blot analysis of MAPKs and NF - κB activation upon S1P stimulation . HeLa NOD1 or NOD2 cells were treated with cytosolic S1P ( 20 µM ) , iE - DAP ( 20 µM ) or MDP ( 20 µM ) for indicated times . Multiplex analysis of CXCL2 ( I ) , CCL5 ( J ) and IL16 ( K ) production in supernatants of WT , Nod1 / 2 and Rip2 KO BMDMs upon S1P ( 10 µM ) or MDP ( 10 µM ) stimulations together with digitonin ( 2 . 5 µg / ml ) for 20 h .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(D) Western blot analysis of MAPKs and NF-κB activation upon S1P stimulation. HeLa NOD1 or NOD2 cells were treated with cytosolic S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) for indicated times.Multiplex analysis of CXCL2 (I), CCL5 (J) and IL16 (K) production in supernatants of WT, Nod1/2 and Rip2 KO BMDMs upon S1P (10 µM) or MDP (10 µM) stimulations together with digitonin (2.5 µg/ml) for 20 h.


Figure 5 ( A , B ) Immunoprecipitation with various lipid - coated beads using HeLa NOD1 - GFP ( A ) and NOD2 - GFP cells ( B ) . Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid - coated beads at room temperature for 2 h . ( E , F ) Single particle tracking of NOD1 - GFP ( E ) or NOD2 - GFP ( F ) with S1P - TAMRA . After doxycycline induction overnight , HeLa NOD1 or NOD2 cells were stimulated with S1P - TAMRA ( 20 µM ) for 1 h together with digitonin . Scale bar : 5 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A, B) Immunoprecipitation with various lipid-coated beads using HeLa NOD1-GFP (A) and NOD2-GFP cells (B). Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid-coated beads at room temperature for 2 h.(E, F) Single particle tracking of NOD1-GFP (E) or NOD2-GFP (F) with S1P-TAMRA. After doxycycline induction overnight, HeLa NOD1 or NOD2 cells were stimulated with S1P-TAMRA (20 µM) for 1 h together with digitonin. Scale bar: 5 µm.


Figure 6 ( D ) MST analysis of direct binding of NOD1 to S1P and iE - DAP . ( E ) MST analysis of direct binding of NOD2 with S1P and MDP . ( F ) ELISA analysis of IL8 in supernatants of HEK293T NOD1 / 2 KO cells transfected with indicated NOD1 or NOD2 mutants . After 24 h transfection , cells were stimulated with S1P ( 20 µM ) , iE - DAP ( 20 µM ) or MDP ( 20 µM ) together with digitonin for 20 h .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(D) MST analysis of direct binding of NOD1 to S1P and iE-DAP. (E) MST analysis of direct binding of NOD2 with S1P and MDP.(F) ELISA analysis of IL8 in supernatants of HEK293T NOD1/2 KO cells transfected with indicated NOD1 or NOD2 mutants. After 24 h transfection, cells were stimulated with S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) together with digitonin for 20 h.


Figure 7 ( A , B ) MST analysis of direct binding of NOD1 ( A ) or NOD2 ( B ) with ADP in the presence of S1P ( 2 µM ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A, B) MST analysis of direct binding of NOD1 (A) or NOD2 (B) with ADP in the presence of S1P (2 µM).


Figure 1Monocyte derived macrophages ( MDM ) were treated with 5μM etoposide ( ETO ) for 18h . Cells were stained for γH2AX and 53BP1 nuclear foci , acquired and analysed using the automated cell ‐ imaging system Hermes WiScan and ImageJ . On average 104 cells were acquired and analysed . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; paired t ‐ test ) . Scale bars , 10μm . Monocyte derived macrophages ( MDM ) were treated with 5μM etoposide ( ETO ) for 18h . Cells were stained for γH2AX and 53BP1 nuclear foci , acquired and analysed using the automated cell ‐ imaging system Hermes WiScan and ImageJ . On average 104 cells were acquired and analysed . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; paired t ‐ test ) . Scale bars , 10μm . MDM were treated with increasing concentrations of ETO and CTH for 66h . Cells were stained to distinguish between live and dead cells using LIVE / DEAD fixable Dead cell stain protocol . Percentages of live / dead cells were determined using automated cell ‐ imaging system Hermes WiScan and ImageJ . Addition of 20 % ethanol for 10min into cells treated with ETO was used as positive control . ( n = 3 , mean ± s . e . m ) . MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV - G pseudotyped HIV - 1 GFP viruses wild type ( wt ) HIV - 1 capsid mutants ( N74D or P90A ) , HIV - 2 , SIV sooty mangabey ( SIVsm E543 ) , replication competent Adenovirus type 5 AdV ( AdV ) , Semliki Forest Virus ( SFV ) and HSV - 1 . The percentages of infected cells were determined using an automated cell ‐ imaging system Hermes WiScan and ImageJ and normalized to untreated control ~ 100 % . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; * * * P ‐ value ≤ 0 . 001 ; ( ns ) non ‐ significant , paired t ‐ test ) . Cells from a representative donor were used for immunoblotting . MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV - G pseudotyped HIV - 1 GFP . 104 cells were recorded and analysed for GFP expression 48h post - infection using an automated cell ‐ imaging system Hermes WiScan and ImageJ . ( n = 4 , mean ± s . e . m ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full - length replication competent macrophage tropic HIV - 1 virus BaL . Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post ‐ infection by FACS ( n = 3 , mean ± s . e . m ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) . G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) . G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . 2LTR circles . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) . G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h and infected with HIV - 1 BaL ( 3 , 6 , 12 ffu / cell ) . Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post ‐ infection by FACS ( n = 3 , mean ± s . e . m ; * * P ‐ value ≤ 0 . 01 ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h , infected with HIV - 1 BaL ( 3 , 6 , 12 ffu / cell ) and DNA isolated 18 h post ‐ infection for qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h , infected with HIV - 1 BaL and DNA isolated 4 , 6 , and 18 h post ‐ infection for qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Monocyte derived macrophages (MDM) were treated with 5μM etoposide (ETO) for 18h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired and analysed. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; paired t‐test). Scale bars, 10μm.Monocyte derived macrophages (MDM) were treated with 5μM etoposide (ETO) for 18h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired and analysed. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; paired t‐test). Scale bars, 10μm.MDM were treated with increasing concentrations of ETO and CTH for 66h. Cells were stained to distinguish between live and dead cells using LIVE/DEAD fixable Dead cell stain protocol. Percentages of live/dead cells were determined using automated cell‐imaging system Hermes WiScan and ImageJ. Addition of 20% ethanol for 10min into cells treated with ETO was used as positive control. (n = 3, mean ± s.e.m).MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP viruses wild type (wt) HIV-1 capsid mutants (N74D or P90A), HIV-2, SIV sooty mangabey (SIVsm E543), replication competent Adenovirus type 5 AdV (AdV), Semliki Forest Virus (SFV) and HSV-1. The percentages of infected cells were determined using an automated cell‐imaging system Hermes WiScan and ImageJ and normalized to untreated control ~100%. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; ***P‐value ≤ 0.001; (ns) non‐significant, paired t‐test). Cells from a representative donor were used for immunoblotting.MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP. 104 cells were recorded and analysed for GFP expression 48h post-infection using an automated cell‐imaging system Hermes WiScan and ImageJ. (n = 4, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full-length replication competent macrophage tropic HIV-1 virus BaL. Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test). G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.  Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).  G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.2LTR circles. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05, paired t‐test).G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; ***P‐value ≤ 0.001, paired t‐test).MDM were treated with 5μM ETO for 18h and infected with HIV-1 BaL (3, 6, 12 ffu/cell). Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ** P‐value ≤ 0.01; ***P‐value ≤ 0.001, paired t‐test).MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL (3, 6, 12 ffu/cell) and DNA isolated 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL and DNA isolated 4, 6, and 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


Figure 2MDM were transfected with control or pool of SAMHD1 ( KD ) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV ‐ G pseudotyped HIV ‐ 1 GFP 18h later . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) or deleted for Vpx ( SIV VLP del Vpx ) or deleted for Vpr ( SIV VLP del Vpr ) . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . ( n = 4 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) . D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) . D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . 2LTR circles . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) . D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . The percentage of infected cells was quantified by Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV‐G pseudotyped HIV‐1 GFP 18h later. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP) or deleted for Vpx (SIV VLP del Vpx) or deleted for Vpr (SIV VLP del Vpr). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 4, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).The percentage of infected cells was quantified by Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).


Figure 3A - B . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx wild type ( WT ) / Vpr or Vpx Q76A mutant / Vpr ( SIV VLP Q76A ) . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . MDM were differentiated and cultured in Human serum instead of FCS . A - B . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx wild type ( WT ) / Vpr or Vpx Q76A mutant / Vpr ( SIV VLP Q76A ) . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . MDM were differentiated and cultured in FCS . A standard culture condition used in all experiments . See Materials and Methods . C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . Cells from a representative donor were used for immunoblotting . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) . C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . 2LTR circles . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) . C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in Human serum instead of FCS.A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in FCS. A standard culture condition used in all experiments. See Materials and Methods. C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.  The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. Cells from a representative donor were used for immunoblotting. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).  C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).


Figure 4MDM were treated with 5μM ETO for 18h , 3μg / ml cGAMP and 10ng / ml IFNβfor 18h . RNA was isolated and qPCR performed for selected genes using TaqMan assays . Expression levels of target genes were normalised to GAPDH . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; ( ns ) non ‐ significant , paired t ‐ test ) . MDM were treated with 5μM ETO or cGAMP for 18h or 100ng / ml LPS for 2h . Cells were stained and analysed for IRF3 translocation into nucleus . Scale bars : 10μm . Quantification of nuclei positive for IRF3 staining . ( n = 3 , mean ± s . e . m . ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4MDM were treated with 5μM ETO for 18h, 3μg/ml cGAMP and 10ng/ml IFNβfor 18h. RNA was isolated and qPCR performed for selected genes using TaqMan assays. Expression levels of target genes were normalised to GAPDH. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; (ns) non‐significant, paired t‐test).MDM were treated with 5μM ETO or cGAMP for 18h or 100ng/ml LPS for 2h. Cells were stained and analysed for IRF3 translocation into nucleus. Scale bars: 10μm.Quantification of nuclei positive for IRF3 staining. (n = 3, mean ± s.e.m.).


Figure 5MDM were treated with increasing concentrations of ETO and CTH . Cells were stained for MCM2 expression , acquired and analysed using the automated cell ‐ imaging system Hermes WiScan and ImageJ . On average 104 cells were acquired . ( n = 3 , mean ± s . e . m . ) . MDM were treated with 5μM ETO , lysed and immunoblotting performed to detect cell cycle / cell cycle arrest and DNA damage associated proteins . MDM were treated with 5μM ETO or 0 . 01μM CTH for 18h , lysed and immunoblotting performed to detect cell cycle / cell cycle arrest and DNA damage associated proteins .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5MDM were treated with increasing concentrations of ETO and CTH. Cells were stained for MCM2 expression, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired. (n = 3, mean ± s.e.m.).MDM were treated with 5μM ETO, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.MDM were treated with 5μM ETO or 0.01μM CTH for 18h, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.


Figure 1 ( F ) Representative images of germ cells immunostained for SYCP3 ( green ) , DMC1 ( red ) , and DDX4 ( magenta ) at each meiotic sub - stage . Nuclear DAPI staining is shown in white . The sub - stages of meiosis were defined based on the staining patterns of SYCP3 , DMC1 , and DAPI as described in Materials and Methods . ( G ) Percentages of germ cells at each meiotic sub - stage as defined in Fig 1F . The average percentages in the outer or inner ovarian cortex on each meiotic sub - stage were calculated from at least three fetal ovaries [ 8 wpf ( n = 4 ) , 10 / 12 / 14 / 16 / 18 wpf ( n = 3 ) ] .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(F) Representative images of germ cells immunostained for SYCP3 (green), DMC1 (red), and DDX4 (magenta) at each meiotic sub-stage. Nuclear DAPI staining is shown in white. The sub-stages of meiosis were defined based on the staining patterns of SYCP3, DMC1, and DAPI as described in Materials and Methods. (G) Percentages of germ cells at each meiotic sub-stage as defined in Fig 1F. The average percentages in the outer or inner ovarian cortex on each meiotic sub-stage were calculated from at least three fetal ovaries [8 wpf (n=4), 10/12/14/16/18 wpf (n=3)].


Figure 2 ( C ) IF for oogonia ( POU5F1 , TFAP2C ) , meiotic ( SYCP3 , SYCP1 ) , and oogenic ( ZP3 , TP63 ) markers in the transplanted cy rOvaries . Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper - right magnified images of each panel show the expression of each marker ( green ) co - stained with DDX4 ( magenta ) . The lower - right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . The numbers written in the upper - left corner indicate weeks after transplantation ( w - ptp ) . Scale bar = 20 μm . ( D ) Percentages of cells positive for individual markers among DDX4 + germ cells from the IF of cy rOvaries at 3 − 15 w - ptp [ 3 w - ptp ( n = 3 ) , 6 / 9 / 12 w - ptp ( n = 4 ) , 15 w - ptp ( n = 6 ) ] . The average values with SDs are shown . ( E ) H & E staining of transplanted cy rOvaries at 21 w - ptp . Dashed lines indicate the transplanted cy rOvaries beneath the kidney capsule of KSN / Slc mice . K , mouse kidney . Scale bars = 200 μm ( left ) and 100 μm ( right ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) IF for oogonia (POU5F1, TFAP2C), meiotic (SYCP3, SYCP1), and oogenic (ZP3, TP63) markers in the transplanted cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of each marker (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate weeks after transplantation (w-ptp). Scale bar = 20 μm. (D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3−15 w-ptp [3 w-ptp (n=3), 6/9/12 w-ptp (n=4), 15 w-ptp (n=6)]. The average values with SDs are shown. (E) H&E staining of transplanted cy rOvaries at 21 w-ptp. Dashed lines indicate the transplanted cy rOvaries beneath the kidney capsule of KSN/Slc mice. K, mouse kidney. Scale bars = 200 μm (left) and 100 μm (right).


Figure 3 ( E ) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries . Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper - right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower - right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . The numbers written in the upper - left corner indicate the period of IVC ( w - ivc ) . Scale bar = 20 μm . ( F ) Percentages of cells positive for individual markers among DDX4 + germ cells from the IF of cy rOvaries at 3 - 15 w - ivc [ 3 / 6 / 9 / 12 w - ivc ( n = 3 ) , 15 w - ivc ( n = 3 or 4 ) ] .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(E) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate the period of IVC (w-ivc). Scale bar = 20 μm. (F) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3-15 w-ivc [3/6/9/12 w-ivc (n=3), 15 w-ivc (n=3 or 4)].


Figure 5 ( A ) Gross appearance ( a ) , H & E staining ( b ) , and IF ( c ) for FOXL2 / DDX4 / DAPI of the human in vivo female gonads ( 11W , left column ) and the cultured rOvaries at 7 and 14 w - ivc . FOXL2 , granulosa cell marker ; DDX4 , germ cell marker ; DAPI , nucleus . Scale bars = 1 mm ( a , left ) , 200 μm ( a , middle / right ) , 50 μm ( b ) , and 20 μm ( c ) . g . a . , gestational age . IF for oogonia ( POU5F1 ) , cell proliferation ( Ki67 ) , meiotic ( SYCP3 , DMC1 , γH2AX , SYCP1 ) , and oogenic ( ZP3 , TP63 ) markers for human in vivo female gonad ( 11W , B ) Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . IF for oogonia ( POU5F1 ) , cell proliferation ( Ki67 ) , meiotic ( SYCP3 , DMC1 , γH2AX , SYCP1 ) , and oogenic ( ZP3 , TP63 ) markers human in vitro cultured rOvaries at 14 w - ivc ( C ) . Germ cells and nuclei were marked with DDX4 ( magenta ) and DAPI ( white ) , respectively . The upper right magnified images of each panel show the expression of key markers ( green ) co - stained with DDX4 ( magenta ) . The lower right magnified images of each panel show DDX4 expression ( magenta ) co - stained with DAPI ( white ) . ( D ) Percentages of cells positive for individual markers among DDX4 + germ cells from the IF of human in vivo female gonads ( 11W ) and human cultured rOvaries at 7 and 14 w - ivc . The mean values from two biological replicates are shown . Analyses for human germ cells by SC3 - seq . ( E ) UHC for human germ cells in vivo and in rOvaries with all expressed genes ( 20 , 048 genes ) and a heatmap of the expression levels of selected marker genes . Color bars under the dendrogram show the in vivo stage ( 1st bar ) , the period of IVC ( 2nd bar ) , and the cell type ( 3rd bar ) . The heatmap color - coding is as indicated . The color coding in bars is as indicated in Fig 5F .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Gross appearance (a), H&E staining (b), and IF (c) for FOXL2/DDX4/DAPI of the human in vivo female gonads (11W, left column) and the cultured rOvaries at 7 and 14 w-ivc. FOXL2, granulosa cell marker; DDX4, germ cell marker; DAPI, nucleus. Scale bars = 1 mm (a, left), 200 μm (a, middle/right), 50 μm (b), and 20 μm (c). g.a., gestational age.IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers for human in vivo female gonad (11W, B) Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white).IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers human in vitro cultured rOvaries at 14 w-ivc (C). Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white).(D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of human in vivo female gonads (11W) and human cultured rOvaries at 7 and 14 w-ivc. The mean values from two biological replicates are shown.Analyses for human germ cells by SC3-seq. (E) UHC for human germ cells in vivo and in rOvaries with all expressed genes (20,048 genes) and a heatmap of the expression levels of selected marker genes. Color bars under the dendrogram show the in vivo stage (1st bar), the period of IVC (2nd bar), and the cell type (3rd bar). The heatmap color-coding is as indicated. The color coding in bars is as indicated in Fig 5F.


Figure 6Analyses performed by 10X scRNA - seq . ( A ) UMAP plot of germ cells from six 10X scRNA - seq datasets ( two for humans [ this study and ( Chitiashvili et al . , 2020 ) ] , one for monkeys [ this study ] , and three for mice [ ( Ge et al . , 2021 , Niu & Spradling , 2020 , Zhao et al . , 2020 ) ] ) integrated by CCA . Meiotic sub - stages were defined after CCA followed by Louvain clustering . Colors indicate the computationally assigned meiotic sub - stages defined by the expression levels of key meiotic markers conserved in all three species ( see Materials and Methods ) . The color - coding is as indicated . M , mitotic ; PL , pre - leptotene ; L , leptotene ; Z , zygotene ; P , pachytene ; D , diplotene . ( B ) The UMAP plot highlighting cells from each dataset . The color - coding for each developmental stage is as indicated . ( I ) IF analyses of CLGN with TP63 ( oogenic marker ) and DDX4 ( germ cell marker ) in a 13 - year - old human oocyte of a primordial follicle . Nuclear DAPI staining is shown in white .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Analyses performed by 10X scRNA-seq. (A) UMAP plot of germ cells from six 10X scRNA-seq datasets (two for humans [this study and (Chitiashvili et al., 2020)], one for monkeys [this study], and three for mice [(Ge et al., 2021, Niu & Spradling, 2020, Zhao et al., 2020)]) integrated by CCA. Meiotic sub-stages were defined after CCA followed by Louvain clustering. Colors indicate the computationally assigned meiotic sub-stages defined by the expression levels of key meiotic markers conserved in all three species (see Materials and Methods). The color-coding is as indicated. M, mitotic; PL, pre-leptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene. (B) The UMAP plot highlighting cells from each dataset. The color-coding for each developmental stage is as indicated.(I) IF analyses of CLGN with TP63 (oogenic marker) and DDX4 (germ cell marker) in a 13-year-old human oocyte of a primordial follicle. Nuclear DAPI staining is shown in white.


Figure 7 ( C ) The Chr . 10 : A and X : A ratios ( top ) , and XIST expression ( bottom ) in human female germ cells in vivo and in cultured rOvaries analyzed by SC3 - seq .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(C) The Chr.10:A and X:A ratios (top), and XIST expression (bottom) in human female germ cells in vivo and in cultured rOvaries analyzed by SC3-seq.


Figure 1A HeLa cells expressing LAMP1 - RFP were treated with vehicle alone ( control ) or LLOMe for 1 h to induce lysosome damage . Cells were fixed 2 h after washout , stained with p62 and galectin - 3 ( Gal3 ) antibodies , and analyzed by confocal microscopy . B p97 translocates to ruptured lysosomes . Cells co - expressing LAMP1 - RFP and GFP - p97 were treated as in ( A ) and stained with the pan - ubiquitin antibody FK2 ( Ub ) . Percentage of cells showing recruitment of p97 in three independent experiments ( mean ± SD ) . C Immunodetection of endogenous p97 after LLOMe treatment as in ( A ) . Percentage of cells showing recruitment of p97 in three independent experiments ( mean ± SD ) . D Clearance of damaged lysosomes depends on p97 . Cells were transfected with control ( Ctrl ) or p97 siRNA oligos , fixed at indicated time points after LLOMe washout and stained for endogenous Gal3 . Quantification of the number of Gal3 - positive vesicles per cell . Data represent mean ± SD of four independent experiments with ≥ 30 cells quantified per condition ( one - sided Welch ' s unequal variances t - tests comparing the siCtrl with sip97 ) . E Cells were chased after LLOMe treatment for 12 h with vehicle alone ( DMSO ) , bortezomib ( Btz , 10 nM ) , NH4Cl ( 20 mM ) or the p97 inhibitor NMS - 873 ( 5 µM ) , and Gal3 vesicles quantified . Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition ( one - sided Welch ' s t - tests ) . F p97 is essential for cell survival after lysosome rupture . Cells were transfected with control ( Ctrl ) , p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h . Cell viability was measured using the MTS assay . Data represent mean ± SD of three independent experiments . G Taufibrils are endocytosed and induce lysosomal damage . Taufibrils were generated in vitro , fluorescently labelled by Dylight488 and fragmented by sonication ( 488 - labelled tau ) . HeLa cells expressing mCherry - Gal3 were incubated with 300 nM taufibrils for 24 h . Arrows indicate compartments that contain 488 - labelled tau and are positive for mCherry - Gal3 indicating lysosome rupture . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD ) . H Accumulation of tau - induced lysosomal damage upon p97 depletion . Cells transfected with control ( Ctrl ) or p97 siRNA oligos were treated as in ( G ) . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD ) . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 . Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A HeLa cells expressing LAMP1-RFP were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with p62 and galectin-3 (Gal3) antibodies, and analyzed by confocal microscopy.B p97 translocates to ruptured lysosomes. Cells co-expressing LAMP1-RFP and GFP-p97 were treated as in (A) and stained with the pan-ubiquitin antibody FK2 (Ub). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).C Immunodetection of endogenous p97 after LLOMe treatment as in (A). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).D Clearance of damaged lysosomes depends on p97. Cells were transfected with control (Ctrl) or p97 siRNA oligos, fixed at indicated time points after LLOMe washout and stained for endogenous Gal3. Quantification of the number of Gal3-positive vesicles per cell. Data represent mean ± SD of four independent experiments with ≥30 cells quantified per condition (one-sided Welch's unequal variances t-tests comparing the siCtrl with sip97).E Cells were chased after LLOMe treatment for 12 h with vehicle alone (DMSO), bortezomib (Btz, 10 nM), NH4Cl (20 mM) or the p97 inhibitor NMS-873 (5 µM), and Gal3 vesicles quantified. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition (one-sided Welch's t-tests).F p97 is essential for cell survival after lysosome rupture. Cells were transfected with control (Ctrl), p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h. Cell viability was measured using the MTS assay. Data represent mean ± SD of three independent experiments.G Taufibrils are endocytosed and induce lysosomal damage. Taufibrils were generated in vitro, fluorescently labelled by Dylight488 and fragmented by sonication (488-labelled tau). HeLa cells expressing mCherry-Gal3 were incubated with 300 nM taufibrils for 24 h. Arrows indicate compartments that contain 488-labelled tau and are positive for mCherry-Gal3 indicating lysosome rupture. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD).H Accumulation of tau-induced lysosomal damage upon p97 depletion. Cells transfected with control (Ctrl) or p97 siRNA oligos were treated as in (G). Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD). * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Scale bars, 10 µm.


Figure 2A Impaired clearance of damaged lysosomes in cells expressing a p97 disease - mutant . Wild - type and p97R155H / + MEFs were LLOMe - treated for 1 h and released for indicated times . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD , Student ' s unpaired t - test ) . Scale bar , 25 µm . B Stable U2OS cell lines were doxycycline - induced to express myc - tagged p97 wild - type or disease - mutants as indicated . Induced parental cells served as control ( U2OS - ) . Cells were treated with LLOMe for 1h and chased for indicated time before fixation . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments . Comparable expression levels were verified ( see Figure EV2A ) . C Stable U2OS cell lines described in ( B ) were treated with LLOMe for 1 h and chased for indicated time . LC3 and GAPDH levels were analyzed by immunoblotting . Quantification is shown in Figure EV2B . D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle . Patient biopsies immunostained for Gal3 and caveolin - 3 ( Cav3 ) . Quantification from 4 random 10X fields for each biopsy and means for controls and patients , respectively . Patients carry p97 mutations R155H ( # 1 - 3 ) or R93C for ( # 4 ) . Patients # 2 and # 3 are cousins . Scale bar , 100 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Impaired clearance of damaged lysosomes in cells expressing a p97 disease-mutant. Wild-type and p97R155H/+MEFs were LLOMe-treated for 1 h and released for indicated times. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD, Student's unpaired t-test). Scale bar, 25 µm.B Stable U2OS cell lines were doxycycline-induced to express myc-tagged p97 wild-type or disease-mutants as indicated. Induced parental cells served as control (U2OS -). Cells were treated with LLOMe for 1h and chased for indicated time before fixation. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments. Comparable expression levels were verified (see Figure EV2A).C Stable U2OS cell lines described in (B) were treated with LLOMe for 1 h and chased for indicated time. LC3 and GAPDH levels were analyzed by immunoblotting. Quantification is shown in Figure EV2B.D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle. Patient biopsies immunostained for Gal3 and caveolin-3 (Cav3). Quantification from 4 random 10X fields for each biopsy and means for controls and patients, respectively. Patients carry p97 mutations R155H (#1-3) or R93C for (#4). Patients #2 and #3 are cousins. Scale bar, 100 µm.


Figure 3A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes . HeLa cells were transfected with indicated siRNA oligos and LLOMe - treated for 1 h . Cells were chased for 12 h , fixed and stained for Gal3 . See Figure EV3 for depletion efficiency . B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition . This threshold was chosen so that less than 1 % of untreated cells were considered positive . Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - sided Welch ' s t - tests ) . C YOD1 , UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes . Cells expressing indicated tagged proteins were LLOMe - treated for 1 h or with vehicle alone ( control ) , fixed 2 h after washout , and stained for endogenous LAMP1 and , in the case of PLAA , with Alexa - 488 conjugated anti - HA antibodies . See Figure EV3B for quantification . D An ATP - stabilized complex of p97 containing both UBXD1 and PLAA . Stable HEK293 cell lines were doxycycline - induced to express p97 wild - type ( wt ) or the ATPase - mutant E578Q ( EQ ) at near endogenous level . Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies . Arrowheads indicate endogenous ( lower band ) and induced p97 ( upper band ) . E Stable HEK293 cells treated as in ( D ) and transiently expressing GFP - tagged YOD1 - wt or the YOD1 - C160S catalytic mutant ( CS ) were processed for co - immunoprecipitation with GFP nanobodies . Asterisk , unspecific band . Note that the YOD1 - CS mutant stabilized p97 binding and , in the p97 - EQ background , also associated with UBXD1 and PLAA . F Control ( IgG ) and UBXD1co - immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP - YOD1 - CS after formaldehyde cross - linking . Ufd1 served as a negative control . Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes. HeLa cells were transfected with indicated siRNA oligos and LLOMe-treated for 1 h. Cells were chased for 12 h, fixed and stained for Gal3. See Figure EV3 for depletion efficiency.B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition. This threshold was chosen so that less than 1% of untreated cells were considered positive. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-sided Welch's t-tests).C YOD1, UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes. Cells expressing indicated tagged proteins were LLOMe-treated for 1 h or with vehicle alone (control), fixed 2 h after washout, and stained for endogenous LAMP1 and, in the case of PLAA, with Alexa-488 conjugated anti-HA antibodies. See Figure EV3B for quantification.D An ATP-stabilized complex of p97 containing both UBXD1 and PLAA. Stable HEK293 cell lines were doxycycline-induced to express p97 wild-type (wt) or the ATPase-mutant E578Q (EQ) at near endogenous level. Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies. Arrowheads indicate endogenous (lower band) and induced p97 (upper band).E Stable HEK293 cells treated as in (D) and transiently expressing GFP-tagged YOD1-wt or the YOD1-C160S catalytic mutant (CS) were processed for co-immunoprecipitation with GFP nanobodies. Asterisk, unspecific band. Note that the YOD1-CS mutant stabilized p97 binding and, in the p97-EQ background, also associated with UBXD1 and PLAA.F Control (IgG) and UBXD1co-immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP-YOD1-CS after formaldehyde cross-linking. Ufd1 served as a negative control. Scale bars, 10 µm.


Figure 4A K63 - and K48 - ubiquitination of damaged lysosomes . Untransfected HeLa cells , or cells expressing mCherry - Gal3 ( Ch - Gal3 ) were LLOMe - treated for 1 h . 2 h after washout , cells were stained with antibodies specific for K63 or K48 chains , and LAMP1 or p62 as indicated . Percentage of K63 - or K48 - ubiquitinated LAMP1 vesicles and percentage of p62 - positive K63 or K48 vesicles were quantified from three independent experiments ( mean ± SD , Student ' s unpaired t - test for LAMP1 ) . * = p < 0 . 05 . B Colocalization of K63 - and K48 - linked ubiquitin conjugates on individual lysosomes . Cells expressing GFP - TAB2 - NZF ( K63 - sensor ) were treated as in ( A ) and stained for K48 chains and LAMP1 . Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure ( van Wijk et al . , 2012 ) . C Cells expressing GFP - p97 - EQ were treated as in ( A ) and stained with antibodies as indicated . D Quantification of the total number of K63 - or K48 - ubiquitinated vesicles per cell , and number of p97 - EQ positive K63 - or K48 - ubiquitinated vesicles ( mean ± SD ) . E Percentage of GFP - p97 - EQ vesicles positive for K63 or K48 chains was determined ( mean ± SD ) . F ELDR components target K48 - conjugates on lysosomes . Detection of GFP - YOD1 - CS or UBXD1 - GFP alone , or co - transfected with PLAA - HA and GFP - p97 - EQ , 2 h after LLOMe washout and co - stained with indicated antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A K63- and K48-ubiquitination of damaged lysosomes. Untransfected HeLa cells, or cells expressing mCherry-Gal3 (Ch-Gal3) were LLOMe-treated for 1 h. 2 h after washout, cells were stained with antibodies specific for K63 or K48 chains, and LAMP1 or p62 as indicated. Percentage of K63- or K48-ubiquitinated LAMP1 vesicles and percentage of p62-positive K63 or K48 vesicles were quantified from three independent experiments (mean ± SD, Student's unpaired t-test for LAMP1). * = p < 0.05.B Colocalization of K63- and K48-linked ubiquitin conjugates on individual lysosomes. Cells expressing GFP-TAB2-NZF (K63-sensor) were treated as in (A) and stained for K48 chains and LAMP1. Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure (van Wijk et al., 2012).C Cells expressing GFP-p97-EQ were treated as in (A) and stained with antibodies as indicated.D Quantification of the total number of K63- or K48-ubiquitinated vesicles per cell, and number of p97-EQ positive K63- or K48-ubiquitinated vesicles (mean ± SD).E Percentage of GFP-p97-EQ vesicles positive for K63 or K48 chains was determined (mean ± SD).F ELDR components target K48-conjugates on lysosomes. Detection of GFP-YOD1-CS or UBXD1-GFP alone, or co-transfected with PLAA-HA and GFP-p97-EQ, 2 h after LLOMe washout and co-stained with indicated antibodies.


Figure 5A Time course of K63 - and K48 - ubiquitination . Control - depleted cells were fixed at indicated time points after LLOMe washout and stained with chain - specific antibodies and LAMP1 . Automated quantification of LAMP1 vesicles positive for K63 or K48 chains . Data represent mean ± SD of three independent experiments . B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes . Experiment and quantification as in ( A ) in cells treated with p97 or YOD1 siRNA oligos . C The p97 - EQ mutant stabilizes K48 - conjugates . Lysosome damage was induced as in ( A ) in cells transfected with GFP - tagged p97 - wt or p97 - EQ . Cells were chased after washout for 12 h and stained for K48 chains . D Quantification of K48 - positive lysosomes 2 h and 12 h after damage in cells treated as in ( A ) . Data represent mean ± SD of three independent experiments . E The catalytically - inactive mutant of YOD1 stabilizes K48 - conjugates . Cells transfected with GFP - tagged YOD1 - wt or the YOD1 - CS were treated and stained as in ( C ) . F Quantification as in ( D ) of cells treated as in ( E ) . * * = p < 0 . 01 * * * = p < 0 . 001 ( Student ' s unpaired t - test ) . ns , not significant . Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Time course of K63- and K48-ubiquitination. Control-depleted cells were fixed at indicated time points after LLOMe washout and stained with chain-specific antibodies and LAMP1. Automated quantification of LAMP1 vesicles positive for K63 or K48 chains. Data represent mean ± SD of three independent experiments.B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes. Experiment and quantification as in (A) in cells treated with p97 or YOD1 siRNA oligos.C The p97-EQ mutant stabilizes K48-conjugates. Lysosome damage was induced as in (A) in cells transfected with GFP-tagged p97-wt or p97-EQ. Cells were chased after washout for 12 h and stained for K48 chains.D Quantification of K48-positive lysosomes 2 h and 12 h after damage in cells treated as in (A). Data represent mean ± SD of three independent experiments.E The catalytically-inactive mutant of YOD1 stabilizes K48-conjugates. Cells transfected with GFP-tagged YOD1-wt or the YOD1-CS were treated and stained as in (C).F Quantification as in (D) of cells treated as in (E). ** = p < 0.01 *** = p < 0.001 (Student's unpaired t-test). ns, not significant. Scale bars, 10 µm.


Figure 6A YOD1 can link p97 to ubiquitin . Ubiquitin - GST ( Ub - GST ) or GST alone was incubated with the indicated purified proteins and isolated . Co - isolated proteins ( pulldown ) were detected in SDS - gels . Note binding of p97 and YOD1 to Ub - GST only in combination . B The S2 site of YOD1 is critical for ubiquitin binding . Experiments as in ( A ) with indicated combinations of p97 and YOD1 - wt or the ubiquitin - binding mutant , MutS2 . C Reconstitution of a complex of ELDR components on ubiquitin . Experiments as in ( A ) with combinations of p97 - wt or p97 - EQ , YOD1 , PLAA and UBXD1 as indicated . Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97 - EQ . D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site . LLOMe - treated cells expressing GFP - YOD1 - CS or the double mutant GFP - YOD1 - CS - MutS2 were chased for 2 h and stained for K48 chains and LAMP1 . Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined . E The S2 site is essential for K48 - conjugate removal . GFP - YOD1 - wt or GFP - YOD1 - MutS2 overexpressing cells treated as in ( D ) were stained for K48 chains . Quantification of the number of K48 - positive vesicles per cell . Data represent mean ± SD of three independent experiments . * * = p < 0 . 01 ( Student ' s unpaired t - test ) . Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A YOD1 can link p97 to ubiquitin. Ubiquitin-GST (Ub-GST) or GST alone was incubated with the indicated purified proteins and isolated. Co-isolated proteins (pulldown) were detected in SDS-gels. Note binding of p97 and YOD1 to Ub-GST only in combination.B The S2 site of YOD1 is critical for ubiquitin binding. Experiments as in (A) with indicated combinations of p97 and YOD1-wt or the ubiquitin-binding mutant, MutS2.C Reconstitution of a complex of ELDR components on ubiquitin. Experiments as in (A) with combinations of p97-wt or p97-EQ, YOD1, PLAA and UBXD1 as indicated. Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97-EQ.D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site. LLOMe-treated cells expressing GFP-YOD1-CS or the double mutant GFP-YOD1-CS-MutS2 were chased for 2 h and stained for K48 chains and LAMP1. Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined.E The S2 site is essential for K48-conjugate removal. GFP-YOD1-wt or GFP-YOD1-MutS2 overexpressing cells treated as in (D) were stained for K48 chains. Quantification of the number of K48-positive vesicles per cell. Data represent mean ± SD of three independent experiments. ** = p < 0.01 (Student's unpaired t-test). Scale bars, 10 µm.


Figure 7A Largely mutually exclusive localization of LC3 and K48 chains . GFP - LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains . Correlation coefficients were calculated for colocalization of LC3 with K63 or K48 . The whiskers represent 10th and 90th percentile . Points below and above the whiskers are drawn as individual dots . The single line indicates the median . * * * = p < 0 . 001 ( Mann - Whitney - U test ) . B Lipofectamine - coated latex beads damage lysosomes and induce their K48 - ubiquitination . Cells expressing mCherry - Gal3 were incubated with Lipofectamine - coated latex beads for 3 h and stained with K48 chain - specific antibodies . C YOD1 association and dissociation precedes LC3 recruitment . Live cell video microscopy stills of cells co - expressing mCherry - YOD1 - wt and GFP - LC3 after treatment with Lipofectamine - coated latex beads for 30 min at 2 min image intervals . D Stabilization of K48 - conjugates on lysosomes impairs recruitment of LC3 . Cells expressing GFP - YOD1 - CS were treated as in ( A ) and stained for LAMP1 and p62 or LC3 . Percentage of GFP - YOD1 vesicles positive for p62 or LC3 was determined ( mean ± SD ) . E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation . Cells were transfected with control ( Ctrl ) , p97 or YOD1 siRNA oligos , treated with LLOMe , fixed after 12 h chase and stained for endogenous LC3 and K48 chains . Quantification of the number of K48 - positive vesicles per cell and number of LC3 - positive K48 vesicles per cell was determined . Data represent mean ± SD of three independent experiments . Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Largely mutually exclusive localization of LC3 and K48 chains. GFP-LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains. Correlation coefficients were calculated for colocalization of LC3 with K63 or K48. The whiskers represent 10th and 90th percentile. Points below and above the whiskers are drawn as individual dots. The single line indicates the median. *** = p < 0.001 (Mann-Whitney-U test).B Lipofectamine-coated latex beads damage lysosomes and induce their K48-ubiquitination. Cells expressing mCherry-Gal3 were incubated with Lipofectamine-coated latex beads for 3 h and stained with K48 chain-specific antibodies.C YOD1 association and dissociation precedes LC3 recruitment. Live cell video microscopy stills of cells co-expressing mCherry-YOD1-wt and GFP-LC3 after treatment with Lipofectamine-coated latex beads for 30 min at 2 min image intervals.D Stabilization of K48-conjugates on lysosomes impairs recruitment of LC3. Cells expressing GFP-YOD1-CS were treated as in (A) and stained for LAMP1 and p62 or LC3. Percentage of GFP-YOD1 vesicles positive for p62 or LC3 was determined (mean ± SD).E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation. Cells were transfected with control (Ctrl), p97 or YOD1 siRNA oligos, treated with LLOMe, fixed after 12 h chase and stained for endogenous LC3 and K48 chains. Quantification of the number of K48-positive vesicles per cell and number of LC3-positive K48 vesicles per cell was determined. Data represent mean ± SD of three independent experiments. Scale bars, 10 µm.


Figure 1Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - WT - FLAG or TFEB - S401A - FLAG treated with 200 μM NaAsO2 or 250 nM Torin - 1 for 1 h . Two different rabbit polyclonal antibodies raised against a TFEB - S401 phospho - specific peptide were tested . Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - WT - FLAG , TFEB - S3A / R4A - FLAG or TFEB - R245 - 247A - FLAG treated with 200 μM NaAsO2 for 1 h . Immunoblots are representative of at least three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG or TFEB-S401A-FLAG treated with 200 μM NaAsO2 or 250 nM Torin-1 for 1 h. Two different rabbit polyclonal antibodies raised against a TFEB-S401 phospho-specific peptide were tested.Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG, TFEB-S3A/R4A-FLAG or TFEB-R245-247A-FLAG treated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.


Figure 2Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h . Quantification of immunoblot data shown in ( A ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , and ( * * * * ) p < 0 . 0001 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) . Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG incubated with either 200 μM NaAsO2 for 1 h , EBSS for 4 h , 100 ng / ml EGF for the indicated times or 37 μM Anisomycin for 1 h . Before the addition of EGF or Anisomycin , cells were serum starved for 8 h . Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG exposed to 30J / m2 of UV - C and allowed to recover in complete medium for the indicated times . Cells were incubated with p38 MAPK inhibitor ( 20 μM , SB203580 ) for 1 h before UV - C irradiation and allowed to recover for 30 min in the presence of the inhibitor . Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG depleted of either p38 MAPK ( α + β ) or JNK1 or JNK2 or JNK ( 1 + 2 ) and incubated with 200 μM NaAsO2 for 1 h . Immunoblots are representative of at least three independent experiments . Immunoblot analysis of in vitro p38 MAPK kinase assay using GST - TFEB - PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP . Quantification of immunoblot data shown in ( F ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( * ) p < 0 . 05 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h. Quantification of immunoblot data shown in (A). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, and (****)p<0.0001 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with either 200 μM NaAsO2 for 1 h, EBSS for 4 h, 100 ng/ml EGF for the indicated times or 37 μM Anisomycin for 1 h. Before the addition of EGF or Anisomycin, cells were serum starved for 8 h.Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG exposed to 30J/m2 of UV-C and allowed to recover in complete medium for the indicated times. Cells were incubated with p38 MAPK inhibitor (20 μM, SB203580) for 1 h before UV-C irradiation and allowed to recover for 30 min in the presence of the inhibitor.Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG depleted of either p38 MAPK (α+β) or JNK1 or JNK2 or JNK(1+2) and incubated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.Immunoblot analysis of in vitro p38 MAPK kinase assay using GST-TFEB-PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Figure 3Immunoblot analysis of protein lysates from Raw 264 . 7 cells stably expressing TFEB - WT - FLAG incubated with 1μg / ml LPS for the indicated times . Immunoblot analysis of protein lysates from Raw 264 . 7 cells stably expressing TFEB - WT - FLAG incubated with either 20 μM SB203580 or 250 nM Torin - 1 for 1 h prior to the addition of 1 μg / ml LPS for 30 min . Quantification of immunoblot data shown in ( B ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , ( * ) p < 0 . 05 and ( * * ) p < 0 . 01 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) . Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg / ml LPS for the indicated times . Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle ( DMSO ) or 20 μM SB203580 for 1 h prior to the addition of 1μg / ml LPS for the indicated times . Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK ( α ) , p38 MAPK ( β ) or p38 MAPK ( α + β ) and incubated with 1 μg / ml LPS for 1 h . Immunoblots are representative of at least three independent experiments . Quantification of immunoblot data shown in ( F ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( * ) p < 0 . 05 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with 1μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with either 20 μM SB203580 or 250 nM Torin-1 for 1 h prior to the addition of 1 μg/ml LPS for 30 min.Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05 and (**)p<0.01 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle (DMSO) or 20 μM SB203580 for 1 h prior to the addition of 1μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK (α), p38 MAPK (β) or p38 MAPK (α+β) and incubated with 1 μg/ml LPS for 1 h. Immunoblots are representative of at least three independent experiments. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Figure 4Immunoblot analysis of protein lysates from naïve THP1 - WT or TFEB - S401A knock - in ( clone M17 ) cells treated with 50 ng / ml PMA for the indicated times , PMA - differentiated THP1 ( Rested ) cells treated without or with 250 nM Torin - 1 for 1 h . Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells treated with 50 ng / ml PMA for the indicated times and PMA - differentiated THP1 ( Rested ) cells . Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA for the indicated times . Immunoblot analysis of proteins from nuclear fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA or 250 nM Torin - 1 for 1 h . Immunoblots are representative of at least three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Immunoblot analysis of protein lysates from naïve THP1-WT or TFEB-S401A knock-in (clone M17) cells treated with 50 ng/ml PMA for the indicated times, PMA-differentiated THP1 (Rested) cells treated without or with 250 nM Torin-1 for 1 h.Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells treated with 50 ng/ml PMA for the indicated times and PMA-differentiated THP1 (Rested) cells.Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for the indicated times.Immunoblot analysis of proteins from nuclear fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA or 250 nM Torin-1 for 1 h. Immunoblots are representative of at least three independent experiments.


Figure 5Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA for 6 h .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for 6 h.


Figure 6Immunoblot analysis of protein lysates and cell culture medium from PMA - differentiated ( Rested ) THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells incubated with 0 . 1 μg / ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min . C - F . Quantification of immunoblot data shown in ( B ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 and ( * * * * ) p < 0 . 0001 from three independent experiments . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Fluorescence confocal microscopy of PMA - differentiated ( Rested ) THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells incubated with 1 μg / ml LPS for 6 h prior to the detection of dead cells with LIVE / DEAD fixable blue dead cell stain . Scale bars , 10 μm . Quantification of the percentage of dead cells shown in ( G ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant and ( * * * * ) p < 0 . 0001 as compared to their corresponding untreated ( Control ) cells , with > 200 cells counted per trial from three independent experiments . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Immunoblot analysis of protein lysates and cell culture medium from PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 0.1 μg/ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min. C-F. Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).Fluorescence confocal microscopy of PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 1 μg/ml LPS for 6 h prior to the detection of dead cells with LIVE/DEAD fixable blue dead cell stain. Scale bars, 10 μm. Quantification of the percentage of dead cells shown in (G). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant and (****)p< 0.0001 as compared to their corresponding untreated (Control) cells, with >200 cells counted per trial from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).


Figure 1A . Blood - stage growth rate of concavin ( - ) parasites in comparison to wild type . Data points represent parasites growing in individual mice , n indicates total number . P - value is calculated using the Mann Whitney test . Shown is the mean ± SEM . B . Gametocyte to ookinete conversion of 3 biological replicates . P - value calculated using the Mann Whitney test . Shown is the mean ± SEM . C . Average speed of moving ookinetes . Data points represent individual ookinetes , n indicates total number of cells from 3 biological replicates . P - values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison . D . Ookinete images of wild type and concavin ( - ) parasites revealing similar cell shapes . Scale bar 5 µm . E . Oocyst development of concavin ( - ) parasites compared to wild type . Data points represent individual midguts observed between d12 - 17 post infection from 3 independent cage feeds . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison . F . Mosquito infections resulted in deformed sporozoites in the salivary gland . Shown are example images of different sporozoites arranged to illustrate their rounding up over time . G . Quantification of sporozoite rounding up over time in the midgut ( MG ) the haemolymph ( HL ) and salivary gland ( SG ) at the indicated days post infection from three biological replicates . Sporozoites were classified as either normal , deformed or round as illustrated in F . H . Average speed of salivary gland sporozoites . Data points represent individual sporozoites , n indicates total number from 3 biological replicates . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test . I . Selected trajectories of manually tracked sporozoites .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O

Figure 1A. Blood-stage growth rate of concavin(-) parasites in comparison to wild type. Data points represent parasites growing in individual mice, n indicates total number. P-value is calculated using the Mann Whitney test. Shown is the mean ± SEM.B. Gametocyte to ookinete conversion of 3 biological replicates. P-value calculated using the Mann Whitney test. Shown is the mean ± SEM. C. Average speed of moving ookinetes. Data points represent individual ookinetes, n indicates total number of cells from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison. D. Ookinete images of wild type and concavin(-) parasites revealing similar cell shapes. Scale bar 5 µm.E. Oocyst development of concavin(-) parasites compared to wild type. Data points represent individual midguts observed between d12-17 post infection from 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison.F. Mosquito infections resulted in deformed sporozoites in the salivary gland. Shown are example images of different sporozoites arranged to illustrate their rounding up over time. G. Quantification of sporozoite rounding up over time in the midgut (MG) the haemolymph (HL) and salivary gland (SG) at the indicated days post infection from three biological replicates. Sporozoites were classified as either normal, deformed or round as illustrated in F.H. Average speed of salivary gland sporozoites. Data points represent individual sporozoites, n indicates total number from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.I. Selected trajectories of manually tracked sporozoites.


Figure 2A . Oocyst development of concavin ( - ) parasites complemented with either the P . berghei gene or the P . falciparum orthologue fused to GFP . Data points represent individual midguts observed between d12 - 17 post infection of 3 independent cage feeds . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test . B . Average speed of salivary gland sporozoites . Data points represent individual sporozoites of 3 independent biological replicates . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test . C . Sum table of mosquito infections of wild type , concavin ( - ) and complemented lines expressing either P . berghei ( Pb ) or P . falciparum ( Pf ) concavin - GFP . Numbers determined on d17 post mosquito infection . Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections . Note the difference of normally shaped and total sporozoites for concavin ( - ) parasites . Images show infection of salivary glands . Scale bar : 100 µmD . Pb concavin - GFP and Pf concavin - GFP localization in blood and mosquito stages . Nuclei ( blue ) stained with Hoechst . Scale bar : 5 µm . E . Localization of P . berghei concavin - GFP in liver stages . Nuclei ( blue ) stained with Hoechst . Scale bar : 5 µm . F . Immunofluorescence images of permeabilized and unpermeabilized concavin - GFP expressing salivary gland sporozoites stained with an anti - GFP antibody . Note that a GFP signal could only be detected after permeabilization , excluding concavin localization on the parasite surface as illustrated in the model . Scale bar : 5 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Oocyst development of concavin(-) parasites complemented with either the P. berghei gene or the P. falciparum orthologue fused to GFP. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.B. Average speed of salivary gland sporozoites. Data points represent individual sporozoites of 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.C. Sum table of mosquito infections of wild type, concavin(-) and complemented lines expressing either P. berghei (Pb) or P. falciparum (Pf) concavin-GFP. Numbers determined on d17 post mosquito infection. Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections. Note the difference of normally shaped and total sporozoites for concavin(-) parasites. Images show infection of salivary glands. Scale bar: 100 µmD. Pb concavin-GFP and Pf concavin-GFP localization in blood and mosquito stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.E. Localization of P. berghei concavin-GFP in liver stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.F. Immunofluorescence images of permeabilized and unpermeabilized concavin-GFP expressing salivary gland sporozoites stained with an anti-GFP antibody. Note that a GFP signal could only be detected after permeabilization, excluding concavin localization on the parasite surface as illustrated in the model. Scale bar: 5 µm.