Figure 2A . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar in MEF Stim1 / Stim2 - / - cells transfected with Stim1 - ( black trace , n = 111 ) , Stim1A ( red trace , n = 116 ) , the combination Stim1 with Stim1A ( blue trace , n = 86 ) or with vector only ( grey trace , n = 79 ) . B . Quantification of changes in ratio of resting , influx rate ( Δratio / time ) , Δpeak and Δplateau measured in A . * * * p < 0 . 001 , Kruskal - Wallis Anova . C . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura 2 ratio ) in HEK293 cells co - transfected with Orai1 and either Stim1 - ( black trace , n = 119 ) or Stim1A IRES - mCherry ( red trace , n = 118 ) . D . Quantification of changes in ratio of resting , influx rate ( Δratio / time ) , Δpeak and Δplateau measured in C . * * p < 0 . 01 * * * p < 0 . 001 Mann - Whitney test . E . Data information : Data were obtained from three biological replicates ( e . g . transfections ) with each three measured dishes ( technical replicates ) with multiple cells each ( yielding a total number n ) and is shown as mean ± s . e . m . I . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura 2 ratio ) in primary astrocytes from C57BL / 6 wt ( CTRL , black trace , n = 49 ) and splice deficient C57BL / 6 10ACon mice ( 10ACon , red trace , n = 82 ) . J . Quantification of parameters determined in [ I ] . * * * p < 0 . 001 Mann - Whitney test . Data information : D were obtained from three biological replicates ( e . g . transfections ) with each three measured dishes ( technical replicates ) with multiple cells each ( yielding a total number n ) and is shown as 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 2A. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar in MEF Stim1/Stim2-/- cells transfected with Stim1- (black trace, n=111), Stim1A (red trace, n=116), the combination Stim1 with Stim1A (blue trace, n=86) or with vector only (grey trace, n=79). B. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in A. *** p<0.001, Kruskal-Wallis Anova. C. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in HEK293 cells co-transfected with Orai1 and either Stim1- (black trace, n=119) or Stim1A IRES-mCherry (red trace, n=118). D. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in C. ** p<0.01 *** p<0.001 Mann-Whitney test. E. Data information: Data were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.I. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in primary astrocytes from C57BL/6 wt (CTRL,black trace, n=49) and splice deficient C57BL/6 10ACon mice (10ACon, red trace, n=82). J. Quantification of parameters determined in [I]. *** p<0.001 Mann-Whitney test. Data information: D were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.


Figure 3C . Images showing representative HEK293 cells expressing Orai1 - GFP ( green = donor molecule ) or either STIM1 - mCherry ( upper panel = acceptor molecule ) or STIM1A - mCherry ( lower panel = acceptor molecule ) after stimulation with TG . Also shown is the overlay of the two channels , as well as the FRET signal .

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

Figure 3C. Images showing representative HEK293 cells expressing Orai1-GFP (green = donor molecule) or either STIM1-mCherry (upper panel = acceptor molecule) or STIM1A-mCherry (lower panel = acceptor molecule) after stimulation with TG. Also shown is the overlay of the two channels, as well as the FRET signal.


Figure 4B . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar with constructs as indicated expressed in HEKO1 . STIM1 ( black , n = 145 ) , STIM1A ( red , n = 157 ) and STIM1A _ D503A ( n = 185 ) . C . Relative fluorescence intensities of mCherry - tagged constructs measured in ( B ) . D . Quantification of changes in resting ratio , TG peak ( ∆ ratio ) and SOCE parameters measured in B . E . Data information : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( each multiple cells ) and shown as mean ± s . e . m for traces and as scatter plots with the underlying boxes showing the means for individual parameters . E . Average traces showing whole - cell current density ( CD ) over time extracted at - 80 mV in HEKO1 cells transfected with STIM1 ( black ) , STIM1A ( red ) or STIM1A _ D503A ( blue ) . F . Average maximum CDs recorded from cells measured in E ( n within bars ) . G Data information : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( each multiple cells ) and shown as mean ± s . e . m for traces and as scatter plots with the underlying boxes showing the means for individual parameters . G . Average current - voltage ( I - V ) relationship of all cells recorded in E .

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

Figure 4B. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKO1. STIM1 (black, n=145), STIM1A (red, n=157) and STIM1A_D503A (n=185). C. Relative fluorescence intensities of mCherry-tagged constructs measured in (B). D. Quantification of changes in resting ratio, TG peak (∆ ratio) and SOCE parameters measured in B. E. Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters.E. Average traces showing whole-cell current density (CD) over time extracted at -80 mV in HEKO1 cells transfected with STIM1 (black), STIM1A (red) or STIM1A_D503A (blue). F. Average maximum CDs recorded from cells measured in E (n within bars). G Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters.G. Average current-voltage (I- V) relationship of all cells recorded in E.


Figure 5F . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar with constructs as indicated expressed in HEKS1 / S2 - / - . G . Quantification of the relative reduction of ratio / time or ratio for cells ( 135 H Data information : , * * * P < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparisons test Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( with multiple cells ) and shown as mean ± s . e . m for traces ( F ) and as scatter plots with the underlying boxes showing the means for individual parameters ( G ) . H , I . Average traces ( mean ± s . e . m . ) showing whole - cell current density ( CD ) over time extracted at - 80 mV in in HEKS1 / S2 - / - cells co - transfected either with STIM1 ( black ) or STIM1A ( red ) and ORAI1 ( H ) or with ORAI1 R77E ( I ) and recorded using extracellular solution containing 2 mM Ca2 + . J . Average maximum CDs recorded from cells measured in [ H , I ] ( n within bars ) . Data information : I , * P < 0 . 05 , * * P < 0 . 01 , unpaired T test with Welch ' s correction . For patch - clamp experiments , number in bars indicate measured cells , which were from three independent transfections .

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 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

Figure 5F. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKS1/S2-/-. G. Quantification of the relative reduction of ratio/time or ratio for cells (135 H Data information: , ***P<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test Data points (total n) were obtained from three biological replicates with each three technical replicates (with multiple cells) and shown as mean±s.e.m for traces (F) and as scatter plots with the underlying boxes showing the means for individual parameters (G).H,I. Average traces (mean±s.e.m.) showing whole- cell current density (CD) over time extracted at -80 mV in in HEKS1/S2-/- cells co-transfected either with STIM1 (black) or STIM1A (red) and ORAI1 (H) or with ORAI1 R77E (I) and recorded using extracellular solution containing 2 mM Ca2+. J. Average maximum CDs recorded from cells measured in [H,I] (n within bars). Data information: I , *P<0.05, **P<0.01, unpaired T test with Welch's correction. For patch-clamp experiments, number in bars indicate measured cells, which were from three independent transfections.


Figure 6B . Hits from ( A ) tested for interaction with commercially available constructs quantified with bimolecular fluorescence complementation ( BiFC ) via flow cytometry . HEKS1 / S2 - / - were transfected with STIM1 - YFPC ( black ) or STIM1A - YFPC ( red ) in combination with POI - YFPN and screened for YFP + cells via FACS , results ( mean ± SD ) from three transfections with each 10 . 000 sorted cells . D . Ratio nuclear NFAT - GFP vs . cytosolic GFP intensity normalized to t = 0 ( mean ± s . e . m . ) after transfection of SH - SY5Y S1 - / - cells with STIM ( n = 42 ) , STIM1A ( n = 82 ) , STIM1A _ D503A ( n = 49 ) or vector only ( ØSTIM , n = 15 ) IRES mCherry and induction of SOCE after stimulation with 1 µM TG . Data ( total n as indicated ) from at least three independent transfections .

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

Figure 6B. Hits from (A) tested for interaction with commercially available constructs quantified with bimolecular fluorescence complementation (BiFC) via flow cytometry. HEKS1/S2-/- were transfected with STIM1-YFPC (black) or STIM1A-YFPC (red) in combination with POI-YFPN and screened for YFP+ cells via FACS, results (mean ± SD) from three transfections with each 10.000 sorted cells.D. Ratio nuclear NFAT-GFP vs. cytosolic GFP intensity normalized to t=0 (mean±s.e.m.) after transfection of SH-SY5Y S1-/- cells with STIM (n=42), STIM1A (n=82), STIM1A_D503A (n=49) or vector only (ØSTIM, n=15) IRES mCherry and induction of SOCE after stimulation with 1 µM TG. Data (total n as indicated) from at least three independent transfections.


Figure 7A . Time course of translocation ( Nuclear NFAT - GFP signal / cytosolic signal normalized to TG addition at t = 0 , mean ± s . e . m , for each time point ) after transfection of indicated constructs and preincubation with 1 µM PF - 04957325 ( analyzed cells : STIM1 + PF n = 75 , STIM1A + PF n = 50 , ØSTIM + PF n = 9 ) Data ( total n as indicated ) from at least three independent transfections . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . B . PF - 04957325 induced difference of the nuclear NFAT - GFP signal vs . cytosolic signal from ( A ) to mean of control without the blocker ( see Fig . 6D ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test of differences . . C Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , 2way Anova Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each . C . Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment , shown as mean ± s . e . m , ØSTIM + 6Bnz - cAMP / 8 - pCPT - cAMP n = 15 / 14 cells ; STIM1 + 6Bnz - cAMP / 8 - pCPT - cAMP : n = 87 / 66 , STIM1A + 6Bnz - cAMP : n = 78 / 67 ; from three independent transfections . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , , Kruskal - Wallis Anova with Dunn ' s multiple comparison test Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each . G . Normalized fluorescence signal change ( mean ± s . e . m ) of R - FlincA , SH - SY5Y S1 - / - cells expressing STIM1 ( n = 20 ) or STIM1A ( n = 22 ) IRES GFP variants and the indicator were stimulated by TG ( 1 μM ) . To avoid photobleaching , values were only recorded after 5 and 30 min , respectively The mutant R - Flinc ( n = 15 ) was measured in SH - SY5Y wt cells after TG stimulation . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparison test Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each .

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 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 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

Figure 7A. Time course of translocation (Nuclear NFAT-GFP signal/cytosolic signal normalized to TG addition at t=0, mean±s.e.m, for each time point) after transfection of indicated constructs and preincubation with 1 µM PF-04957325 (analyzed cells: STIM1 + PF n = 75, STIM1A + PF n = 50, ØSTIM + PF n = 9) Data (total n as indicated) from at least three independent transfections. * p< 0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test. B. PF-04957325 induced difference of the nuclear NFAT-GFP signal vs. cytosolic signal from (A) to mean of control without the blocker (see Fig. 6D). * p< 0.05, **p<0.01, ***p<0.001,Mann-Whitney test of differences.. C Data information: *p<0.05, **p<0.01, ***p<0.001, 2way Anova Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.C. Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment, shown as mean±s.e.m , ØSTIM + 6Bnz-cAMP/8-pCPT-cAMP n = 15/14 cells; STIM1 + 6Bnz-cAMP/8-pCPT-cAMP: n = 87/66, STIM1A + 6Bnz-cAMP : n = 78/67; from three independent transfections. Data information: *p<0.05, **p<0.01, ***p<0.001, , Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.G. Normalized fluorescence signal change (mean±s.e.m) of R-FlincA, SH-SY5Y S1-/- cells expressing STIM1 (n=20) or STIM1A (n=22) IRES GFP variants and the indicator were stimulated by TG (1 μM). To avoid photobleaching, values were only recorded after 5 and 30 min, respectively The mutant R-Flinc (n=15) was measured in SH-SY5Y wt cells after TG stimulation. Data information: *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.


Figure 2Relative expression and co - localization of IL6 mRNA and IL - 6 Signaling Scoring in integrated skin cells by blended UMAPs . Quantification by real - time qPCR analysis of Il6 mRNA in the skin of naïve and irradiated ( 15Gy ) WT mice at indicated times , ( n = 3 - 6 ) . Data information : Data represent mean ± SD ( B , * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by one - way ANOVA with Dunnet ' s multiple comparisons test ( B ) , five independent experiments . Quantification of ventral hair loss area 21 days post - IR ( 15Gy ) in WT and IL - 6 - / - mice , ( n = 11 ) . Photographic images showing hair loss ( area demarked by yellow dashed line ) 21 days post - IR ( 15 Gy ) in wild type ( WT ) and IL - 6 - / - mice . Data information : Data represent mean ± SD C , * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by , two - tailed Mann - Whitney test ( C , ; five independent experiments . Histochemical ( H & E ) and immunostaining ( red ) showing skin morphology , and T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration in WT or IL - 6 - / - mice before ( naïve ) , and 14 days ( H & E , Ly6b + ) , or 21 days ( CD3 + ) post - IR . Infiltrating CD3 + T cells ( yellow arrows ) within degenerating hair follicles ( HF ; dashed lines ) are indicated . Scale bars , 100 μm . Quantification of epidermal thickness at 14 days post - IR ( n = 5 - 8 ) . Kinetics of neutrophil ( Ly6b + ) ( n = 3 - 9 ) and T cell ( CD3 + ) ( n = 3 ) infiltration . Data information : Data represent mean ± SD F , mean ± SD ( G ) . * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by two - tailed Mann - Whitney test F , or two - way ANOVA with Bonferroni ' s posthoc multiple comparisons test ( G ) ; five independent experiments . Quantification by real - time qPCR analysis of Tnfa , Ccl3 , and Ifng mRNAs in whole skin of WT and IL - 6 - / - mice at indicated times post - IR ( 15Gy ) , ( n = 5 - 7 ) . Data information : Data represent mean ± SD H ) * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by , two - tailed Mann - Whitney test H ) , five 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 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 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 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 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

Figure 2Relative expression and co-localization of IL6 mRNA and IL-6 Signaling Scoring in integrated skin cells by blended UMAPs.Quantification by real-time qPCR analysis of Il6 mRNA in the skin of naïve and irradiated (15Gy) WT mice at indicated times, (n=3-6). Data information: Data represent mean ± SD (B, *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by one-way ANOVA with Dunnet's multiple comparisons test (B), five independent experiments.Quantification of ventral hair loss area 21 days post-IR (15Gy) in WT and IL-6-/- mice, (n=11). Photographic images showing hair loss (area demarked by yellow dashed line) 21 days post-IR (15 Gy) in wild type (WT) and IL-6-/- mice. Data information: Data represent mean ± SD C, *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by , two-tailed Mann-Whitney test (C, ; five independent experiments.Histochemical (H&E) and immunostaining (red) showing skin morphology, and T cell (CD3+) and neutrophil (Ly6b+) infiltration in WT or IL-6-/- mice before (naïve), and 14 days (H&E, Ly6b+), or 21 days (CD3+) post-IR. Infiltrating CD3+ T cells (yellow arrows) within degenerating hair follicles (HF; dashed lines) are indicated. Scale bars, 100 μm. Quantification of epidermal thickness at 14 days post-IR (n = 5-8). Kinetics of neutrophil (Ly6b+) (n = 3-9) and T cell (CD3+) (n = 3) infiltration. Data information: Data represent mean ± SD F, mean ± SD (G). *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by two-tailed Mann-Whitney test F, or two-way ANOVA with Bonferroni's posthoc multiple comparisons test (G); five independent experiments.Quantification by real-time qPCR analysis of Tnfa, Ccl3, and Ifng mRNAs in whole skin of WT and IL-6-/- mice at indicated times post-IR (15Gy), (n = 5-7). Data information: Data represent mean ± SD H) *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by , two-tailed Mann-Whitney test H), five independent experiments.


Figure 3Western blot analysis shows phosphorylated STAT3 ( p - Stat3 ) , Akt ( p - Akt ) ) , Erk1 and Erk2 ( p - Erk1 / 2 ) ) , and β - Actin in the skin of WT , IL - 6 - / - , and IL - 1R - / - mice at indicated times ( days ) post - IR ( 15Gy ) . Histochemical ( H & E ) and immunostaining ( red ) staining of T cells ( CD3 + ) , neutrophils ( Ly6b + ) and phosphorylated Stat3 ( p - Stat3 ) ( red nuclear staining , black arrows ) in skin thin sections prepared at 14 days post - IR from mice treated by daily topical application of acquosum ( control ) , ruxolitinib or tofacitinib following irradiation ( 15 Gy ) . Scale bar , 100 μm . ( CD3 and Ly6b immunostaining images from Tofacitinib treated mice represent seperate analyses performed on serial thin sections . ) Quantification of epidermal thickness , T cells ( CD3 + ) and neutrophils ( Ly6b + ) , and p - STAT3 + cells in ( B ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed Student ' s t test ; two independent experiments . Photographic images showing hair loss in mice at 14 days post - IR ( 15 Gy ) following treatment by daily topical application of acquosum ( carrier control ) or JAK inhibitors , ruxolitinib or tofacitinib . Chest and neck hairs of mice in all groups were clipped prior to irradiation to enhance drug absorption . Quantification of the area of ventral hair loss ( in pixels ) in mice in ( D ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed Student ' s t test ; two 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 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 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

Figure 3Western blot analysis shows phosphorylated STAT3 (p-Stat3), Akt (p-Akt)), Erk1 and Erk2 (p-Erk1/2)), and β-Actin in the skin of WT, IL-6-/-, and IL-1R-/- mice at indicated times (days) post-IR (15Gy).Histochemical (H&E) and immunostaining (red) staining of T cells (CD3+), neutrophils (Ly6b+) and phosphorylated Stat3 (p-Stat3) (red nuclear staining, black arrows) in skin thin sections prepared at 14 days post-IR from mice treated by daily topical application of acquosum (control), ruxolitinib or tofacitinib following irradiation (15 Gy). Scale bar, 100 μm. (CD3 and Ly6b immunostaining images from Tofacitinib treated mice represent seperate analyses performed on serial thin sections.) Quantification of epidermal thickness, T cells (CD3+) and neutrophils (Ly6b+), and p-STAT3+ cells in (B), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.Photographic images showing hair loss in mice at 14 days post-IR (15 Gy) following treatment by daily topical application of acquosum (carrier control) or JAK inhibitors, ruxolitinib or tofacitinib. Chest and neck hairs of mice in all groups were clipped prior to irradiation to enhance drug absorption. Quantification of the area of ventral hair loss (in pixels) in mice in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.


Figure 4UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin - derived cell clusters . Photographic images showing hair loss 14 days post - IR ( 15 Gy ) in WT and IL - 1R - / - mice . Quantification of ventral hair loss area 14 days post - IR ( 15Gy ) in WT and IL - 1R - / - mice , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , by two - tailed Mann - Whitney test ( C ) five independent experiments . Histochemical ( H & E ) and immunostaining ( red ) showing morphology and neutrophil ( Ly6b ) infiltration in the skin of mice in ( B ) . Scale bar , 50 μm . Quantification of epidermal thickness , neutrophils ( Ly6b ) and T cells ( CD3 + ) in specimens in ( D ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , by Student ' s t test ( E ) ; five 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 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 O O O O O O O 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 4UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin-derived cell clusters.Photographic images showing hair loss 14 days post-IR (15 Gy) in WT and IL-1R-/- mice. Quantification of ventral hair loss area 14 days post-IR (15Gy) in WT and IL-1R-/- mice, (n = 4). Data information: Data represent mean ± SD. *P<0.05, by two-tailed Mann-Whitney test (C) five independent experiments.Histochemical (H&E) and immunostaining (red) showing morphology and neutrophil (Ly6b) infiltration in the skin of mice in (B). Scale bar, 50 μm. Quantification of epidermal thickness, neutrophils (Ly6b) and T cells (CD3+) in specimens in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, by Student's t test (E); five independent experiments.


Figure 5UMAP plots depicting relative expression and localization of Ccr6 and Ccl20 mRNAs in irradiated and naïve skin - derived cell clusters . Quantification by real - time qPCR analysis of Ccr6 and Ccl20 mRNAs in whole skin of naïve and irradiated ( 15Gy ) WT and IL - 6 - / - mice at indicated times post - IR , ( n = 5 - 7 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments . Photographic images showing the development of radiodermatitis and alopecia in irradiated ( 15 Gy ) WT and CCR6 - / - mice at indicated times post - IR . Escalating radiodermatitis , which manifests on day 7 - 10 as serous exudates on the chin and neck , is visible in both strains and progresses in WT mice to alopecia by day 14 post - IR but resolves spontaneously in the CCR6 - / - mice . Quantification ( lower right ) of the area of hair loss at 14 days post - IR , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments . Histochemical staining ( H & E ) and immunostaining ( red ) show morphology and infiltration of neutrophils ( Ly6b + ) and T cells ( CD3 + ) ( red arrows ) to the skin and to the hair follicle ( HF ) ( dashed lines ) in WT or CCR6 - / - mice 14 days post - IR . Scale bars , 50 μm ( black ) and 25 μm ( red ) . Quantification of epidermal thickness , neutrophils ( Ly6b + ) , total T cell ( CD3 + ) infiltration to the skin , and ( F ) CD3 + cells infiltration to the hair follicle in ( D ) , ( n = 5 - 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t test ( E - Ly6b , by Mann - Whitney test Two independent experiments . MHC class I immunostaining ( red ) of skin thin sections prepared at 14 days post - IR from mice in ( C ) , and quantification ( right ) of MHC I positive hair follicles , ( n = 4 - 5 ) . Scale bar , 50 μm . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments . Quantification by real - time qPCR analysis of Il6 , Il17a , Ccl20 , and Ccl3 mRNAs in the skin of naïve WT and irradiated ( 15Gy ) WT and CCR6 - / - mice at 14 days post - IR , ( n = 3 - 5 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two 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 O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O

Figure 5UMAP plots depicting relative expression and localization of Ccr6 and Ccl20 mRNAs in irradiated and naïve skin-derived cell clusters. Quantification by real-time qPCR analysis of Ccr6 and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) WT and IL-6-/- mice at indicated times post-IR, (n=5-7). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.Photographic images showing the development of radiodermatitis and alopecia in irradiated (15 Gy) WT and CCR6-/- mice at indicated times post-IR. Escalating radiodermatitis, which manifests on day 7-10 as serous exudates on the chin and neck, is visible in both strains and progresses in WT mice to alopecia by day 14 post-IR but resolves spontaneously in the CCR6-/- mice. Quantification (lower right) of the area of hair loss at 14 days post-IR, (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.Histochemical staining (H&E) and immunostaining (red) show morphology and infiltration of neutrophils (Ly6b+) and T cells (CD3+) (red arrows) to the skin and to the hair follicle (HF) (dashed lines) in WT or CCR6-/- mice 14 days post-IR. Scale bars, 50 μm (black) and 25 μm (red). Quantification of epidermal thickness, neutrophils (Ly6b+), total T cell (CD3+) infiltration to the skin, and (F) CD3+ cells infiltration to the hair follicle in (D), (n = 5-6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t test (E-Ly6b, by Mann-Whitney test Two independent experiments.MHC class I immunostaining (red) of skin thin sections prepared at 14 days post-IR from mice in (C), and quantification (right) of MHC I positive hair follicles, (n = 4-5). Scale bar, 50 μm. Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.Quantification by real-time qPCR analysis of Il6, Il17a, Ccl20, and Ccl3 mRNAs in the skin of naïve WT and irradiated (15Gy) WT and CCR6-/- mice at 14 days post-IR, (n = 3-5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Figure 6Photographic images showing hair loss in control ( PBS ) and cyclosporine ( CsA ) treated WT mice at 14 days post - IR ( 15 Gy ) . Quantification of the area of ventral hair loss in mice in ( A ) , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two independent experiments . Histochemical ( H & E ) and immunostaining ( red ) show T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration in the skin of mice in ( A ) . Scale bar , 100 μm . ( Images of CD3 and Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections . ) Quantification of epidermal thickness , and T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration ( red staining ) in skin thin sections from ( C ) , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two independent experiments . Quantification by real - time qPCR analysis of Il6 , Il17a , II22 , Ccl3 , Ccr6 , and Ccl20 mRNAs in whole skin of naïve and irradiated ( 15Gy ) control ( PBS ) and CSA treated WT mice at 14 days post - IR , ( n = 5 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two 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 O O O O O O O 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

Figure 6Photographic images showing hair loss in control (PBS) and cyclosporine (CsA) treated WT mice at 14 days post-IR (15 Gy). Quantification of the area of ventral hair loss in mice in (A), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.Histochemical (H&E) and immunostaining (red) show T cell (CD3+) and neutrophil (Ly6b+) infiltration in the skin of mice in (A). Scale bar, 100 μm. (Images of CD3 and Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections.) Quantification of epidermal thickness, and T cell (CD3+) and neutrophil (Ly6b+) infiltration (red staining) in skin thin sections from (C), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.Quantification by real-time qPCR analysis of Il6, Il17a, II22, Ccl3, Ccr6, and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) control (PBS) and CSA treated WT mice at 14 days post-IR, (n = 5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.


Figure 1 ( A - B ) Immunofluorescence ( IF ) analysis of IκBα in sections from murine small intestine of 2 month - old WT ( A ) and Lgr5 - GFP reporter mice ( B ) . Scale bars , 50μm . IF analysis of P - IκBα in the intestine of 2 month - old WT ( C ) and quantification of P - IκBα positivity Scale bars in C , 25 μmIF analysis of P - IκBα in the intestine of 2 month - old Lgr5 - GFP reporter mice ( D ) , and quantification of P - IκBα positivity inside the Lgr5 - GFP population . A minimum of 30 crypts was counted in 3 Lgr5 - GFP mice . Scale bars in D , 50 μm . ( E ) Distribution of P - IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted . ( F ) Double IF analysis of 2 month - old murine small intestine sections with the indicated antibodies and quantification of P - IκBα positivity inside the EphB2 + population . 70 crypts were counted . Bars represent mean values ± standard error of the mean ( s . e . m ) . Scale bars , 50 μm . ( G ) Double IF analysis of murine small intestine sections with the indicated antibodies . Scale bars , 50 μm . ( H ) Western blot analysis of cytoplasmic ( CYT ) and nuclear ( NUC ) extracts from isolated intestinal crypt cells . ( I ) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti - SUMO2 / 3 antibody . ( J ) IF analysis of developing intestines from E12 . 5 to E17 . 5 with the indicated antibodies . Scale bars , 25 μm . ( K ) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP - sequencing analysis ( n = 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 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 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 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 B-PANEL_START 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 B-PANEL_START 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 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

Figure 1(A-B) Immunofluorescence (IF) analysis of IκBα in sections from murine small intestine of 2 month-old WT (A) and Lgr5-GFP reporter mice (B). Scale bars, 50μm.IF analysis of P-IκBα in the intestine of 2 month-old WT (C) and quantification of P-IκBα positivity Scale bars in C, 25 μmIF analysis of P-IκBα in the intestine of 2 month-old Lgr5-GFP reporter mice (D), and quantification of P-IκBα positivity inside the Lgr5-GFP population. A minimum of 30 crypts was counted in 3 Lgr5-GFP mice. Scale bars in D, 50 μm.(E) Distribution of P-IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted.(F) Double IF analysis of 2 month-old murine small intestine sections with the indicated antibodies and quantification of P-IκBα positivity inside the EphB2+ population. 70 crypts were counted. Bars represent mean values ± standard error of the mean (s.e.m). Scale bars, 50 μm.(G) Double IF analysis of murine small intestine sections with the indicated antibodies. Scale bars, 50 μm.(H) Western blot analysis of cytoplasmic (CYT) and nuclear (NUC) extracts from isolated intestinal crypt cells.(I) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti-SUMO2/3 antibody.(J) IF analysis of developing intestines from E12.5 to E17.5 with the indicated antibodies. Scale bars, 25 μm.(K) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP-sequencing analysis (n=4).


Figure 2 ( A ) Representative images of P4 and P6 mice of the indicated IκBα genotypes . Quantification of the animal weight ( B ) from 50 WT / HET and 15 KO mice analyzed . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant . Quantification of the intestinal length ( C ) from 50 WT / HET and 15 KO mice analyzed . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant . D - G ( D ) Immunohistochemical ( IHC ) , IF and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines ( D ) , 2 month - old IκBβ ; IκBε KO ( E ) and 2 month - old IκBαNES mice ( F and G ) . A minimum of 50 crypts / villus was counted per genotype ( 3 mice ) . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant . Scale bars in D , E , F and G , 50 μm .

O O B-PANEL_START 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 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 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

Figure 2(A) Representative images of P4 and P6 mice of the indicated IκBα genotypes.Quantification of the animal weight (B) from 50 WT/HET and 15 KO mice analyzed. Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant.Quantification of the intestinal length (C) from 50 WT/HET and 15 KO mice analyzed. Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant.D-G (D) Immunohistochemical (IHC), IF and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines (D), 2 month-old IκBβ;IκBε KO (E) and 2 month-old IκBαNES mice (F and G). A minimum of 50 crypts/villus was counted per genotype (3 mice). Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant. Scale bars in D, E, F and G, 50 μm.


Figure 3 ( A ) Representative image of the intestinal populations purified in the cell sorting experiments . ( B ) IF analysis of EphB2 in P6 WT and IκBα KO intestine . Data information : Scale bars , 25 μm . ( C ) P - IκBα IF of EphB2 - high or negative sorted cells . Data information : Scale bars , 25 μm . ( D ) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA - seq . 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq . ( E ) Correlation plot of the indicated differentially expressed gene ( DEG ) sets . Values for DEG in fetal and adult ISC was obtained from Nusse et al , 2012 . ( F ) Table indicating the top 15 up - regulated genes in the IκBα KO EphB2 - high signature including 11 genes in the fetal ISC signature ( in orange ) . ( G ) GSEA of EphB2 - high cells indicating the enrichment of relevant ISC - related pathways . ( H ) Representative image of the Lgr5 + population purified in the cell sorting experiments . ( I ) qPCR analysis of the indicated genes in purified Lgr5 + cells from the different IκBα backgrounds . 4 WT and 1 KO mice were analyzed with at least three technical replicates . Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

O O B-PANEL_START 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 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 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 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 B-PANEL_START 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 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

Figure 3(A) Representative image of the intestinal populations purified in the cell sorting experiments.(B) IF analysis of EphB2 in P6 WT and IκBα KO intestine. Data information: Scale bars , 25 μm.(C) P-IκBα IF of EphB2-high or negative sorted cells. Data information: Scale bars , 25 μm.(D) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA-seq. 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq.(E) Correlation plot of the indicated differentially expressed gene (DEG) sets. Values for DEG in fetal and adult ISC was obtained from Nusse et al, 2012.(F) Table indicating the top 15 up-regulated genes in the IκBα KO EphB2-high signature including 11 genes in the fetal ISC signature (in orange).(G) GSEA of EphB2-high cells indicating the enrichment of relevant ISC-related pathways.(H) Representative image of the Lgr5+ population purified in the cell sorting experiments.(I) qPCR analysis of the indicated genes in purified Lgr5+ cells from the different IκBα backgrounds. 4 WT and 1 KO mice were analyzed with at least three technical replicates. Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, *p-value<0.05, n.s. no significant.


Figure 4 ( A ) IF analysis of c - Rel and RelA / p65 in P6 WT and IκBα KO intestines . Scale bars in 25 μm . ( B ) Number of peaks from p65 ChIP - sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition ( n = 2 P6 WT and n = 2 P6 IκBα KO intestinal crypt cells ) . Data from individual experiments have been deposited at GSE131187 . IF analysis of active Notch1 ( ICN1 ) ( C ) in the indicated IκBα backgrounds at P6 intestines . Scale bars in 25 μm . IF analysis of β - catenin ( D ) in the indicated IκBα backgrounds at P6 intestines . Scale bars 25 μm . ( E ) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n = 3 P6 animals per genotype were analyzed ( a minimum of 15 crypts was counted per genotype ) . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 . Scale bars 25 μm . ( F ) Table indicating the methylation - related genes ( PRC2 members and KDM6 demethylase ) differentially express between IκBα WT and KO EphB2 - high sorted cells ( RNA - seq ) . ( G ) Western blot analysis of IκBα IP from crypt nuclear extracts . ( I ) Representation of H3K27me3 distribution at embryonic E14 . 5 ( n = 2 WT and n = 2 KO mice ) and postnatal P6 ( n = 2 WT and n = 2 KO mice ) ( from ChIP - sequencing ) and expression levels ( from RNA - sequencing ) in the genomic region of the indicated fetal ISC genes . ( J ) Representation of IκBα ( n = 4 ) and H3K27me3 ( n = 2 ) distribution ( from ChIP - sequencing ) and expression levels ( from RNA - sequencing ) in the genomic region of Litaf .

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 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 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 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 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 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 B-PANEL_START 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 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 4(A) IF analysis of c-Rel and RelA/p65 in P6 WT and IκBα KO intestines. Scale bars in 25 μm.(B) Number of peaks from p65 ChIP-sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition (n=2 P6 WT and n=2 P6 IκBα KO intestinal crypt cells). Data from individual experiments have been deposited at GSE131187.IF analysis of active Notch1 (ICN1) (C) in the indicated IκBα backgrounds at P6 intestines. Scale bars in 25 μm.IF analysis of β-catenin (D) in the indicated IκBα backgrounds at P6 intestines. Scale bars 25 μm.(E) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n=3 P6 animals per genotype were analyzed (a minimum of 15 crypts was counted per genotype). Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001. Scale bars 25 μm.(F) Table indicating the methylation-related genes (PRC2 members and KDM6 demethylase) differentially express between IκBα WT and KO EphB2-high sorted cells (RNA-seq).(G) Western blot analysis of IκBα IP from crypt nuclear extracts.(I) Representation of H3K27me3 distribution at embryonic E14.5 (n=2 WT and n=2 KO mice) and postnatal P6 (n=2 WT and n=2 KO mice) (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of the indicated fetal ISC genes.(J) Representation of IκBα (n=4) and H3K27me3 (n=2) distribution (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of Litaf.


Figure 5 ( A ) Representative stereoscopic image of P6 WT and IκBα KO - derived intestinal organoids . Scale bars in A , 100 μm ( B ) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO - derived organoids . Scale bars in B 50 μm . ( C ) qPCR analysis of the indicated ISC genes in WT and IκBα KO - derived organoids . Data information : 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 . ( D ) Correlation plots of the indicated differentially expressed gene ( DEG ) sets obtained in expression microarray ( 5 technical replicates of a minimum of two organoids per genotype were analyzed ) . Values for DEG in fetal and adult organoids were obtained from Mustata et al , 2013 . ( E ) qPCR analysis of the indicated fetal genes in WT and IκBα KO - derived organoids . Data information : 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 . ( F ) Western blot analysis of control and IκBα - deleted organoids using CRISPR - Cas9 technology . ( G ) Representative stereoscopic image of WT and IκBα - deleted organoids . Scale bars in G , 50 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Representative stereoscopic image of P6 WT and IκBα KO-derived intestinal organoids. Scale bars in A, 100 μm(B) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO-derived organoids. Scale bars in B 50 μm.(C) qPCR analysis of the indicated ISC genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05.(D) Correlation plots of the indicated differentially expressed gene (DEG) sets obtained in expression microarray (5 technical replicates of a minimum of two organoids per genotype were analyzed). Values for DEG in fetal and adult organoids were obtained from Mustata et al, 2013.(E) qPCR analysis of the indicated fetal genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05.(F) Western blot analysis of control and IκBα-deleted organoids using CRISPR-Cas9 technology.(G) Representative stereoscopic image of WT and IκBα-deleted organoids. Scale bars in G, 50 μm.


Figure 6A ) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh - RNA for NF - κB elements . Data information Scale bars in A , 100 μm , ( B ) qPCR analysis of the indicated ISC and differentiation genes in the different sh - RNA treated organoids . The canonical NF - κB target gene Cxcl10 is shown as positive control of the experiment . Data information 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant . ( C ) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ - 6438 . Data information Scale bars in 50 μm . ( D - E ) qPCR analysis of the indicated adult and fetal ISC genes ( D ) and elements of the Notch and IFN pathway ( E ) ( differentially expressed in IκBα KO organoids ) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ - 6438 . Data information : 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant . ( F ) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor . Data information Scale bars in F , 50 μm . ( G ) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT . Data information : 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh-RNA for NF-κB elements. Data information Scale bars in A, 100 μm,(B) qPCR analysis of the indicated ISC and differentiation genes in the different sh-RNA treated organoids. The canonical NF-κB target gene Cxcl10 is shown as positive control of the experiment. Data information 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.(C) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information Scale bars in 50 μm.(D-E) qPCR analysis of the indicated adult and fetal ISC genes (D) and elements of the Notch and IFN pathway (E) (differentially expressed in IκBα KO organoids) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.(F) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor. Data information Scale bars in F, 50 μm.(G) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.


Figure 7 ( A ) IF analysis of colonic tissue from control and DSS - treated animals including acute damaged ( AD ) areas and recovery ( R ) areas . Data information : Scale bars 100 μmIF analysis of P - IκBα and the indicated markers in the areas of acute damage and recovery ( B ) Asterisks indicate the presence of rare cells with nuclear P - IκBα . Data information : Scale bars 50 μm . IF analysis of P - IκBα and colocalization of P - IκBα with the ISC marker Olfm4 in acute damaged areas ( C ) . Asterisks indicate the presence of rare cells with nuclear P - IκBα . Data information : Scale bars 50 μm . ( D ) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time - points . 3 technical replicates of organoids from each genotype were analyzed . ( E - F ) Representative images of the colonic tissue from 2 months - old DSS - treated WT and IκBα KO mice are shown . Alcian Blue staining was used to identify the mucus - secreting goblet cells in the colonic glands and Ki67 as proliferation marker . Nuclear counterstain is Fast Red . The table shows number of ulcerations present in the intestines of mice analysed ( 3 WT , 2 IκBα KO ) . Data information : Scale bars in E and F , 100

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 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 O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O 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) IF analysis of colonic tissue from control and DSS-treated animals including acute damaged (AD) areas and recovery (R) areas. Data information: Scale bars 100 μmIF analysis of P-IκBα and the indicated markers in the areas of acute damage and recovery (B) Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm.IF analysis of P-IκBα and colocalization of P-IκBα with the ISC marker Olfm4 in acute damaged areas (C). Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm.(D) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time-points. 3 technical replicates of organoids from each genotype were analyzed.(E-F) Representative images of the colonic tissue from 2 months-old DSS-treated WT and IκBα KO mice are shown. Alcian Blue staining was used to identify the mucus-secreting goblet cells in the colonic glands and Ki67 as proliferation marker. Nuclear counterstain is Fast Red. The table shows number of ulcerations present in the intestines of mice analysed (3 WT, 2 IκBα KO). Data information: Scale bars in E and F, 100 μm


Figure 1A and B ) Confocal microscopy images showing mitochondrial fragmentation in HBV - and HBx - expressing cells , respectively . Huh7 cells were transiently transfected with HBV ( A ) or the HBx - flag construct ( B ) . At 2 days post - transfection , cells prestained with MitoTracker ( Mito , white ) were immunostained with anti - HBsAg ( A , green ) and anti - flag ( B , green ) antibodies , respectively . In the zoomed images , typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown . C ) Whole cell lysates extracted from Huh7 cells with empty vector ( Mock ) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins . β - actin was used as an internal loading control . Mitochondrial fraction ( bottom panel ) isolated from HepAD38 cells ( see the purity in Figure 2B ) was analyzed by Western blotting with phospho - Drp1 ( Ser616 ) antibody . ( D - F ) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV - and HBx - expressing cells , respectively . Huh7 cells transfected with HBV ( D and E ) or HBx - flag construct ( F ) were immunostained with antibodies specific to TOM20 ( red ) , Drp1 ( D , green ) , and phospho - Drp1 ( Ser616 ) ( E and F , green ) , respectively . HBV and HBx gene expression ( white ) was verified using anti - HBsAg ( D and E ) and anti - flag ( F ) antibodies , respectively . In the zoomed images , the yellow color indicates the merge of Drp1 or phospho - Drp1 ( Ser616 ) with mitochondria . ( A , B , D , E , and F ) Transfected ( + ) and untransfected ( − ) cells are marked . Nuclei were stained with DAPI ( blue ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Confocal microscopy images showing mitochondrial fragmentation in HBV- and HBx-expressing cells, respectively. Huh7 cells were transiently transfected with HBV (A) or the HBx-flag construct (B). At 2 days post-transfection, cells prestained with MitoTracker (Mito, white) were immunostained with anti-HBsAg (A, green) and anti-flag (B, green) antibodies, respectively. In the zoomed images, typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown.C) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins. β-actin was used as an internal loading control. Mitochondrial fraction (bottom panel) isolated from HepAD38 cells (see the purity in Figure 2B) was analyzed by Western blotting with phospho-Drp1 (Ser616) antibody.(D-F) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV- and HBx-expressing cells, respectively. Huh7 cells transfected with HBV (D and E) or HBx-flag construct (F) were immunostained with antibodies specific to TOM20 (red), Drp1 (D, green), and phospho-Drp1 (Ser616) (E and F, green), respectively. HBV and HBx gene expression (white) was verified using anti-HBsAg (D and E) and anti-flag (F) antibodies, respectively. In the zoomed images, the yellow color indicates the merge of Drp1 or phospho-Drp1 (Ser616) with mitochondria. (A, B, D, E, and F) Transfected (+) and untransfected (−) cells are marked. Nuclei were stained with DAPI (blue).


Figure 2 ( A ) Huh7 cells transfected with HBV construct were prestained with MitoTracker ( Mito , red ) and subsequently , immunostained with anti - Parkin ( green ) and anti - HBsAg ( white ) antibodies . Nuclei are demarcated with white dots . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria . Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV - expressing cells . ( B ) The cytosolic ( Cyto ) and mitochondrial ( Mito ) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h . Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins . Fractions : WCL , whole cell lysates ; Cyto , purified cytoplasm ; Mito , purified mitochondria . Organelle markers : VDAC1 , mitochondria ; GAPDH , cytoplasm . ( C ) Parkin protein in whole cell lysates of HBV - expressing cells was immunoprecipitated by anti - Parkin antibody , followed by immunoblotting ( IB ) with anti - ubiquitin ( Ub ) antibody . Normal rabbit IgG was used as a control for immunoprecipitation ( IP ) . Western blot analysis for β - actin indicates equivalent amount of cell lysates for IP ( C and D ) . ( D ) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non - targeting ( NT ) and Parkin ( P ) siRNA , respectively , for 48 h was immunoprecipitated by anti - Mfn2 antibody , followed by immunoblotting ( IB ) with anti - ubiquitin ( Ub ) antibody . Normal mouse IgG was used as a control for immunoprecipitation ( IP ) . The protein expression was analyzed by Western blotting with antibodies specific to Mfn2 , Parkin , and β - actin proteins . ( ( E ) Intracellular mRNA levels of Parkin , PINK1 , and LC3B were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in Parkin , PINK1 , and LC3B gene expression . ( F ) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins . β - actin was used as a loading cont ( F ) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins . β - actin was used as a loading control ( F and G ) . The relative intensity of ATF4 , Parkin , PINK1 , and LC3B protein expression normalized to β - actin was analyzed by ImageJ software . . ( G ) Whole cell lysates extracted from Huh7 cells with empty vector ( Mock ) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins .

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 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 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 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

Figure 2(A) Huh7 cells transfected with HBV construct were prestained with MitoTracker (Mito, red) and subsequently, immunostained with anti-Parkin (green) and anti-HBsAg (white) antibodies. Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria. Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV-expressing cells.(B) The cytosolic (Cyto) and mitochondrial (Mito) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h. Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins. Fractions: WCL, whole cell lysates; Cyto, purified cytoplasm; Mito, purified mitochondria. Organelle markers: VDAC1, mitochondria; GAPDH, cytoplasm.(C) Parkin protein in whole cell lysates of HBV-expressing cells was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a control for immunoprecipitation (IP). Western blot analysis for β-actin indicates equivalent amount of cell lysates for IP (C and D).(D) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non-targeting (NT) and Parkin (P) siRNA, respectively, for 48 h was immunoprecipitated by anti-Mfn2 antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal mouse IgG was used as a control for immunoprecipitation (IP). The protein expression was analyzed by Western blotting with antibodies specific to Mfn2, Parkin, and β-actin proteins. ((E) Intracellular mRNA levels of Parkin, PINK1, and LC3B were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin, PINK1, and LC3B gene expression.(F) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading cont(F) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading control (F and G). The relative intensity of ATF4, Parkin, PINK1, and LC3B protein expression normalized to β-actin was analyzed by ImageJ software.. (G) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins.


Figure 3 ( A ) Huh7 cells transiently expressing GFP - LC3 protein were transfected with HBV construct in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 8 h before fixation . At 2 days post - transfection , cells were immunostained with antibodies against HBsAg ( white ) , TOM20 ( blue ) , and Parkin ( red ) . Nuclei are demarcated with white dots . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , the arrows ( white puncta ) indicate GFP - LC3 puncta ( green ) colocalized with TOM20 and Parkin . ( B and C ) Quantification of the number of total GFP - LC3 puncta ( B ) and GFP - LC3 puncta colocalized with TOM20 ( C ) in the panel ( A ) . ( D ) Quantitative analysis of the area of GFP - LC3 puncta ( white ) representing merge of GFP - LC3 puncta , TOM20 , and Parkin in the panel ( A ) .

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 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

Figure 3(A) Huh7 cells transiently expressing GFP-LC3 protein were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells were immunostained with antibodies against HBsAg (white), TOM20 (blue), and Parkin (red). Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin.(B and C) Quantification of the number of total GFP-LC3 puncta (B) and GFP-LC3 puncta colocalized with TOM20 (C) in the panel (A). (D) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A).


Figure 4 ( B ) Huh7 cells transiently expressing Mito - mRFP - EGFP were transfected with HBV , HBx - flag , and HBV - ΔX constructs , respectively , for 48 h . Cells were immunostained with antibodies specific to HBsAg and flag ( white ) , respectively . Nuclei were stained with DAPI ( blue ) . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , fluorescence signals indicate the expression of Mito - mRFP - EGFP targeting mitochondria : yellow color , no mitophagy ; red color , mitophagy . ( C ) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel ( B ) . ( D ) GFP - LC3 - expressing Huh7 cells were transfected with HBV construct in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 8 h before fixation . At 2 days post - transfection , cells prestained with LysoTracker ( Lyso , red ) were immunostained with antibodies against HBsAg ( white ) and TOM20 ( blue ) . Nuclei are demarcated with white dot circles . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoom images , the arrows denoting white puncta indicate GFP - LC3 puncta ( green ) colocalized with TOM20 and lysosome . ( E ) Quantification of the colocalization of GFP - LC3 puncta containing lysosome with mitochondria in the panel ( D ) .

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 B-PANEL_START 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

Figure 4(B) Huh7 cells transiently expressing Mito-mRFP-EGFP were transfected with HBV, HBx-flag, and HBV-ΔX constructs, respectively, for 48 h. Cells were immunostained with antibodies specific to HBsAg and flag (white), respectively. Nuclei were stained with DAPI (blue). Transfected (+) and untransfected (−) cells are marked. In the zoomed images, fluorescence signals indicate the expression of Mito-mRFP-EGFP targeting mitochondria: yellow color, no mitophagy; red color, mitophagy.(C) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel (B).(D) GFP-LC3-expressing Huh7 cells were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells prestained with LysoTracker (Lyso, red) were immunostained with antibodies against HBsAg (white) and TOM20 (blue). Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (−) cells are marked. In the zoom images, the arrows denoting white puncta indicate GFP-LC3 puncta (green) colocalized with TOM20 and lysosome. (E) Quantification of the colocalization of GFP-LC3 puncta containing lysosome with mitochondria in the panel (D).


Figure 5 ( A - C ) Parkin silencing accelerates HBV - induced mitochondrial apoptotic signaling . HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non - targeting ( NT ) and Parkin siRNA , respectively , for 72 h . Cells were used for Western blot analysis using antibodies specific to the indicated proteins ( A ) , caspase - 3 / 7 activity assay ( B ) , and TUNEL assay ( C )

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

Figure 5(A-C) Parkin silencing accelerates HBV-induced mitochondrial apoptotic signaling. HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non-targeting (NT) and Parkin siRNA, respectively, for 72 h. Cells were used for Western blot analysis using antibodies specific to the indicated proteins (A), caspase-3/7 activity assay (B), and TUNEL assay (C)


figf1 ( a - c ) HeLa cells were either left untreated , amino - acid ( aa ) / FBS starved for 5 h , or starved and then recovered in amino - acid / FBS - containing medium , then immunostained , or immunoblotted using antibodies as shown . Co - localization panels show an overlap between mTOR and LAMP1 signals . Note that changes in the positioning of lysosomal mTOR ( quantified as the percentage of cells with predominantly peripheral localization of LAMP1 - positive vesicles ; a , c ) correlate with mTORC1 activity ( levels of phosphorylated S6K relative to the total S6K ; b ) . ( d ) Visualization of Akt activated in response to recovery after serum starvation . After nutrient recovery , LAMP1 - positive vesicles localize to peripheral regions with higher concentrations of phospho - Akt . DAPI , 4 , 6 − diamidino − 2 − phenylindole . For all panels , values are means ± s . e . m . of three independent experiments carried out in triplicate . * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; other comparisons are not significant ( n . s . ) . Representative maximum - intensity projections of serial confocal optical sections are shown . Uncropped images of blots are shown in Supplementary Fig . S7 .

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 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

figf1(a-c) HeLa cells were either left untreated, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunostained, or immunoblotted using antibodies as shown. Co-localization panels show an overlap between mTOR and LAMP1 signals. Note that changes in the positioning of lysosomal mTOR (quantified as the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles; a,c) correlate with mTORC1 activity (levels of phosphorylated S6K relative to the total S6K; b).(d) Visualization of Akt activated in response to recovery after serum starvation. After nutrient recovery, LAMP1-positive vesicles localize to peripheral regions with higher concentrations of phospho-Akt. DAPI, 4,6−diamidino−2−phenylindole. For all panels, values are means ± s.e.m. of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons are not significant (n.s.). Representative maximum-intensity projections of serial confocal optical sections are shown. Uncropped images of blots are shown in Supplementary Fig. S7.


figf2 ( a , b ) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability . Cells were treated as in Fig . 1a followed by incubation with dimethylsulphoxide ( vehicle ) or with nocodazole during the last 2 h before fixation / lysis . Samples were analysed by immunofluorescence ( a ) or immunoblotting ( b ) . Quantification of phopho - S6K levels is shown in ( b ) . ( c - e ) Changes in lysosomal positioning induced by kinesin - or small GTPase - family members ( c , d ) correlate with changes in mTORC1 activity ( e ) . HeLa cells were transfected with overexpression constructs ( OE ) or with siRNA as shown , followed by immunofluorescence ( c , d ) or by immunoblotting ( e ) analyses . Values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a,b) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability. Cells were treated as in Fig. 1a followed by incubation with dimethylsulphoxide (vehicle) or with nocodazole during the last 2 h before fixation/lysis. Samples were analysed by immunofluorescence (a) or immunoblotting (b). Quantification of phopho-S6K levels is shown in (b).(c-e) Changes in lysosomal positioning induced by kinesin- or small GTPase-family members (c,d) correlate with changes in mTORC1 activity (e). HeLa cells were transfected with overexpression constructs (OE) or with siRNA as shown, followed by immunofluorescence (c,d) or by immunoblotting (e) analyses. Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.


figf3 ( a - c ) HeLa cells transfected with ARL8B or KIF2 siRNA , or with ARL8B overexpression construct ( non - targeting siRNA and empty pcDNA vector used as transfection controls ) , were either left untreated , serum / amino - acid starved for 5 h , or starved and then recovered in amino - acid - and FBS - containing medium for 30 min . Cells were immunostained using LAMP1 antibody ( a ) and the percentage of cells with predominantly peripheral localization of LAMP1 - positive vesicles was quantified ( b ) or subjected to immunoblotting ( c ) using antibodies as shown . Quantification of phospho - S6K levels relative to the total S6K is shown in ( c ) . Values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf3(a-c) HeLa cells transfected with ARL8B or KIF2 siRNA, or with ARL8B overexpression construct (non-targeting siRNA and empty pcDNA vector used as transfection controls), were either left untreated, serum/amino-acid starved for 5 h, or starved and then recovered in amino-acid- and FBS-containing medium for 30 min. Cells were immunostained using LAMP1 antibody (a) and the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles was quantified (b) or subjected to immunoblotting (c) using antibodies as shown. Quantification of phospho-S6K levels relative to the total S6K is shown in (c). Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.


figf4 ( a ) Starvation increases pHi in HeLa cells allowed to starve using three different protocols ( see Methods ) with or without subsequent recovery . ( b - d ) Changing pHi from 7 . 1 to 7 . 7 is sufficient to affect localization of lysosomes and mTORC1 activity . pHi was titrated in full tissue - culture medium containing nigericin , which enables changes in pHi to be forced by altering pH in the medium , followed by immunostaining ( b , c ) or western blotting ( d ) using antibodies as shown . ( e ) Changes in lysosomal localization have no effect on pHi . ARL8B and KIF2 were overexpressed or knocked down in HeLa cells followed by pHi measurement . ( f , g ) Nutrients and pHi affect levels of ARL8 and KIF2 in lysosomal fractions . Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation - recovery ( f ) or to 1 h changes of pHi in full tissue - culture medium containing nigericin ( g ) are shown . ( h , i ) Effect of nutrients and pHi on binding of ARL8 and KIF2 to polymerized microtubules . HeLa cells treated as in ( f ) and ( g ) , followed by isolation of polymerized microtubule fraction and western blotting . The asterisk indicates a nonspecific band . For all panels values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

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 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 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 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

figf4(a) Starvation increases pHi in HeLa cells allowed to starve using three different protocols (see Methods) with or without subsequent recovery.(b-d) Changing pHi from 7.1 to 7.7 is sufficient to affect localization of lysosomes and mTORC1 activity. pHi was titrated in full tissue-culture medium containing nigericin, which enables changes in pHi to be forced by altering pH in the medium, followed by immunostaining (b,c) or western blotting (d) using antibodies as shown.(e) Changes in lysosomal localization have no effect on pHi. ARL8B and KIF2 were overexpressed or knocked down in HeLa cells followed by pHi measurement.(f,g) Nutrients and pHi affect levels of ARL8 and KIF2 in lysosomal fractions. Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation-recovery (f) or to 1 h changes of pHi in full tissue-culture medium containing nigericin (g) are shown.(h,i) Effect of nutrients and pHi on binding of ARL8 and KIF2 to polymerized microtubules. HeLa cells treated as in (f) and (g), followed by isolation of polymerized microtubule fraction and western blotting. The asterisk indicates a nonspecific band. For all panels values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.