Figure 4 ( A ) HeLa cells left unstimulated ( CTR ) or infected with Listeria WT for 1 - 4 h , or with Listeria TKO for 4 h and with analysed by IF using an antibody against NDP52 . ( B ) Higher magnification view of the selected area from ( A ) showing that NDP52 + granules generally form at the vicinity of cytosolic Listeria . ( C ) Percentage of cells infected with Listeria WT for 1 - 4 h , or with Listeria TKO for 4 h , displaying one or several NDP52 + granules . Values are means ± s . e . m . n = 3 . ( D ) Percentage of cells infected for 4 h with different Listeria mutants as indicated displaying one or several NDP52 + granules . Values are means ± s . e . m . n = 3 . ( E ) HeLa cells infected with Listeria WT , PlcA / B − or PlcA / B − ectopically expressing PlcA for 3 h , analysed by IF using an antibody against NDP52 . ( F , G ) Percentage of cells infected for 3 h with Listeria WT , PlcA / B − or PlcA / B − ectopically expressing PlcA displaying one or several NDP52 + granules ( F ) or bacteria entrapped in NDP52 + autophagosomes ( G ) . Values are means ± s . e . m . n = 2 . Scale bars : 5 μm .

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Figure 4(A) HeLa cells left unstimulated (CTR) or infected with Listeria WT for 1-4 h, or with Listeria TKO for 4 h and with analysed by IF using an antibody against NDP52.(B) Higher magnification view of the selected area from (A) showing that NDP52+ granules generally form at the vicinity of cytosolic Listeria.(C) Percentage of cells infected with Listeria WT for 1-4 h, or with Listeria TKO for 4 h, displaying one or several NDP52+ granules. Values are means±s.e.m. n=3.(D) Percentage of cells infected for 4 h with different Listeria mutants as indicated displaying one or several NDP52+ granules. Values are means±s.e.m. n=3.(E) HeLa cells infected with Listeria WT, PlcA/B− or PlcA/B− ectopically expressing PlcA for 3 h, analysed by IF using an antibody against NDP52.(F, G) Percentage of cells infected for 3 h with Listeria WT, PlcA/B− or PlcA/B− ectopically expressing PlcA displaying one or several NDP52+ granules (F) or bacteria entrapped in NDP52+ autophagosomes (G). Values are means±s.e.m. n=2. Scale bars: 5 μm.


Figure 5 ( A ) HeLa cells transfected with GFP ‐ LC3 and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52 . ( B ) Murine embryonic fibroblasts ( MEFs ) from WT or ATG16L1 ‐ deficient ( ATG16L1 KO ) mice infected with Listeria WT for 4 h were analysed by IF using an antibody against ubiquitinated proteins ( Ubi ) . ( C , D ) HeLa cells infected with Listeria WT for 4 h analysed by IF using antibodies against NDP52 and WIPI ‐ 2 ( C ) or Galectin ‐ 8 ( Gal ‐ 8 ) and calreticulin ( D ) . ( E ) HeLa cells infected with Listeria WT for 4 h , in the absence or presence of Wortmannin added during the last hour of infection , analysed by IF using antibodies against NDP52 and WIPI ‐ 2 . Scale bars : 5 μm for all panels except ( D ) ( 1 . 5 μm ) .

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Figure 5(A) HeLa cells transfected with GFP‐LC3 and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52.(B) Murine embryonic fibroblasts (MEFs) from WT or ATG16L1‐deficient (ATG16L1 KO) mice infected with Listeria WT for 4 h were analysed by IF using an antibody against ubiquitinated proteins (Ubi).(C, D) HeLa cells infected with Listeria WT for 4 h analysed by IF using antibodies against NDP52 and WIPI‐2 (C) or Galectin‐8 (Gal‐8) and calreticulin (D).(E) HeLa cells infected with Listeria WT for 4 h, in the absence or presence of Wortmannin added during the last hour of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. Scale bars: 5 μm for all panels except (D) (1.5 μm).


Figure 6 ( A ) HeLa cells were transfected with GFP ‐ RFP ‐ LC3 and either were left uninfected ( t = 0 ) or were infected with Listeria WT or PlcA / B − for 1 - 4 h . GFP + RFP + and GFP − RFP + puncta appear yellow and red , respectively . Percentage of cells infected with Listeria WT or PlcA / B − for 1 - 4 h displaying GFP − RFP + red puncta were quantified . Values are means ± s . e . m . n = 3 . * * P0 . 01 ; * * * P0 . 001 . ( B ) HeLa cells left unstimulated ( CTR ) or infected with Listeria WT or PlcA / B − for 4 h were lysed and PI3P levels measured by competitive ELISA . ( C ) HeLa cells transfected with GFP ‐ PKCδ and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52 . The arrow indicates an NDP52 + granule . ( D ) HeLa cells infected with Listeria PlcA / B − for 3 h , in the absence or presence of Wortmannin added during the last 10 min of infection , analysed by IF using antibodies against NDP52 and WIPI ‐ 2 . The arrow indicates an NDP52 + granule . Scale bars : 5 μm .

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Figure 6(A) HeLa cells were transfected with GFP‐RFP‐LC3 and either were left uninfected (t=0) or were infected with Listeria WT or PlcA/B− for 1-4 h. GFP+ RFP+ and GFP− RFP+ puncta appear yellow and red, respectively. Percentage of cells infected with Listeria WT or PlcA/B− for 1-4 h displaying GFP− RFP+ red puncta were quantified. Values are means±s.e.m. n=3. **P0.01; ***P0.001.(B) HeLa cells left unstimulated (CTR) or infected with Listeria WT or PlcA/B− for 4 h were lysed and PI3P levels measured by competitive ELISA.(C) HeLa cells transfected with GFP‐PKCδ and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52. The arrow indicates an NDP52+ granule.(D) HeLa cells infected with Listeria PlcA/B− for 3 h, in the absence or presence of Wortmannin added during the last 10 min of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. The arrow indicates an NDP52+ granule. Scale bars: 5 μm.


Figure 7 ( A , B ) HeLa cells infected for 4 h with Listeria WT expressing GFP ( WT GFP ) , Listeria PlcA / B − expressing mCherry ( PlcA / B − mCherry ) , or both Listeria strains at a 1 : 1 ratio , were analysed by IF using an antibody against NDP52 ( A ) . ( B ) Quantification of the percentage of cells displaying NDP52 + PlcA / B − mCherry from the experiments described in ( A ) , in cells infected with either PlcA / B − mCherry alone or WT GFP and PlcA / B − mCherry ( co ‐ infection ) . Values are means ± s . e . m . n = 3 . * * P0 . 01 . ( C ) Murine embryonic fibroblasts ( MEFs ) from WT or ATG16L1 ‐ deficient ( ATG16L1 KO ) mice infected for 4 h with WT GFP or Listeria PlcA / B − Listeria , either separately ( Single ) or in combination ( Mix ) were lysed and colony forming units ( CFUs ) determined on agar plates containing appropriate selection markers . Each point represents the value from individual independent experiments . * * P0 . 01 ; * P0 . 05 . Scale bars : 2 . 5 μm .

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

Figure 7(A, B) HeLa cells infected for 4 h with Listeria WT expressing GFP (WT GFP), Listeria PlcA/B− expressing mCherry (PlcA/B− mCherry), or both Listeria strains at a 1:1 ratio, were analysed by IF using an antibody against NDP52 (A). (B) Quantification of the percentage of cells displaying NDP52+ PlcA/B− mCherry from the experiments described in (A), in cells infected with either PlcA/B− mCherry alone or WT GFP and PlcA/B−mCherry (co‐infection). Values are means±s.e.m. n=3. **P0.01.(C) Murine embryonic fibroblasts (MEFs) from WT or ATG16L1‐deficient (ATG16L1 KO) mice infected for 4 h with WT GFP or Listeria PlcA/B− Listeria, either separately (Single) or in combination (Mix) were lysed and colony forming units (CFUs) determined on agar plates containing appropriate selection markers. Each point represents the value from individual independent experiments. **P0.01; *P0.05. Scale bars: 2.5 μm.


Figure 1METTL14 is arginine methylated at its C - terminal IDR in cells . HEK293 cells expressing Flag - tagged FL or C - terminal IDR - truncated ( 1 - 400 ) METTL14 were lysed and immunoprecipitated with an anti - Flag antibody . Arginine methylation of immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA . the black triangles indicate arginine methylated - METTL14 ; open triangles indicate the immunoprecipitated METTL14 proteins .

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Figure 1METTL14 is arginine methylated at its C-terminal IDR in cells. HEK293 cells expressing Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14 were lysed and immunoprecipitated with an anti-Flag antibody. Arginine methylation of immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA. the black triangles indicate arginine methylated-METTL14; open triangles indicate the immunoprecipitated METTL14 proteins.


Figure 2The C - terminal IDR of METTL14 is essential for its interaction with PRMT1 . HEK293 cells were transfected with GFP - tagged PRMT1 and Flag - tagged FL or C - terminal IDR - truncated ( 1 - 400 ) METTL14 . IP was performed using an anti - GFP antibody , and Western blot analysis was performed using anti - GFP and anti - Flag antibodies . Knockdown of PRMT1 expression reduces METTL14 arginine methylation . HEK293 cells were transfected with control siRNA ( siCtrl ) and the siRNA targeting PRMT1 ( siPRMT1 ) . METTL14 was immunoprecipitated from these cells , and its methylation level was detected by Western blot analysis using an anti - ASYM26 antibody . * indicates the location of the IgG heavy chain .

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Figure 2The C-terminal IDR of METTL14 is essential for its interaction with PRMT1. HEK293 cells were transfected with GFP-tagged PRMT1 and Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14. IP was performed using an anti-GFP antibody, and Western blot analysis was performed using anti-GFP and anti-Flag antibodies.Knockdown of PRMT1 expression reduces METTL14 arginine methylation. HEK293 cells were transfected with control siRNA (siCtrl) and the siRNA targeting PRMT1 (siPRMT1). METTL14 was immunoprecipitated from these cells, and its methylation level was detected by Western blot analysis using an anti-ASYM26 antibody. * indicates the location of the IgG heavy chain.


Figure 3Arginine methylation of the C - terminal IDR enhances the RNA methylation activity of the METTL14 / METTL3 complex in vitro . In vitro RNA methylation assays were performed by incubating biotin - labeled RNA substrates with METTL3 / METTL14 methyltransferase complexes containing WT , hypomethylated ( MS023 ) , and arginine methylation - deficient ( RK ) mutant METTL14 in various concentrations ( 10 - 100 nM ) . The methylation status of the METTL3 / METTL14 complex was confirmed by Western blot analysis using an anti - ADMA antibody . Coomassie staining shows the purification of the enzyme complex . Enzymatic activity was measured in counts per minute ( c . p . m . ) using a scintillation counter . Data from three replicates were analyzed by Student ' s t - test and shown as mean ± SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; ns , not significant .

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Figure 3Arginine methylation of the C-terminal IDR enhances the RNA methylation activity of the METTL14/METTL3 complex in vitro. In vitro RNA methylation assays were performed by incubating biotin-labeled RNA substrates with METTL3/METTL14 methyltransferase complexes containing WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14 in various concentrations (10-100 nM). The methylation status of the METTL3/METTL14 complex was confirmed by Western blot analysis using an anti-ADMA antibody. Coomassie staining shows the purification of the enzyme complex. Enzymatic activity was measured in counts per minute (c.p.m.) using a scintillation counter. Data from three replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01; ns, not significant.


Figure 4Co - IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023 - treated HEK293 cells . Cells were treated with either DMSO or MS023 ( 1 μM ) for 48 h before they were lysed . IP was performed using control IgG and METTL14 antibodies , respectively . Western blot analysis was performed using anti - RNAPII , anti - ADMA , anti - METTL14 , and anti - METTL3 antibodies . Co - IP assays were performed to examine the involvement of RNA in the METTL14 - RNAPII interaction . Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP . Western blot analysis was performed using anti - RNAPII , anti - METTL3 , and anti - METTL14 antibodies .

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Figure 4Co-IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023-treated HEK293 cells. Cells were treated with either DMSO or MS023 (1 μM) for 48 h before they were lysed. IP was performed using control IgG and METTL14 antibodies, respectively. Western blot analysis was performed using anti-RNAPII, anti-ADMA, anti-METTL14, and anti-METTL3 antibodies.   Co-IP assays were performed to examine the involvement of RNA in the METTL14-RNAPII interaction. Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP. Western blot analysis was performed using anti-RNAPII, anti-METTL3, and anti-METTL14 antibodies.


Figure 5m6A levels are reduced in mESCs expressing arginine methylation - deficient ( RK ) mutant METTL14 . The mRNA purified from WT , Mettl14 KO , KO + WT , and KO + RK mESCs was subjected to LC - MS / MS analysis to quantify m6A levels ( presented as the m6A / A ratio ) . data from three independent replicates were analyzed by Student ' s t - test and shown as mean ± SD . * , p < 0 . 05 ; * * , p < 0 . 01 , * * * , p < 0 . 001 .

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Figure 5m6A levels are reduced in mESCs expressing arginine methylation-deficient (RK) mutant METTL14. The mRNA purified from WT, Mettl14 KO, KO+WT, and KO+RK mESCs was subjected to LC-MS/MS analysis to quantify m6A levels (presented as the m6A/A ratio). data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.


Figure 6MeRIP ( m6A - IP ) - qPCR assays were performed for WT , Mettl14 KO , KO + WT , and KO + RK mESCs to validate the MeRIP - seq results . m6A - negative regions of the transcripts ( blue ) were included as negative controls . : In ( B ) , data from three independent replicates were analyzed by Student ' s t - test and shown as mean ± SD . * , p < 0 . 05 ; * * , p < 0 . 01 , * * * , p < 0 . 001 . The expression of ICL repair genes is reduced in mESCs expressing arginine methylation - deficient mutant ( RK ) METTL14 . Total cell lysates from WT , Mettl14 KO , KO + WT , and KO + RK mESCs were subjected to Western blot analysis using the indicated antibodies .

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Figure 6MeRIP (m6A-IP)-qPCR assays were performed for WT, Mettl14 KO, KO+WT, and KO+RK mESCs to validate the MeRIP-seq results. m6A-negative regions of the transcripts (blue) were included as negative controls. : In (B) , data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.The expression of ICL repair genes is reduced in mESCs expressing arginine methylation-deficient mutant (RK) METTL14. Total cell lysates from WT, Mettl14 KO, KO+WT, and KO+RK mESCs were subjected to Western blot analysis using the indicated antibodies.


Figure 7mESCs expressing arginine methylation - deficient mutant ( RK ) METTL14 are sensitive to ICL damage induced by MMC . WT , Mettl14 KO , KO + WT , and KO + RK mESCs were treated with various concentrations of MMC for 4 days before cell viability was measured . Similar to ( A ) , except that mESCs were treated with cisplatin , another ICL - inducing chemical , at various concentrations . In both ( A ) and ( B ) , each point represents the average of three independent replicates and error bars represent standard deviation ( SD ) . Statistical analysis was performed using Student ' s t - test . * , p < 0 . 05 .

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Figure 7mESCs expressing arginine methylation-deficient mutant (RK) METTL14 are sensitive to ICL damage induced by MMC. WT, Mettl14 KO, KO+WT, and KO+RK mESCs were treated with various concentrations of MMC for 4 days before cell viability was measured.   Similar to (A), except that mESCs were treated with cisplatin, another ICL-inducing chemical, at various concentrations.   In both (A) and (B), each point represents the average of three independent replicates and error bars represent standard deviation (SD). Statistical analysis was performed using Student's t-test. *, p < 0.05.


Figure 1Phenotypic analysis . Wild - type , uvr8 , and rup1 rup2 seedlings were grown in 1 / 2 MS with or without UV - B ( 1 W / m2 ) for 2 weeks . Images are shown in ( A ) ; scale bars = 5 mm . Phenotypic analysis . Wild - type , uvr8 , and rup1 rup2 seedlings were grown in 1 / 2 MS with or without UV - B ( 1 W / m2 ) for 2 weeks . The lateral root density ( number of lateral roots / length of primary root ) ( B ) and average length of lateral roots ( each plant ) ( C ) of the indicated genotypes were measured . SDs ( n > 8 independent seedlings ) are indicated . Distribution of lateral root primordia stages . Seedlings of the indicated genotypes were grown in 1 / 2 MS treatment with or without UV - B ( 1 W / m2 ) for 2 weeks . SDs ( n > 8 ) are indicated . GUS staining of seedlings expressing DR5p : : GUS transgene in the WT , uvr8 and rup1 rup2 background . Seedlings were grown in 1 / 2 MS with or without UV - B for 2 weeks . Images of lateral root primordia ( E ) and lateral roots ( F ) are shown . Scale bars = 100 and 50 μm , respectively .

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Figure 1Phenotypic analysis. Wild-type, uvr8, and rup1 rup2 seedlings were grown in 1/2 MS with or without UV-B (1 W/m2) for 2 weeks. Images are shown in (A); scale bars = 5 mm.Phenotypic analysis. Wild-type, uvr8, and rup1 rup2 seedlings were grown in 1/2 MS with or without UV-B (1 W/m2) for 2 weeks. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (each plant) (C) of the indicated genotypes were measured. SDs (n > 8 independent seedlings) are indicated.Distribution of lateral root primordia stages. Seedlings of the indicated genotypes were grown in 1/2 MS treatment with or without UV-B (1 W/m2) for 2 weeks. SDs (n > 8) are indicated.GUS staining of seedlings expressing DR5p::GUS transgene in the WT, uvr8 and rup1 rup2 background. Seedlings were grown in 1/2 MS with or without UV-B for 2 weeks. Images of lateral root primordia (E) and lateral roots (F) are shown. Scale bars = 100 and 50 μm, respectively.


Figure 2Phenotypic analysis . Seedlings of the indicated genotypes were grown in 1 / 2 MS with the addition of a series of concentrations of NAA under continuous white light or white plus UV - B ( 1 W / m2 ) light for 2 weeks . The lateral root density ( number of lateral roots / length of primary root ) ( A ) and average length of lateral roots ( B ) of the indicated genotypes were measured . UV - B inhibits the response of Arabidopsis seedlings to exogenously added NAA in a UVR8 - dependent manner , since the response to NAA was not repressed in uvr8 . Seedlings of the indicated genotypes were grown in LD ( 16 - h light / 8 - h dark ) conditions for 5 days , then transplanted to new plates containing 0 . 4 μM NAA and kept in continuous white light or white light plus UV - B ( 1 W / m2 ) for 7 days . Images are shown in ( C ) ; scale bars = 2 mm . The lateral root density ( number of lateral roots / length of primary root ) ( D ) and average length of lateral roots ( E ) of the indicated genotypes were measured .

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Figure 2Phenotypic analysis. Seedlings of the indicated genotypes were grown in 1/2 MS with the addition of a series of concentrations of NAA under continuous white light or white plus UV-B (1 W/m2) light for 2 weeks. The lateral root density (number of lateral roots/length of primary root) (A) and average length of lateral roots (B) of the indicated genotypes were measured.UV-B inhibits the response of Arabidopsis seedlings to exogenously added NAA in a UVR8-dependent manner, since the response to NAA was not repressed in uvr8. Seedlings of the indicated genotypes were grown in LD (16-h light/ 8-h dark) conditions for 5 days, then transplanted to new plates containing 0.4 μM NAA and kept in continuous white light or white light plus UV-B (1 W/m2) for 7 days. Images are shown in (C); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (D) and average length of lateral roots (E) of the indicated genotypes were measured.


Figure 3Phenotypic analysis . WT , GR - UVR8R338A / uvr8 and GR - UVR8W285F / uvr8 seedlings were grown in LD ( 16 - h light / 8 - h dark ) for 5 days , then transplanted to new medium with 0 . 4 μM NAA and with or without 20 μM DEX and kept in white light plus UV - B ( 1 W / m2 ) for 7 days . Images are shown in ( A ) ; scale bars = 2 mm . The lateral root density ( number of lateral roots / length of primary root ) ( B ) and average length of lateral roots ( C ) of indicated genotypes were measured . SDs ( n > 8 ) are indicated . UVR8 inhibited auxin responses under UV - B in a tissue - autonomous way . WT and uvr8 seedlings grown in LD for 5 days were used for reciprocal grafting . Seedlings were kept in LD for 7 days after grafting , then transplanted to new medium containing 0 . 4 μM NAA and kept in UV - B light ( 1 W / m2 ) for 10 days . Images are shown in ( D ) ; scale bar = 2 mm . The lateral root density ( number of lateral roots / length of primary root ) ( E ) and average length of lateral roots ( F ) of indicated genotypes were measured . SDs ( n > 8 ) are indicated .

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 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

Figure 3Phenotypic analysis. WT, GR-UVR8R338A/uvr8 and GR-UVR8W285F/uvr8 seedlings were grown in LD (16-h light/ 8-h dark) for 5 days, then transplanted to new medium with 0.4 μM NAA and with or without 20 μM DEX and kept in white light plus UV-B (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of indicated genotypes were measured. SDs (n > 8) are indicated.UVR8 inhibited auxin responses under UV-B in a tissue-autonomous way. WT and uvr8 seedlings grown in LD for 5 days were used for reciprocal grafting. Seedlings were kept in LD for 7 days after grafting, then transplanted to new medium containing 0.4 μM NAA and kept in UV-B light (1 W/m2) for 10 days. Images are shown in (D); scale bar = 2 mm. The lateral root density (number of lateral roots/length of primary root) (E) and average length of lateral roots (F) of indicated genotypes were measured. SDs (n > 8) are indicated.


Figure 4Transcriptome analysis of gene expression regulated by auxin , UV - B , and UVR8 . ( A ) Heat map of UV - B - , UVR8 - , and auxin - regulated genes . The parameter measured by color key shows the Log - fold change . ( B ) Auxin signaling genes are up - regulated by auxin treatment but down - regulated by UV - B in a UVR8 - dependent manner . Three biological replicates were analyzed and final Log fold - change is shown . Quantitative RT - PCR results showing that UV - B represses the expression of auxin signaling genes . Six - day - old WT seedlings grown in continuous white light were transferred to UV - B light ( 2 W / m2 ) for the indicated period . Error bars are SD of three biological replicates . Quantitative RT - PCR analysis of auxin signaling gene expression in wild - type ( Col - 0 ) , uvr8 , and rup1 rup2 seedlings . Seedlings grown in continuous white light for 5 days were kept in white light ( D ) or transferred to white light plus UV - B ( E ) for 1 day with the addition of 1 μM IAA . Error bars are SD of three biological replicates . qPCR analysis of auxin signaling gene expression in roots of WT , uvr8 , and rup1 rup2 seedlings . Seedlings grown in continuous white light for 12 days were kept in white light ( F ) or transferred to white light plus UV - B ( G ) for 1 day with the addition of 0 . 4 μM NAA . Error bars are SD of three biological replicates .

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

Figure 4Transcriptome analysis of gene expression regulated by auxin, UV-B, and UVR8. (A) Heat map of UV-B-, UVR8-, and auxin-regulated genes. The parameter measured by color key shows the Log-fold change. (B) Auxin signaling genes are up-regulated by auxin treatment but down-regulated by UV-B in a UVR8-dependent manner. Three biological replicates were analyzed and final Log fold-change is shown.Quantitative RT-PCR results showing that UV-B represses the expression of auxin signaling genes. Six-day-old WT seedlings grown in continuous white light were transferred to UV-B light (2 W/m2) for the indicated period. Error bars are SD of three biological replicates.Quantitative RT-PCR analysis of auxin signaling gene expression in wild-type (Col-0), uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 5 days were kept in white light (D) or transferred to white light plus UV-B (E) for 1 day with the addition of 1 μM IAA. Error bars are SD of three biological replicates.qPCR analysis of auxin signaling gene expression in roots of WT, uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 12 days were kept in white light (F) or transferred to white light plus UV-B (G) for 1 day with the addition of 0.4 μM NAA. Error bars are SD of three biological replicates.


Figure 5In vitro pull - down assay showing that UV - B treatment promotes the interaction between MYB73 and UVR8 . The bound proteins were eluted and analyzed by probing immunoblots with an anti - His antibody . In vitro pull - down assay showing that UV - B treatment promotes the interaction between MYB77 ( B ) and UVR8 . The bound proteins were eluted and analyzed by probing immunoblots with an anti - His antibody . BiFC assay showing that UVR8 interacts with MYB73 / MYB77 in white light condition . N . benthamiana was co - transformed with MYB73 - nYFP / MYB77 - nYFP and cCFP , nYFP , and UVR8 - cCFP , or MYB73 - nYFP / MYB77 - nYFP and UVR8 - cCFP . BF , bright field . Merge , overlay of the YFP and bright field images . Scale bar = 20 μm . Co - IP assays showing that MYB73 interact with UVR8 in a UV - B - dependent manner in whole seedlings and roots Input : immunoblots showing the levels of UVR8 , MYB73 - TAP in the total protein extract . UVR8 IP : the IP products precipitated by the UVR8 antibody . Total proteins ( Input ) or IP product were probed , on immunoblots , with an anti - UVR8 or anti - Myc antibody , which detects the TAP tag . Co - IP assays showing that MYB77 interact with UVR8 in a UV - B - dependent manner in whole seedlings and roots Input : immunoblots showing the levels of UVR8 , and MYB77 - TAP in the total protein extract . UVR8 IP : the IP products precipitated by the UVR8 antibody . Total proteins ( Input ) or IP product were probed , on immunoblots , with an anti - UVR8 or anti - Myc antibody , which detects the TAP tag .

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

Figure 5In vitro pull-down assay showing that UV-B treatment promotes the interaction between MYB73 and UVR8. The bound proteins were eluted and analyzed by probing immunoblots with an anti-His antibody.In vitro pull-down assay showing that UV-B treatment promotes the interaction between MYB77 (B) and UVR8. The bound proteins were eluted and analyzed by probing immunoblots with an anti-His antibody.BiFC assay showing that UVR8 interacts with MYB73/MYB77 in white light condition. N. benthamiana was co-transformed with MYB73-nYFP/MYB77-nYFP and cCFP, nYFP, and UVR8-cCFP, or MYB73-nYFP/MYB77-nYFP and UVR8-cCFP. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bar = 20 μm.Co-IP assays showing that MYB73 interact with UVR8 in a UV-B-dependent manner in whole seedlings and roots Input: immunoblots showing the levels of UVR8, MYB73-TAP in the total protein extract. UVR8 IP: the IP products precipitated by the UVR8 antibody. Total proteins (Input) or IP product were probed, on immunoblots, with an anti-UVR8 or anti-Myc antibody, which detects the TAP tag.Co-IP assays showing that MYB77 interact with UVR8 in a UV-B-dependent manner in whole seedlings and roots Input: immunoblots showing the levels of UVR8 , and MYB77-TAP in the total protein extract. UVR8 IP: the IP products precipitated by the UVR8 antibody. Total proteins (Input) or IP product were probed, on immunoblots, with an anti-UVR8 or anti-Myc antibody, which detects the TAP tag.


Figure 6Phenotypic analysis . Seedlings of the indicated genotypes were grown in LD conditions ( 16 - h light / 8 - h dark ) for 5 days , and then transplanted to new medium containing 0 . 4 μM NAA and kept in continuous white light or white plus UV - B light ( 1 W / m2 ) for 7 days . Images are shown in ( A ) ; scale bars = 2 mm . Lateral root density ( number of lateral roots / length of primary root ) ( B ) and average length of lateral roots ( C ) of the indicated genotypes were measured . SDs ( n > 8 ) are indicated . Quantitative RT - PCR analysis of auxin signaling gene expression in roots of wild type ( Col - 0 ) , uvr8 , myb73 myb77 , and myb73 myb77 uvr8 . ( D ) Results under white light and ( E ) under white plus UV - B light . Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV - B for 1 day with the addition of 0 . 4 μM NAA , and roots were collected to analyze auxin signaling gene expression . Error bars represent SD of three biological replicates .

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 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 6Phenotypic analysis. Seedlings of the indicated genotypes were grown in LD conditions (16-h light/ 8-h dark) for 5 days, and then transplanted to new medium containing 0.4 μM NAA and kept in continuous white light or white plus UV-B light (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. Lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of the indicated genotypes were measured. SDs (n > 8) are indicated.Quantitative RT-PCR analysis of auxin signaling gene expression in roots of wild type (Col-0), uvr8, myb73 myb77, and myb73 myb77 uvr8. (D) Results under white light and (E) under white plus UV-B light. Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV-B for 1 day with the addition of 0.4 μM NAA, and roots were collected to analyze auxin signaling gene expression. Error bars represent SD of three biological replicates.


Figure 7Electrophoretic mobility - shift assay ( EMSA ) showing that MYB73 / MYB77 binds to the MBSI motif in vitro . Cold probe was added as a competitor . EMSA showing that MYB73 / MYB77 binds to the promoter of IAA19 . Cold IAA19p or IAA19mp ( MBSI motif mutated ) was added as a competitor . EMSA results showing that monomeric UVR8 inhibits the DNA - binding activity of MYB73 ( C ) EMSA results showing that monomeric UVR8 inhibits the DNA - binding activity of MYB77 ( D ) . ChIP - qPCR analysis showed that UV - B inhibits the DNA - binding activity of MYB73 . ChIP - qPCR assays were performed using transgenic seedlings expressing 35S : : MYB73 - TAP , treated with or without UV - B for 5 h before harvesting samples . Chromatin fragments ( ~ 500 bp ) were immunoprecipitated by anti - Myc antibody , and the precipitated DNA was analyzed by qPCR using the primer pairs indicated . The IP / Input ratios are shown with SDs ( n = 3 ) . ChIP - qPCR showing that UV - B inhibits the DNA - binding activity of MYB73 in a UVR8 - dependent manner . The IP / Input ratios are shown with SDs ( n = 3 ) . ChIP - qPCR showing that UV - B inhibits the DNA - binding activity of MYB73 in roots ( G ) in a UVR8 - dependent manner ( H ) . The IP / Input ratios are shown with SDs ( n = 3 ) . ChIP - qPCR showing that UV - B inhibits MYB77 from binding to the indicated promoters in a UVR8 - dependent manner . The IP / Input ratios are shown with SDs ( n = 3 ) .

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 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 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 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

Figure 7Electrophoretic mobility-shift assay (EMSA) showing that MYB73/MYB77 binds to the MBSI motif in vitro. Cold probe was added as a competitor.EMSA showing that MYB73/MYB77 binds to the promoter of IAA19. Cold IAA19p or IAA19mp (MBSI motif mutated) was added as a competitor.EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB73(C)EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB77(D).ChIP-qPCR analysis showed that UV-B inhibits the DNA-binding activity of MYB73. ChIP-qPCR assays were performed using transgenic seedlings expressing 35S::MYB73-TAP, treated with or without UV-B for 5 h before harvesting samples. Chromatin fragments (~500 bp) were immunoprecipitated by anti-Myc antibody, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/Input ratios are shown with SDs (n=3). ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).ChIP-qPCR showing that UV-B inhibits the DNA-binding activity of MYB73 in roots (G) in a UVR8-dependent manner (H). The IP/Input ratios are shown with SDs (n=3).ChIP-qPCR showing that UV-B inhibits MYB77 from binding to the indicated promoters in a UVR8-dependent manner. The IP/Input ratios are shown with SDs (n=3).


Figure 2Immunoblot with anti - HA Abs of lysates from parental ( Ctrl ) and tagged lines ( TgCRMPa - HA3 and TgCRMPb - HA3 ) together with inducible - knockdown lines ( TgCRMPa - HA3 _ iKD and TgCRMPb - HA3 _ iKD ) treated with ATc for 0 , 24 or 48h . TgROP5 was used as loading control . Two close bands around 300kDa were detected for TgCRMPa . A ~ 820kDa protein , corresponding to the predicted size for TgCRMPb , was observed together with a ~ 130kDa band . Quantification of microneme secretion in TgCRMPa - and TgCRMPb - depleted tachyzoites was measured by detecting the processed form ( arrowhead ) of TgMIC2 ( arrow ) in the media . Control , TgCRMPa _ iKD and TgCRMPb _ iKD parasites , ATc - treated ( + ) and untreated ( - ) , were stimulated with propranolol to release microneme contents . Blots were probed with anti - MIC2 ( secretion of micronemes ) and anti - GRA3 ( constitutive secretion of dense granules ) . P : Parasites pellet . Sup : Supernatant from untreated parasites . Sup + Prop : Supernatant from parasites treated with propranolol . The results are representative of 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 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 2Immunoblot with anti-HA Abs of lysates from parental (Ctrl) and tagged lines (TgCRMPa-HA3 and TgCRMPb-HA3) together with inducible-knockdown lines (TgCRMPa-HA3_iKD and TgCRMPb-HA3_iKD) treated with ATc for 0, 24 or 48h. TgROP5 was used as loading control. Two close bands around 300kDa were detected for TgCRMPa. A ~820kDa protein, corresponding to the predicted size for TgCRMPb, was observed together with a ~130kDa band.Quantification of microneme secretion in TgCRMPa- and TgCRMPb-depleted tachyzoites was measured by detecting the processed form (arrowhead) of TgMIC2 (arrow) in the media. Control, TgCRMPa_iKD and TgCRMPb_iKD parasites, ATc-treated (+) and untreated (-), were stimulated with propranolol to release microneme contents. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.


Figure 3Co - immunoprecipitation of TgCRMPa and TgCRMPb . Lysates from parasites co - expressing TgCRMPa - FLAG3 and TgCRMPb - HA3 were split and incubated with either anti - FLAG ( left panels ) or anti - HA beads ( right panels ) . Eluates were subjected to SDS - PAGE and immunoblotted with anti - HA and anti - FLAG Abs . Untagged , TgCRMPa - FLAG3 and TgCRMPb - HA3 parasites were used as controls . TgCRMPs robustly associate with each other . Quantification of microneme secretion in control and Tg277910 - depleted tachyzoites was measured as in Fig 2H . Blots were probed with anti - MIC2 ( secretion of micronemes ) and anti - GRA3 ( constitutive secretion of dense granules ) . P : Parasites pellet . Sup : Supernatant from untreated parasites . Sup + Prop : Supernatant from parasites treated with propranolol . The results are representative of 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 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

Figure 3Co-immunoprecipitation of TgCRMPa and TgCRMPb. Lysates from parasites co-expressing TgCRMPa-FLAG3 and TgCRMPb-HA3 were split and incubated with either anti-FLAG (left panels) or anti-HA beads (right panels). Eluates were subjected to SDS-PAGE and immunoblotted with anti-HA and anti-FLAG Abs. Untagged, TgCRMPa-FLAG3 and TgCRMPb-HA3 parasites were used as controls. TgCRMPs robustly associate with each other.Quantification of microneme secretion in control and Tg277910-depleted tachyzoites was measured as in Fig 2H. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.


Figure 5Immunofluorescence images of extracellular tachyzoites of untagged , TgCRMPa - HA3 and TgCRMPb - HA3 parasites , incubated either with host cell monolayers for 2min , or with ionophore A23187 , to induce natural or artificial conoid extrusion , respectively . Parasites were immunostained with anti - HA Abs ; DNA was labeled by Hoechst . CRMPa and CRMPb consistently accumulate at the tip of extruded conoids ( arrows ) . The apexes of A23187 - treated parasites were magnified on the right , and increased in brightness and contrast to highlight the apical dots . DIC : differential interference contrast . Quantification of the dot pattern upon A23187 treatment shown in ( A ) . Values are expressed as percentage of parasites showing ( dot ) or lacking ( no dot ) the apical accumulation of TgCRMPa and TgCRMPb ; n = number of parasites analyzed per line . Immunofluorescence images of extracellular TgCRMPb - HA3 tachyzoites in presence ( - ATc ) or absence ( + 48h ATc ) of TgCRMPa - FLAG3 ( iKD line ) . Parasites were stained with anti - HA Abs to visualize CRMPb . TgCRMPb localization at the tip of the extruded conoid ( arrow ) disappears upon TgCRMPa depletion , but it is still detected in the cytoplasm ( lower panel ) . DNA is labeled by Hoechst . Single focal planes are shown . DIC : differential interference contrast . Quantification of the dot pattern shown in ( C ) . The values are reported as in ( B ) . Whole cell lysates from TIR1 - expressing parental line ( Ctrl ) , HA3 - miniAID - TgCRMPa _ iKD ( N - term ) and TgCRMPa - miniAID - HA3 _ iKD ( C - term ) lines were immunoblotted with anti - HA Abs to visualize tagged CRMPa in IAA - treated and untreated samples . CRMPa was undetectable upon 24h incubation with IAA when C - terminally , but not N - terminally , tagged with the miniAID - HA3 , suggesting that the C - terminus is the one exposed towards the cytosol . TgMIC2 was used as loading control and detected with anti - MIC2 Abs . MW : molecular weight standards . Immunofluorescence images of extracellular A23187 - treated parasites expressing either N - or C - terminally HA3 - tagged TgCRMPa , and immunostained as in ( A ) . TgCRMPa accumulates at the tip of extruded conoids ( arrows ) in triton - permeabilized ( + ) or non - permeabilized ( - ) parasites . DIC : differential interference contrast . Single focal planes are shown . Quantification of the dot pattern upon natural ( + H F Fibroblasts ) or artificial ( + A23187 ) conoid extrusion in parasites expressing either N - or C - terminally HA3 - tagged TgCRMPa . Values are reported as in ( B ) .

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

Figure 5Immunofluorescence images of extracellular tachyzoites of untagged, TgCRMPa-HA3 and TgCRMPb-HA3 parasites, incubated either with host cell monolayers for 2min, or with ionophore A23187, to induce natural or artificial conoid extrusion, respectively. Parasites were immunostained with anti-HA Abs; DNA was labeled by Hoechst. CRMPa and CRMPb consistently accumulate at the tip of extruded conoids (arrows). The apexes of A23187-treated parasites were magnified on the right, and increased in brightness and contrast to highlight the apical dots. DIC: differential interference contrast. Quantification of the dot pattern upon A23187 treatment shown in (A). Values are expressed as percentage of parasites showing (dot) or lacking (no dot) the apical accumulation of TgCRMPa and TgCRMPb; n= number of parasites analyzed per line.Immunofluorescence images of extracellular TgCRMPb-HA3 tachyzoites in presence (-ATc) or absence (+48h ATc) of TgCRMPa-FLAG3 (iKD line). Parasites were stained with anti-HA Abs to visualize CRMPb. TgCRMPb localization at the tip of the extruded conoid (arrow) disappears upon TgCRMPa depletion, but it is still detected in the cytoplasm (lower panel). DNA is labeled by Hoechst. Single focal planes are shown. DIC: differential interference contrast.   Quantification of the dot pattern shown in (C). The values are reported as in (B).  Whole cell lysates from TIR1-expressing parental line (Ctrl), HA3-miniAID-TgCRMPa_iKD (N-term) and TgCRMPa-miniAID-HA3_iKD (C-term) lines were immunoblotted with anti-HA Abs to visualize tagged CRMPa in IAA-treated and untreated samples. CRMPa was undetectable upon 24h incubation with IAA when C-terminally, but not N-terminally, tagged with the miniAID-HA3, suggesting that the C-terminus is the one exposed towards the cytosol. TgMIC2 was used as loading control and detected with anti-MIC2 Abs. MW: molecular weight standards.Immunofluorescence images of extracellular A23187-treated parasites expressing either N-or C-terminally HA3-tagged TgCRMPa, and immunostained as in (A). TgCRMPa accumulates at the tip of extruded conoids (arrows) in triton-permeabilized (+) or non-permeabilized (-) parasites. DIC: differential interference contrast. Single focal planes are shown.   Quantification of the dot pattern upon natural (+H F Fibroblasts) or artificial (+A23187) conoid extrusion in parasites expressing either N- or C-terminally HA3-tagged TgCRMPa. Values are reported as in (B).


Figure 6Ultrastructure Expansion Microscopy of extracellular tachyzoites , either untagged or co - expressing TgCRMPa - HA3 / TgCRMPa - TY2 and TgCRMPb - HA3 together or in pairwise combination with TgNd6 - TY2 . Parasites were treated with A23187 prior to fixation and preparation for U - ExM , and stained with anti - HA , anti - TY and anti - α / β tubulin Abs to label CRMPs , Nd6 / CRMPa and microtubules , respectively . Shown are maximum intensity projections of z - stack confocal images . CRMPs overlap with Nd6 at the tip of the extruded conoid ( yellow selection ) . A magnified image of the apical tip of each parasite is shown on the right . Extent of colocalization between TgCRMPa - TY2 and TgCRMPb - HA3 , and TgCRMPs - HA3 with TgNd6 - TY2 in the apical dot shown in B . Pearson ' s correlation coefficient was measured using the Fiji - JACoP plugin . Values are expressed as mean ± SD . Three - five parasites per line were analyzed . Quantification of the dot pattern for TgCRMPa - HA3 and TgCRMPb - HA3 in TgNd9 _ iKD lines . CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A . TgCRMPs were still found in the apical dot in absence of TgNd9 ( + ATc ) . Numbers are expressed as percentage of parasites showing ( dot ) or lacking ( no dot ) the tip accumulation of TgCRMPa and TgCRMPb . The number of parasites ( n ) analyzed for each line is reported at the top of the column .

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

Figure 6Ultrastructure Expansion Microscopy of extracellular tachyzoites, either untagged or co-expressing TgCRMPa-HA3/TgCRMPa-TY2 and TgCRMPb-HA3 together or in pairwise combination with TgNd6-TY2. Parasites were treated with A23187 prior to fixation and preparation for U-ExM, and stained with anti-HA, anti-TY and anti-α/β tubulin Abs to label CRMPs, Nd6/CRMPa and microtubules, respectively. Shown are maximum intensity projections of z-stack confocal images. CRMPs overlap with Nd6 at the tip of the extruded conoid (yellow selection). A magnified image of the apical tip of each parasite is shown on the right.Extent of colocalization between TgCRMPa-TY2 and TgCRMPb-HA3, and TgCRMPs-HA3 with TgNd6-TY2 in the apical dot shown in B. Pearson's correlation coefficient was measured using the Fiji-JACoP plugin. Values are expressed as mean ± SD. Three-five parasites per line were analyzed. Quantification of the dot pattern for TgCRMPa-HA3 and TgCRMPb-HA3 in TgNd9_iKD lines. CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A. TgCRMPs were still found in the apical dot in absence of TgNd9 (+ATc). Numbers are expressed as percentage of parasites showing (dot) or lacking (no dot) the tip accumulation of TgCRMPa and TgCRMPb. The number of parasites (n) analyzed for each line is reported at the top of the column.


Figure 1 ( A - B ) Western - Blotting ( WB ) of p53 - negative lung cancer H1299 cells stably expressing ( A ) or transiently expressing ( B ) the indicated constructs . Post - translationally modified Δ133p53 and Δ160p53 isoforms are indicated with * . ( C ) The same WB membrane containing lysates from R273Hp53 - expressing A431cells was incubated with rabbit polyclonal CM - 1 antibody and mouse monoclonal 1801 antibody against the N - terminus of p53 . Detection using anti - rabbit IRDye 680LT ( red ) and anti - mouse IRDye 800CW ( green ) secondary antibodies confirmed that the Δ160p53 band corresponds to a C - terminal isoform of p53 . ( D ) WB of H1299 cells transiently expressing the indicated constructs . ( E ) WB of cell lines expressing wild - type ( HCT116 , U2OS , A549 ) or mutant ( SW480 [ R273H / P309S ] , MDAMB231 [ R280K ] , A431 [ R273H ] , HT29 [ R273H ] and SKBR3 [ R175H ] ) p53 . ( F ) Cell lines harbouring endogenous wild - type ( A549 ) or R273H mutant ( A431 ) p53 were treated or not with control siRNA ( ctl ) or siRNA targeting both p53 transcripts ( ex6 and ex7 ) , full - length mRNA only ( ex2 / 3 ) or Δ133p53 mRNA only ( in4 ) before lysis and WB . ( G ) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO , proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide ( CHX ) . Shown are representative data of three independent experiments . The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α - tubulin or GAPDH and relative to the value indicated in bold and set to 1 . 0 . In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53 - null H1299 cells .

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Figure 1(A-B) Western-Blotting (WB) of p53-negative lung cancer H1299 cells stably expressing (A) or transiently expressing (B) the indicated constructs. Post-translationally modified Δ133p53 and Δ160p53 isoforms are indicated with *.(C) The same WB membrane containing lysates from R273Hp53-expressing A431cells was incubated with rabbit polyclonal CM-1 antibody and mouse monoclonal 1801 antibody against the N-terminus of p53. Detection using anti-rabbit IRDye 680LT (red) and anti-mouse IRDye 800CW (green) secondary antibodies confirmed that the Δ160p53 band corresponds to a C-terminal isoform of p53.(D) WB of H1299 cells transiently expressing the indicated constructs.(E) WB of cell lines expressing wild-type (HCT116, U2OS, A549) or mutant (SW480 [R273H/P309S], MDAMB231 [R280K], A431 [R273H], HT29 [R273H] and SKBR3 [R175H]) p53.(F) Cell lines harbouring endogenous wild-type (A549) or R273H mutant (A431) p53 were treated or not with control siRNA (ctl) or siRNA targeting both p53 transcripts (ex6 and ex7), full-length mRNA only (ex2/3) or Δ133p53 mRNA only (in4) before lysis and WB.(G) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO, proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide (CHX). Shown are representative data of three independent experiments. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.


Figure 2 ( A ) Cell lines endogenously expressing mutant R273Hp53 ( A431 and HT29 ) were treated with control siRNA ( ctl ) or siRNA targeting exon 7 of p53 , submitted to endoplasmic reticulum ( ER ) stress by exposure to thapsigargin ( Th ) and then analysed for apoptosis by incubation with propidium - iodide ( PI ) and FACS analysis . Data was normalized against si ctl condition , which was set to 100 . ( B and C ) A549 ( B ) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli ( over - confluency ( O - C ) for A549 ) . Data was normalized against 1st day values . Δ160p53 shows similar pro - proliferative capacities as mutant R273Hp53 . On the other hand R273Hp53 lost pro - proliferative functions when deficient for Δ160p53 expression ( M160AR273Hp53 ) . Also shown is the WB of A549 cells used in ( B ) and stably expressing the indicated constructs . ( B and C ) H1299 ( C ) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli ( thapsigargin ( Th ) for H1299 ) . Data was normalized against 1st day values . Δ160p53 shows similar pro - proliferative capacities as mutant R273Hp53 . On the other hand R273Hp53 lost pro - proliferative functions when deficient for Δ160p53 expression ( M160AR273Hp53 ) . ( D to G ) H1299 cells transfected with the indicated constructs were submitted to ER stress ( E and G ) or cultured under regular conditions ( D ) . Data was normalized against empty vector ( E ) or p53 ( D ) condition , which were set to 100 . Cells were then analysed for apoptosis as in ( A ) . Shown are representative data or averages + s . d . of three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 and ^ P > 0 . 05 compared to control ( " Th + si ctl " or " - " ) or as indicated ) . ( D to G ) H1299 cells transfected with the indicated constructs were submitted to ER stress ( E and G ) , DNA - damage by 36h 3 μM etoposide ( Eto ) treatment ( F ) . Data was normalized against empty vector ( E ) or p53 ( D ) condition , which were set to 100 . Cells were then analysed for apoptosis as in ( A ) . Shown are representative data or averages + s . d . of three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 and ^ P > 0 . 05 compared to control ( " Th + si ctl " or " - " ) or as indicated ) .

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

Figure 2(A) Cell lines endogenously expressing mutant R273Hp53 (A431 and HT29) were treated with control siRNA (ctl) or siRNA targeting exon 7 of p53, submitted to endoplasmic reticulum (ER) stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. Data was normalized against si ctl condition, which was set to 100.(B and C) A549 (B) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (over-confluency (O-C) for A549). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). Also shown is the WB of A549 cells used in (B) and stably expressing the indicated constructs.(B and C) H1299 (C) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (thapsigargin (Th) for H1299). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53).(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G) or cultured under regular conditions (D). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G), DNA-damage by 36h 3 μM etoposide (Eto) treatment (F). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).


Figure 3 ( A ) Adhesion assay shows that MCF10A cells stably expressing Δ133p53 , Δ160p53 , R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells . Excluding Δ160p53 expression from the mutant background ( M160A / R273Hp53 ) rescued control - cell phenotype . Scale bar represents 200 μm . ( B ) WB of MCF10A cells used in ( A ) and stably expressing the indicated constructs . Shown are representative data of three independent experiments and quantifications . ( C and D ) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel . Control - and Δ133p53 - expressing cells formed regular three - dimensional ( 3D ) mammary acinar structures with hollow lumina but Δ160p53 - , R273HΔ160p53 and to a smaller extent mutant R273Hp53 - expressing cells formed acini with filled lumen and long invasive structures ( n > 30 acini per experiment per condition ) . ( C and D ) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel . Control - and Δ133p53 - expressing cells formed regular three - dimensional ( 3D ) mammary acinar structures with hollow lumina but Δ160p53 - , R273HΔ160p53 and to a smaller extent mutant R273Hp53 - expressing cells formed acini with filled lumen and long invasive structures ( n > 30 acini per experiment per condition ) . ( E ) H1299 cells stably expressing empty vector ( control ) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel - fibronectin matrix towards EGF - supplemented growth media . The 30 μm plane is indicated and scale bars represent 100 μm . Quantifications are shown in the right panel .

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

Figure 3(A) Adhesion assay shows that MCF10A cells stably expressing Δ133p53, Δ160p53, R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells. Excluding Δ160p53 expression from the mutant background (M160A/R273Hp53) rescued control-cell phenotype. Scale bar represents 200 μm.(B) WB of MCF10A cells used in (A) and stably expressing the indicated constructs. Shown are representative data of three independent experiments and quantifications.(C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition).(C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition).(E) H1299 cells stably expressing empty vector (control) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.


Figure 4 ( A ) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos ( Ctl - 1 or Ctl - 2 ) or antisense morpholino oligo targeting Δ160p53 ' s translation initiation ( MO ) ( B ) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos ( Ctl - 2 or Ctl - 1 ) or antisense morpholino oligo targeting Δ160p53 ' s translation initiation site ( MO ) before lysis and WB . wtp53 - expressing A549 cells were used as reference for endogenous wtp53 expression levels . Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53 - null H1299 cells . ( C ) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino ( Ctl - 1 ) or morpholino targeting 160p53 ' s translation initiation site ( MO ) . MO efficiently targets 160p53 in endogenous R273Hp53 cell lines . See also figure EV3 for the raw data . ( D - E ) A431 ( D ) and HT29 ( E ) cells endogenously expressing mutant R273Hp53 were treated with control siRNA ( si Ctl ) or control MO ( MO Ctl - 2 or MO Ctl - 1 ) or siRNA targeting exon 7 of p53 ( si ) or MO targeting Δ160p53 ' s translation initiation site ( MO ) , as indicated , submitted to endoplasmic reticulum stress by exposure to thapsigargin ( Th ) and then analysed for apoptosis by incubation with propidium - iodide ( PI ) and FACS analysis . wtp53 - expressing A549 cells were similarly control - treated and used as comparison . Data was normalized against A549 cell condition , which was set to 100 . ( F ) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl - 1 or morpholino targeting Δ160p53 ' s translation initiation site ( MO ) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel - fibronectin matrix towards EGF - supplemented growth media . The 30 μm plane is indicated and scale bars represent 100 μm . Quantifications are shown in the right panel .

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

Figure 4(A) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos (Ctl-1 or Ctl-2) or antisense morpholino oligo targeting Δ160p53's translation initiation (MO)(B) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos (Ctl-2 or Ctl-1) or antisense morpholino oligo targeting Δ160p53's translation initiation site (MO) before lysis and WB. wtp53-expressing A549 cells were used as reference for endogenous wtp53 expression levels. Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.(C) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino (Ctl-1) or morpholino targeting 160p53's translation initiation site (MO). MO efficiently targets 160p53 in endogenous R273Hp53 cell lines. See also figure EV3 for the raw data.(D-E) A431 (D) and HT29 (E) cells endogenously expressing mutant R273Hp53 were treated with control siRNA (si Ctl) or control MO (MO Ctl-2 or MO Ctl-1) or siRNA targeting exon 7 of p53 (si) or MO targeting Δ160p53's translation initiation site (MO), as indicated, submitted to endoplasmic reticulum stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. wtp53-expressing A549 cells were similarly control-treated and used as comparison. Data was normalized against A549 cell condition, which was set to 100.(F) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl-1 or morpholino targeting Δ160p53's translation initiation site (MO) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.


Figure 2A . Western blot analysis of reporter HslO - FLAG protein produced with a custom - made PURExpress ® coupled in vitro transcription - translation system supplemented with the indicated EF - G proteins and incubated for the indicated times . Blot was developed using anti - FLAG antibodies . Shown is a representative from at least two independent experiments . Eco EF - G : E . coli EF - G .

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Figure 2A. Western blot analysis of reporter HslO-FLAG protein produced with a custom-made PURExpress® coupled in vitro transcription-translation system supplemented with the indicated EF-G proteins and incubated for the indicated times. Blot was developed using anti-FLAG antibodies. Shown is a representative from at least two independent experiments. Eco EF-G: E. coli EF-G.


Figure 4B . Western blot analysis of reporter HslO - FLAG protein synthesized in vitro using a custom - made PURExpress ® system with EF - G1 , EF - G2 , and two engineered EF - G2 variants : one missing the 26 - amino acid insert , EF - G2 ( ∆ 514 - 539 ) and one missing the beta hairpin in the insert , EF - G2 ( ∆ 516 - 527 ) . Shown is a representative experiment from at least two independent experiments . D . SDS - PAGE analysis of co - sedimentation assays monitoring factor ( EF - G1 EF - G2 or EF - G2 variant EF - G2 ( ∆ 516 - 527 ) or EF - G2 ( ∆ 514 - 539 ) ) binding to E . coli 70S ribosomes . Binding reactions were centrifuged through a sucrose cushion to recover ribosome and associated factor in the pellets . The positions of EF - G proteins and ribosomal proteins and molecular weight markers are indicated . E . Western blot analysis of reporter HisC - FLAG protein synthesized in vitro using a custom - made PURExpress ® system with the EF - G1 , EF - G2 or EF - G2 ( S84H ) proteins . Shown is a representative from at least 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Western blot analysis of reporter HslO-FLAG protein synthesized in vitro using a custom-made PURExpress® system with EF-G1, EF-G2, and two engineered EF-G2 variants: one missing the 26-amino acid insert, EF-G2 (∆514-539) and one missing the beta hairpin in the insert, EF-G2 (∆516-527). Shown is a representative experiment from at least two independent experiments.D. SDS-PAGE analysis of co-sedimentation assays monitoring factor (EF-G1 EF-G2 or EF-G2 variant EF-G2 (∆516-527) or EF-G2 (∆514-539)) binding to E. coli 70S ribosomes. Binding reactions were centrifuged through a sucrose cushion to recover ribosome and associated factor in the pellets. The positions of EF-G proteins and ribosomal proteins and molecular weight markers are indicated.E. Western blot analysis of reporter HisC-FLAG protein synthesized in vitro using a custom-made PURExpress® system with the EF-G1, EF-G2 or EF-G2(S84H) proteins. Shown is a representative from at least two independent experiments.


Figure 6Western blot analysis of B . thetaiotaomicron strains expressing C - terminally FLAG - tagged EF - G1 ( WH405 ) ( A ) , or EF - G2 ( GT1301 ) ( B ) , from their normal promoters and chromosomal locations , grown in Tryptone Yeast Extract Glucose liquid medium ( TYG ) and sampled at the indicated times . E - F . Western blot of crude extracts from wild - type B . thetaiotaomicron harvested from the ceca of mice ( representative blot for 2 out of 4 mice tested ) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF - G1 and EF - G2 proteins at the indicated amounts ( E ) . Blots were developed with anti - EF - G1 or EF - G2 polyclonal antibodies ; anti - GroEL antibodies were used as loading controls . Western blot quantifications of all four biological replicates and their average are shown in ( F ) , where EF - G1 and EF - G2 abundances were estimated by normalizing the signal of bacterial sample to the closest signal of known amount of purified protein , and the derived absolute amount ( pmol ) was normalized to the signal of GroEL .

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Figure 6Western blot analysis of B. thetaiotaomicron strains expressing C-terminally FLAG-tagged EF-G1 (WH405) (A), or EF-G2 (GT1301) (B), from their normal promoters and chromosomal locations, grown in Tryptone Yeast Extract Glucose liquid medium (TYG) and sampled at the indicated times.E-F. Western blot of crude extracts from wild-type B. thetaiotaomicron harvested from the ceca of mice (representative blot for 2 out of 4 mice tested) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF-G1 and EF-G2 proteins at the indicated amounts (E). Blots were developed with anti-EF-G1 or EF-G2 polyclonal antibodies; anti-GroEL antibodies were used as loading controls. Western blot quantifications of all four biological replicates and their average are shown in (F), where EF-G1 and EF-G2 abundances were estimated by normalizing the signal of bacterial sample to the closest signal of known amount of purified protein, and the derived absolute amount (pmol) was normalized to the signal of GroEL.


Fig . 1 : Three - dimensional structure of 2019 - nCoV Mpro , in two different views . One protomer of the dimer is shown in light blue , the other one in orange . Amino - acid residues of the catalytic site are indicated as yellow and blue spheres , for Cys145 and His41 , respectively . Black spheres indicate the positions of Ala285 of each of the two domains III ( see text ) . Chain termini are labeled N and C for molecule A ( light blue ) and N * and C * for molecule B ( orange ) .

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Fig. 1: Three-dimensional structure of 2019-nCoV Mpro, in two different views. One protomer of the dimer is shown in light blue, the other one in orange. Amino-acid residues of the catalytic site are indicated as yellow and blue spheres, for Cys145 and His41, respectively. Black spheres indicate the positions of Ala285 of each of the two domains III (see text). Chain termini are labeled N and C for molecule A (light blue) and N* and C* for molecule B (orange).


Fig . 2 : Compound 13b in the substrate - binding cleft located between domains I and II of the Mpro , in the monoclinic crystal form ( space group C2 ) . 2Fo - Fc electron density is shown for the inhibitor ( contouring level : 1σ ) . Carbon atoms of the inhibitor are magenta , oxygens red , nitrogens blue , and sulfur yellow . Note the interaction between the N - terminal residue of chain B , S1 * , and E166 of chain A .

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Fig. 2: Compound 13b in the substrate-binding cleft located between domains I and II of the Mpro, in the monoclinic crystal form (space group C2). 2Fo-Fc electron density is shown for the inhibitor (contouring level: 1σ). Carbon atoms of the inhibitor are magenta, oxygens red, nitrogens blue, and sulfur yellow. Note the interaction between the N-terminal residue of chain B, S1*, and E166 of chain A.


Figure 1 ( C ) Western blot analysis of STAT2 protein levels in mock - , WT - RCMV - E - , ΔE27 - RCMV - E - or UV - inactivated RCMV - E - infected REF . 24 h p . i . , cells were either left untreated or treated for 5 . 5 h with DMSO , 2 . 5 µM MLN4924 for blocking CRL activity or 10 µM MG132 for proteasome inhibition . IE1 and β - tubulin served as infection and loading controls , respectively .

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Figure 1(C) Western blot analysis of STAT2 protein levels in mock-, WT-RCMV-E-, ΔE27-RCMV-E- or UV-inactivated RCMV-E-infected REF. 24 h p.i., cells were either left untreated or treated for 5.5 h with DMSO, 2.5 µM MLN4924 for blocking CRL activity or 10 µM MG132 for proteasome inhibition. IE1 and β-tubulin served as infection and loading controls, respectively.


Figure 2 ( A , B ) GF analysis of in vitro reconstitutions containing the indicated protein combinations . Coomassie blue - stained SDS - PAGE analyses of fractions collected during the GF runs are shown next to the chromatograms . The red * indicates a contaminant , which we identified by cryo - EM analysis as E . coli ArnA , a notorious contaminant in E . coli Ni - NTA protein purifications ( Andersen et al . , 2013 ) ( Appendix Fig S2 , S3 ) . Note that ArnA is present in GF fraction 6 ( Ve ~ 10 . 2 ml ) of all runs containing E27 , and migrates similarly to E27 on the SDS - PAGE . * - additional contaminant from E27 preparation ; * * - contaminant from STAT2 preparation .

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Figure 2(A, B) GF analysis of in vitro reconstitutions containing the indicated protein combinations. Coomassie blue-stained SDS-PAGE analyses of fractions collected during the GF runs are shown next to the chromatograms. The red * indicates a contaminant, which we identified by cryo-EM analysis as E. coli ArnA, a notorious contaminant in E. coli Ni-NTA protein purifications (Andersen et al., 2013) (Appendix Fig S2, S3). Note that ArnA is present in GF fraction 6 (Ve ~ 10.2 ml) of all runs containing E27, and migrates similarly to E27 on the SDS-PAGE. * - additional contaminant from E27 preparation; ** - contaminant from STAT2 preparation.


Figure 4 ( E ) Pre - formed DDB1 / DCAF1 protein complex was incubated with indicated amounts of E27 and analysed by GF . SDS - PAGE analyses of GF fractions are shown next to the chromatogram . The red * indicates the ArnA contaminant ( see Fig 2 ) . * - additional contaminant from E27 preparation .

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Figure 4(E) Pre-formed DDB1/DCAF1 protein complex was incubated with indicated amounts of E27 and analysed by GF. SDS-PAGE analyses of GF fractions are shown next to the chromatogram. The red * indicates the ArnA contaminant (see Fig 2). * - additional contaminant from E27 preparation.


Figure 7 ( B ) 293T cells were transfected with expression plasmids encoding E27 - HA or indicated variants . 24 h after transfection , cells were lysed and E27 - HA was precipitated using an HA - specific antibody . Precipitates were separated by SDS - PAGE , blotted , and probed using the indicated antibodies . ( E ) 293T cells were transfected with expression plasmids encoding M27 - FLAG or the indicated M27 - FLAG variants . 24 h after transfection , cells were lysed and M27 - FLAG was precipitated using a FLAG - specific antibody . Precipitates were separated by SDS - PAGE , blotted and probed using the indicated antibodies . ( F ) Mouse fibroblasts were infected with MCMV or the indicated MCMV mutants . At the indicated time points , cells were lysed , proteins were separated by SDS - PAGE and blotted . Blots were probed using the indicated antibodies . pIE1 and GAPDH were used as infection and loading controls . ( G ) NIH3T3 reporter cells stably expressing ISRE - Luc were infected with the indicated MCMV strains , treated with IFNβ or IFNλ , and luciferase activity was measured . The error bars represent the standard deviation , N = 4 biological replicates , * * * - P ≤ 0 . 001 ( ANOVA ) . ( H ) BALB / c mice were infected i . p . with WT - MCMV and indicated MCMV mutants . At 3 and 21 d p . i . , salivary glands were collected and frozen . Virus titres were determined from organ homogenates by plaque titration . Titrations were performed in quadruplicates . Bars depict the geometric mean , dots show titres of individual mice ( N = 5 biological replicates ) . DL - detection limit .

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Figure 7(B) 293T cells were transfected with expression plasmids encoding E27-HA or indicated variants. 24 h after transfection, cells were lysed and E27-HA was precipitated using an HA-specific antibody. Precipitates were separated by SDS-PAGE, blotted, and probed using the indicated antibodies.(E) 293T cells were transfected with expression plasmids encoding M27-FLAG or the indicated M27-FLAG variants. 24 h after transfection, cells were lysed and M27-FLAG was precipitated using a FLAG-specific antibody. Precipitates were separated by SDS-PAGE, blotted and probed using the indicated antibodies.(F) Mouse fibroblasts were infected with MCMV or the indicated MCMV mutants. At the indicated time points, cells were lysed, proteins were separated by SDS-PAGE and blotted. Blots were probed using the indicated antibodies. pIE1 and GAPDH were used as infection and loading controls.(G) NIH3T3 reporter cells stably expressing ISRE-Luc were infected with the indicated MCMV strains, treated with IFNβ or IFNλ, and luciferase activity was measured. The error bars represent the standard deviation, N=4 biological replicates, *** - P ≤ 0.001 (ANOVA).(H) BALB/c mice were infected i.p. with WT-MCMV and indicated MCMV mutants. At 3 and 21 d p.i., salivary glands were collected and frozen. Virus titres were determined from organ homogenates by plaque titration. Titrations were performed in quadruplicates. Bars depict the geometric mean, dots show titres of individual mice (N=5 biological replicates). DL - detection limit.


Figure 1A . Western blot analysis of mTORC1 activity in C2C12 - Vevtor ( VC ) and Aster - C KO ( KO ) cells in complete medium ( CM ) and in response to nutrient starvation or stimulation with amino acids for 30 min . B and C . Statistical analysis of phosphorylated level of S6K ( B ) and 4E - BP1 ( C ) in C2C12 - Vector and Aster - C KO cells in CM , under nutrient starvation ( - AA ) and in response to stimulation with amino acids ( + AA ) using ImageJ software . Data are represented as mean ± SD ( n = 3 biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test . D . Immunostaining of endogenous mTOR and LAMP1 in C2C12 - Vector and Aster - C KO cells under nutrient starvation ( - AA ) and in response to AA stimulation ( + AA ) . LAMP1 was immunostained as a lysosome marker . Arrows highlight the co - localization of mTOR with LAMP1 . Scale bar , 40 μm ( Row 1 ) and 10 μm ( Rows 2 , 3 , 4 ) . E . Statistical analysis of the Pearson ' s correlation coefficient of mTOR and LAMP1 in Figure 1F ( n = 12 - 15 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one way ANOVA . F . Western blot analysis of mTORC1 activity in Aster - C KO cells stably re - expressing exogenous GFP - Aster - C under nutrient starvation and AA stimulation . Aster - C KO cells were transfected with GFP - Aster - C and selected with G418 for 2 weeks . G . Immunostaining of endogenous mTOR and LAMP1 in C2C12 - Vector and Aster - C KO cells under cholesterol depletion by treatment with 0 . 5 % MCD for 2 hrs ( - CHOL ) or cholesterol ( 50 µM ) re - stimulation for 1 hr ( + CHOL ) . Arrows highlight the co - localization of mTOR with LAMP1 . Scale bar , 40 μm ( Row 1 ) and 10 μm ( Rows 2 , 3 , 4 ) . H . Western blot analysis of mTORC1 activity in C2C12 - Vector and Aster - C KO cells in CM , under starvation , cholesterol depletion by treatment with 0 . 5 % MCD for 2 hrs or cholesterol ( 50 µM ) re - stimulation for the indicated time .

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Figure 1A. Western blot analysis of mTORC1 activity in C2C12-Vevtor (VC) and Aster-C KO (KO) cells in complete medium (CM) and in response to nutrient starvation or stimulation with amino acids for 30 min. B and C. Statistical analysis of phosphorylated level of S6K (B) and 4E-BP1 (C) in C2C12-Vector and Aster-C KO cells in CM, under nutrient starvation (-AA) and in response to stimulation with amino acids (+AA) using ImageJ software. Data are represented as mean ± SD (n=3 biological replicates). *p < 0.05, **p<0.01 by Student's t test.D. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under nutrient starvation (-AA) and in response to AA stimulation (+AA). LAMP1 was immunostained as a lysosome marker. Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).E. Statistical analysis of the Pearson's correlation coefficient of mTOR and LAMP1 in Figure 1F (n=12-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one way ANOVA.F. Western blot analysis of mTORC1 activity in Aster-C KO cells stably re-expressing exogenous GFP-Aster-C under nutrient starvation and AA stimulation. Aster-C KO cells were transfected with GFP-Aster-C and selected with G418 for 2 weeks.G. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under cholesterol depletion by treatment with 0.5% MCD for 2 hrs (-CHOL) or cholesterol (50 µM) re-stimulation for 1 hr (+CHOL). Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).H. Western blot analysis of mTORC1 activity in C2C12-Vector and Aster-C KO cells in CM, under starvation, cholesterol depletion by treatment with 0.5% MCD for 2 hrs or cholesterol (50 µM) re-stimulation for the indicated time.


Figure 2A . Confocal image analysis depicting the co - localization of Aster - C with the ER in C2C12 cells transiently expressing GFP - Aster - C and DsRed - ER under starvation and in response to AA stimulation . Scale bar , 5 μm . B . Subcellular fractionation analysis of mTOR , GATOR2 complex and Aster - C localization in HEK293T cells transiently expressing FLAG - Aster - C under starvation and in response to AA stimulation . LAMP1 , Sec63 and GAPDH were used as lysosomal ( Lyso ) , rough ER ( RER ) and cytosol ( Cyto ) protein markers , respectively . Microsomal fraction ( Micro ) was also used in the blot . C . Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12 - Vector and Aster - C KO cells under starvation and in response to AA stimulation . LAMP1 , Sec63 and GAPDH were used as lysosomal ( Lyso ) , rough ER ( RER ) and cytosol ( Cyto ) protein markers , respectively . D . Co - IP analysis of the interaction of Aster - C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins . Anti - Myc antibody was used for immunoprecipitation . E . Co - IP analysis of the interaction of Aster - C with endogenous mTOR and the GATOR2 complex using FLAG - Sestrin2 ( SESN2 ) as a positive control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation . F . Co - IP analysis of the interaction of Aster - C with mTOR , the GATOR2 complex , GATOR1 complex and the Ragulator - Rag complex using FLAG - DEPDC5 as a positive control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation . Black arrow indicates the RagC protein .

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

Figure 2A. Confocal image analysis depicting the co-localization of Aster-C with the ER in C2C12 cells transiently expressing GFP-Aster-C and DsRed-ER under starvation and in response to AA stimulation. Scale bar, 5 μm.B. Subcellular fractionation analysis of mTOR, GATOR2 complex and Aster-C localization in HEK293T cells transiently expressing FLAG-Aster-C under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively. Microsomal fraction (Micro) was also used in the blot.C. Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12-Vector and Aster-C KO cells under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively.D. Co-IP analysis of the interaction of Aster-C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins. Anti-Myc antibody was used for immunoprecipitation.E. Co-IP analysis of the interaction of Aster-C with endogenous mTOR and the GATOR2 complex using FLAG-Sestrin2 (SESN2) as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation.F. Co-IP analysis of the interaction of Aster-C with mTOR, the GATOR2 complex, GATOR1 complex and the Ragulator-Rag complex using FLAG-DEPDC5 as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the RagC protein.