Figure 2A Immunoblot analysis of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K in NHF , Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF cells , three independent experiments . B Immunoblot analysis of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K in WT ( Ocrl + / y ) and IOB MEFs grown in complete medium or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF cells , three independent experiments . C Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test unless otherwise stated . D NHF and Lowe 1676 cells were grown in complete media , amino - acid ( aa ) / FBS starved for 5 h , or starved and then recovered in amino - acid / FBS - containing medium , then immunoblotted using antibodies against endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K . Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test unless otherwise stated .

O B-PANEL_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 B-PANEL_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

Figure 2A Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. B Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in WT (Ocrl+/y) and IOB MEFs grown in complete medium or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.D NHF and Lowe 1676 cells were grown in complete media, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunoblotted using antibodies against endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.


Figure 3A Representative images of NHF , Lowe 1676 and Lowe 3265 cells immunostained with anti - α - tubulin ( red ) and anti - γ - tubulin ( green ) antibodies . Scale bar , 10 μm . B Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in the experiments shown in A . n = 3 cells per group , three independent experiments . C Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons . C WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) and IOB MEFs derived from the corresponding mice were costained with anti - α - tubulin and anti - γ - tubulin antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . D Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in the experiments shown in C . n = 3 per group in which 30 cells were counted per condition per experiment . E Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons . E Microtubule regrowth assay was performed on NHF , Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice , followed by rinse with PBS , and addition of pre - warmed medium to induce microtubule regrowth . Cells were then immunostained with anti - α - tubulin and anti - γ - tubulin antibodies at the indicated time point after addition of pre - warmed medium . The magnified view shows the cytoplasmic microtubules organization center . Scale bar , 10 μm . F Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center , MTOC ) measured in the experiments shown in E . n = 3 per group in which 30 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

O B-PANEL_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 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

Figure 3A Representative images of NHF, Lowe 1676 and Lowe 3265 cells immunostained with anti-α-tubulin (red) and anti-γ-tubulin (green) antibodies. Scale bar, 10 μm. B Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in A. n = 3 cells per group, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.C WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs derived from the corresponding mice were costained with anti-α-tubulin and anti- γ-tubulin antibodies. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. D Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in C. n = 3 per group in which 30 cells were counted per condition per experiment. E Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.E Microtubule regrowth assay was performed on NHF, Lowe 1676 and Lowe 3265 cells with 20 μM nocodazole for 2 h on ice, followed by rinse with PBS, and addition of pre-warmed medium to induce microtubule regrowth. Cells were then immunostained with anti-α-tubulin and anti-γ-tubulin antibodies at the indicated time point after addition of pre-warmed medium. The magnified view shows the cytoplasmic microtubules organization center. Scale bar, 10 μm. F Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center, MTOC) measured in the experiments shown in E. n = 3 per group in which 30 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.


Figure 4E Super - resolution images of centrosome stained with anti - CEP164 ( red , labeling distal appendages of mother centriole ) and anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 cells by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . F Super - resolution images of centrosome stained with anti - CNAP1 ( red , labeling proximal ends of centriole ) and anti - OCRL ( blue ) were acquired using Centrin2 - GFP - RPE - 1 cells by DeltaVision OMX V4 BLAZE system . Scale bar , 0 . 5 μm . G H Representative images of the co - localization of OCRL - WT , GFP - OCRL - △ RhoGAP and GFP - OCRL - △ ASH + RhoGAP with γ - tubulin . Insets , enlargements of the outlined areas . Scale bars , 10 μm . I Quantitative analysis of cells with and without centrosomal OCRL ( centrosomal GFP signal ) in the experiments shown in A . n = 3 per group in which 10 cells were counted per condition per experiment . Means ± SEM , * * * * P < 0 . 0001 , n . s . , not significant , calculated by two - way ANOVA , multiple comparisons .

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

Figure 4E Super-resolution images of centrosome stained with anti-CEP164 (red, labeling distal appendages of mother centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. F Super-resolution images of centrosome stained with anti-CNAP1 (red, labeling proximal ends of centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. G H Representative images of the co-localization of OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP with γ-tubulin. Insets, enlargements of the outlined areas. Scale bars, 10 μm. I Quantitative analysis of cells with and without centrosomal OCRL (centrosomal GFP signal) in the experiments shown in A. n = 3 per group in which 10 cells were counted per condition per experiment. Means ±SEM, ****P < 0.0001, n.s., not significant, calculated by two-way ANOVA, multiple comparisons.


Figure 5A RPE - 1 cells transfected with nontargeting control siRNA or OCRL siRNA , were left for 48 h , then subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , γ - tubulin ( red ) and nuclei ( blue , DAPI staining ) . Scale bar , 10 μm . B Quantitative analyses of centrosomal PI ( 4 , 5 ) P2 fluorescence intensity in the experiments shown in A . n = 3 per group in which 50 cells were counted per condition per experiment . C Data information : data are presented as mean ± SEM . n . s . , not significant , calculated by calculated by two - tailed Student ' s t - test . C NHF , Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI ( 4 , 5 ) P2 ( green ) , gamma - tubulin ( red ) and nuclei ( blue , DAPI staining ) . Scale bar , 10 μm . D The fluorescence intensity of PI ( 4 , 5 ) P2 on centrosomes in C was measured . n = 3 per group in which 50 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . n . s . , not significant , calculated by calculated by two - tailed Student ' s t - test .

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

Figure 5A RPE-1 cells transfected with nontargeting control siRNA or OCRL siRNA, were left for 48 h, then subjected to IF microscopy to visualize PI(4,5)P2 (green), γ-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. B Quantitative analyses of centrosomal PI(4,5)P2 fluorescence intensity in the experiments shown in A. n = 3 per group in which 50 cells were counted per condition per experiment. C Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.C NHF, Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI(4,5)P2 (green), gamma-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. D The fluorescence intensity of PI (4,5)P2 on centrosomes in C was measured. n = 3 per group in which 50 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.


Figure 6A SSX2IP interacts with OCRL . Plasmids carrying GFP - OCRL - WT , GFP - OCRL - △ RhoGAP and GFP - OCRL - △ ASH + RhoGAP were transfected into RPE - 1 cells . Immunoprecipitation was performed with GFP - Trap , followed by blotting with the indicated antibodies . D NHF , Lowe 1676 and Lowe 3265 cells were transfected with wildtype GFP - SSX2IP or GFP - PACT - SSX2IP for 48 h , then immunostained with anti - β - tubulin ( red ) antibody . Nuclei ( blue ) were stained with DAPI . The centrosomal microtubule nucleation in GFP - PACT - SSX2IP transfected cells was indicated by white arrowhead . Scale bar , 5 μm . F Western blot analysis of GFP , endogenous phospho - mTOR , total mTOR , in NHF and Lowe 1676 cells . NHF and Lowe 1676 cells were left untransfected , transfected with GFP - SSX2IP - WT or GFP - PACT - SSX2IP , then immunoblotted with indicated antibodies .

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

Figure 6A SSX2IP interacts with OCRL. Plasmids carrying GFP-OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP were transfected into RPE-1 cells. Immunoprecipitation was performed with GFP-Trap, followed by blotting with the indicated antibodies.D NHF, Lowe 1676 and Lowe 3265 cells were transfected with wildtype GFP-SSX2IP or GFP-PACT-SSX2IP for 48 h, then immunostained with anti-β-tubulin (red) antibody. Nuclei (blue) were stained with DAPI. The centrosomal microtubule nucleation in GFP-PACT-SSX2IP transfected cells was indicated by white arrowhead. Scale bar, 5 μm.F Western blot analysis of GFP, endogenous phospho-mTOR, total mTOR, in NHF and Lowe 1676 cells. NHF and Lowe 1676 cells were left untransfected, transfected with GFP-SSX2IP-WT or GFP-PACT-SSX2IP, then immunoblotted with indicated antibodies.


Figure 1 ( A ) HeLa cells expressing GFP ‐ tagged UBQLN1 or UBQLN2 ( or GFP for controls ) were transferred to starvation medium ( PBS ) for the indicated time . Where specified , only serum was removed from the culture medium . Viability of transfected cells was assessed by PI staining and analysed by flow cytometry . The graph represents the mean of three experiments ± s . e . ( B ) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection ; depletion was verified by Western blot . Cells were starved for increasing periods of time , and their viability was analysed as in ( A ) . The graph represents the mean of three experiments ± s . e ; * P0 . 05 between control and UBQLN2 ‐ KD at 6 h ( paired Student ' s t ‐ test ) . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; GFP , green fluorescent protein ; KD , knockdown ; PI , propidium iodide ; siRNA , short interfering RNA ; UBQLN , ubiquilin .

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

Figure 1(A) HeLa cells expressing GFP‐tagged UBQLN1 or UBQLN2 (or GFP for controls) were transferred to starvation medium (PBS) for the indicated time. Where specified, only serum was removed from the culture medium. Viability of transfected cells was assessed by PI staining and analysed by flow cytometry. The graph represents the mean of three experiments±s.e.(B) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection; depletion was verified by Western blot. Cells were starved for increasing periods of time, and their viability was analysed as in (A). The graph represents the mean of three experiments±s.e; *P0.05 between control and UBQLN2‐KD at 6 h (paired Student's t‐test). GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GFP, green fluorescent protein; KD, knockdown; PI, propidium iodide; siRNA, short interfering RNA; UBQLN, ubiquilin.


Figure 2 ( A ) HeLa cells were transfected with siRNAs for autophagy - related gene ATG5 ( siATG5 ) or with a non - silencing siRNA ( siC ) . Depletion of ATG5 , which decreased the levels of the ATG5 - ATG12 covalent complex , was confirmed by Western blot . Loading control : transferrin receptor ( TfR ) . ( B ) Control or ATG5 ‐ KD cells transfected with UBQLN1 were starved , and the cell viability was assessed as in Fig 1 ; the graph represents the mean of three experiments ± s . e . ( C ) HeLa cells were stably transfected with control ( shC ) or ATG7 ( shATG7 ) shRNA ; depletion was verified by Western blot ( loading control : GAPDH ) . ( D ) Control or ATG7 ‐ KD cells transfected with UBQLN1 were starved for 3 or 6 h and assessed for their viability . The graph represents the mean of three experiments ± s . e . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; KD , knockdown ; Pl , propidium iodide ; shRNA , short hairpin RNA ; siRNA , short interfering RNA ; UBQLN , ubiquilin .

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 B-PANEL_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 B-PANEL_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

Figure 2(A) HeLa cells were transfected with siRNAs for autophagy-related gene ATG5 (siATG5) or with a non-silencing siRNA (siC). Depletion of ATG5, which decreased the levels of the ATG5-ATG12 covalent complex, was confirmed by Western blot. Loading control: transferrin receptor (TfR).(B) Control or ATG5‐KD cells transfected with UBQLN1 were starved, and the cell viability was assessed as in Fig 1; the graph represents the mean of three experiments±s.e.(C) HeLa cells were stably transfected with control (shC) or ATG7 (shATG7) shRNA; depletion was verified by Western blot (loading control: GAPDH).(D) Control or ATG7‐KD cells transfected with UBQLN1 were starved for 3 or 6 h and assessed for their viability. The graph represents the mean of three experiments±s.e. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; KD, knockdown; Pl, propidium iodide; shRNA, short hairpin RNA; siRNA, short interfering RNA; UBQLN, ubiquilin.


Figure 3 ( A ) Immunolocalization of transfected Myc ‐ UBQLN ( 1 or 2 ) and GFP ‐ tagged microtubule ‐ associated protein 1 light chain 3 ( GFP ‐ LC3 ) was analysed by microscopy . Inset : magnification of UBQLN1 vesicular staining ( arrow ) , which , after acquisition of Z ‐ stacks , was found in all UBQLN structures analysed . ( B ) Cells were starved for 2 h , with ( top row ) or without BAF A1 ( bottom row ) . Cells were stained for endogenous UBQLN2 ( magenta ) and LC3 ( green ) . ( C ) Control cells ( untransfected , right panel ) or cells overexpressing Myc ‐ UBQLN2 and GFP ‐ LC3 ( left panel ) were transferred into starvation medium for 2 h . Localization of Myc ‐ UBQLN2 and GFP ‐ LC3 was analysed by immunoelectron microscopy using Myc ( arrow ) and GFP ( arrowhead ) antibodies coupled to 10 and 15 nm gold particles , respectively . Double ‐ membrane vacuoles ( autophagosomes ) in transfected cells ( left panel ) show increased staining by both antibodies compared with autophagosomes in untransfected cells ( right panel ) . Scale bar , 0 . 5 μm . BAF A , bafilomycin A1 ; GFP , green fluorescent protein ; UBQLN , ubiquilin .

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 B-PANEL_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 O O O O O O O O O O O O O O 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) Immunolocalization of transfected Myc‐UBQLN (1 or 2) and GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) was analysed by microscopy. Inset: magnification of UBQLN1 vesicular staining (arrow), which, after acquisition of Z‐stacks, was found in all UBQLN structures analysed.(B) Cells were starved for 2 h, with (top row) or without BAF A1 (bottom row). Cells were stained for endogenous UBQLN2 (magenta) and LC3 (green).(C) Control cells (untransfected, right panel) or cells overexpressing Myc‐UBQLN2 and GFP‐LC3 (left panel) were transferred into starvation medium for 2 h. Localization of Myc‐UBQLN2 and GFP‐LC3 was analysed by immunoelectron microscopy using Myc (arrow) and GFP (arrowhead) antibodies coupled to 10 and 15 nm gold particles, respectively. Double‐membrane vacuoles (autophagosomes) in transfected cells (left panel) show increased staining by both antibodies compared with autophagosomes in untransfected cells (right panel). Scale bar, 0.5 μm. BAF A, bafilomycin A1; GFP, green fluorescent protein; UBQLN, ubiquilin.


Figure 4 ( B ) Each mutant was co ‐ transfected with GFP ‐ tagged microtubule ‐ associated protein 1 light chain 3 ( GFP ‐ LC3 ) and the localization of each protein was analysed by microscopy . ( C ) Starvation ‐ induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA . The graph represents the mean of three experiments ± s . e . , with * P ⩽ 0 . 05 ( P = 0 . 0017 by one ‐ way ANOVA test , comparing control , UBQLN1 and UBQLN1ΔUBA ) . No difference was found between control and UBQLN1ΔUBA at any time point . ANOVA , analysis of variance ; GFP , green fluorescent protein ; Pl , propidium iodide ; UBA , ubiquitin ‐ associated ; UBQLN , ubiquilin .

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

Figure 4(B) Each mutant was co‐transfected with GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) and the localization of each protein was analysed by microscopy.(C) Starvation‐induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA. The graph represents the mean of three experiments±s.e., with *P⩽0.05 (P=0.0017 by one‐way ANOVA test, comparing control, UBQLN1 and UBQLN1ΔUBA). No difference was found between control and UBQLN1ΔUBA at any time point. ANOVA, analysis of variance; GFP, green fluorescent protein; Pl, propidium iodide; UBA, ubiquitin‐associated; UBQLN, ubiquilin.


Figure 5 ( A ) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN ‐ or autophagy ‐ related gene ATG5 ‐ KD cells expressing GFP ‐ tagged microtubule associated protein 1 light chain 3 ( GFP ‐ LC3 ) . ( B ) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus ( in pixels ) and by analysing the frequency distribution . ( C ) Control or UBQLN ‐ KD cells expressing GFP ‐ LC3 were starved and stained with the mitochondria dye Mitotracker ™ . Dual staining of LC3 ( green ) with Mitotracker ( magenta ) was analysed by microscopy . ( D ) Control and UBQLN ‐ KD cells stained with Mitotracker were starved for 6 h ; BAF A was added where indicated . Mitotracker fluorescence was measured by flow cytometry . Unless specified otherwise , values for UBQLN ‐ KD and control + BAF A were compared with that of controls at the same time points , with * * * P0 . 001 , * * P0 . 01 and * P0 . 05 ( paired Student ' s t ‐ test ) . ( E ) Cells were transfected with pShooter ™ together with control or UBQLN ( 1 + 2 ) siRNA . Expression of pShooter was comparable in control and UBQLN ‐ KD cells ( top ) . Cells were starved for 3 or 6 h , and the intensity of GFP was quantified by flow cytometry ( bottom ) . The fluorescence value obtained with controls ( no starvation ) was considered as 100 % , and the value obtained for the 3 ‐ and 6 ‐ h time points was normalized to controls . The graph represents the mean ± s . e . of four assessments . ( F ) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA , and starvation was induced for the indicated times . Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour . The graph represents the mean of three experiments ± s . e . The number of mCherry single ‐ positive puncta in UBQLN ‐ KD cells was compared with that of controls for each time point ; * * P0 . 01 and * P0 . 05 ( paired Student ' s t ‐ test ) . ( G ) Control or UBQLN ‐ KD cells were starved for increasing periods . Where indicated , BAF A was added to the starvation medium ( + B ) . Cell lysates were analysed by Western blot for their content in LC3 ‐ II . Loading control : transferrin receptor ( TfR ) . BAF A , bafilomycin A1 ; GFP , green fluorescent protein ; KD , knockdown ; siRNA , short interfering RNA ; UBQLN , ubiquilin .

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 B-PANEL_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 B-PANEL_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 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 B-PANEL_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) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN‐ or autophagy‐related gene ATG5‐KD cells expressing GFP‐tagged microtubule associated protein 1 light chain 3 (GFP‐LC3).(B) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus (in pixels) and by analysing the frequency distribution.(C) Control or UBQLN‐KD cells expressing GFP‐LC3 were starved and stained with the mitochondria dye Mitotracker™. Dual staining of LC3 (green) with Mitotracker (magenta) was analysed by microscopy.(D) Control and UBQLN‐KD cells stained with Mitotracker were starved for 6 h; BAF A was added where indicated. Mitotracker fluorescence was measured by flow cytometry. Unless specified otherwise, values for UBQLN‐KD and control+BAF A were compared with that of controls at the same time points, with ***P0.001, **P0.01 and *P0.05 (paired Student's t‐test).(E) Cells were transfected with pShooter™ together with control or UBQLN (1+2) siRNA. Expression of pShooter was comparable in control and UBQLN‐KD cells (top). Cells were starved for 3 or 6 h, and the intensity of GFP was quantified by flow cytometry (bottom). The fluorescence value obtained with controls (no starvation) was considered as 100%, and the value obtained for the 3‐ and 6‐h time points was normalized to controls. The graph represents the mean±s.e. of four assessments.(F) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA, and starvation was induced for the indicated times. Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour. The graph represents the mean of three experiments±s.e. The number of mCherry single‐positive puncta in UBQLN‐KD cells was compared with that of controls for each time point; **P0.01 and *P0.05 (paired Student's t‐test).(G) Control or UBQLN‐KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3‐II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.


Figure 1 ( A , B ) BMDCs were infected with Pru parasites ( at a MOI of 4 parasites / BMDC ) or treated with tunicamycin ( TN ) for 16h and the expression of UPR target genes were analyzed by RT - qPCR . The results are expressed as ( Log2 ) Fold change ( A ) or relative mRNA level ( B ) compared to non - infected ( NI ) conditions . IRE1α / XBP1s pathway : Ern1 ( IRE1α ) , Xbp1s , Dnajb9 ( ERdj4 ) , Sec24D , Bloc1s1 and Sec61a1 ; PERK / ATF4 pathway : Atf4 , Ddit3 ( CHOP ) and Ppp1r15a ( GADD34 ) ; ATF6 pathway : Atf6 and Herpud1 ; classical ER chaperons : Hspa5 ( BIP ) and Hsp90b1 ( GRP94 ) . Results are normalized to the housekeeping gene Gapdh . Unpaired Student T - test , ns : p > 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; mean ± S . E . M ( n = 2 - 5 independent experiments depending of the studied gene , 3 mice / independent experiment ) . ( C , D ) Immunoblot analysis of IRE1α , PERK , Phospho - PERK ( detected by the presence of a second upper band after migration on a 6 % acrylamide gel ) , Phospho - eIF2α , CHOP and XBP1s protein expression in non - infected BMDCs , BMDCs treated with TN and Pru infected BMDCs ( C ) and in non - infected and Pru infected Flt3 / Notch - DCs ( D ) for the indicated times . GAPDH and tubulin were used as loading controls . The same blots are shown in Figure EV3B - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1(A, B) BMDCs were infected with Pru parasites (at a MOI of 4 parasites/BMDC) or treated with tunicamycin (TN) for 16h and the expression of UPR target genes were analyzed by RT-qPCR. The results are expressed as (Log2) Fold change (A) or relative mRNA level (B) compared to non-infected (NI) conditions. IRE1α/XBP1s pathway: Ern1 (IRE1α), Xbp1s, Dnajb9 (ERdj4), Sec24D, Bloc1s1 and Sec61a1; PERK/ATF4 pathway: Atf4, Ddit3 (CHOP) and Ppp1r15a (GADD34); ATF6 pathway: Atf6 and Herpud1; classical ER chaperons: Hspa5 (BIP) and Hsp90b1 (GRP94). Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=2-5 independent experiments depending of the studied gene, 3 mice / independent experiment).(C, D) Immunoblot analysis of IRE1α, PERK, Phospho-PERK (detected by the presence of a second upper band after migration on a 6% acrylamide gel), Phospho-eIF2α, CHOP and XBP1s protein expression in non-infected BMDCs, BMDCs treated with TN and Pru infected BMDCs (C) and in non-infected and Pru infected Flt3/Notch-DCs (D) for the indicated times. GAPDH and tubulin were used as loading controls. The same blots are shown in Figure EV3B-C.


Figure 2 ( D ) mRNA relative expression level of Xbp1s , Dnajb9 ( ERdj4 ) , Ddit3 ( CHOP ) and Ppp1r15a ( GADD34 ) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non - infected ( NI ) conditions . Results are normalized to the housekeeping gene Gapdh . Two - way ANOVA Tukey ' s multiple comparisons , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 , * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 4 - 5 mice pooled from 2 independent experiments ) .

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Figure 2(D) mRNA relative expression level of Xbp1s, Dnajb9 (ERdj4), Ddit3 (CHOP) and Ppp1r15a (GADD34) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001; mean ± S.E.M (n=4-5 mice pooled from 2 independent experiments).


Figure 3 ( A ) BMDCs ( CD11c + ) were incubated with Pru - Tomato parasites for 16h . Infected cells ( red gating ) and non - infected bystander cells ( green gating ) were sorted - purified and further analyzed . ( B ) The expression of UPR target genes were analyzed by RT - qPCR in infected and non - infected bystander cells compared to non - infected BMDCs ( NI ) . The results are expressed as relative mRNA level compared to non - infected conditions . Results are normalized to the housekeeping gene Gapdh . Unpaired Student T - test , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; mean ± S . E . M ( n = 6 mice pooled from 2 independent experiments ) .

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 3(A) BMDCs (CD11c+) were incubated with Pru-Tomato parasites for 16h. Infected cells (red gating) and non-infected bystander cells (green gating) were sorted-purified and further analyzed. (B) The expression of UPR target genes were analyzed by RT-qPCR in infected and non-infected bystander cells compared to non-infected BMDCs (NI). The results are expressed as relative mRNA level compared to non-infected conditions. Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=6 mice pooled from 2 independent experiments).


Figure 4 ( E ) mRNA relative expression level of Il - 6 , Il - 12 and Il - 23 from XBP1fl / flIRE1fl / fl , XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non - infected ( NI ) conditions . Results are normalized to the housekeeping gene Gapdh . Two - way ANOVA Tukey ' s multiple comparisons , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 3 - 5 mice pooled from 2 independent experiments ) . ( I ) Graph depicts the expression ( indicated as the Mean Fluorescence Intensity ) of H - 2Kb by Pru Tomato SAG1 ‐ OVA infected XBP1fl / fl IRE1fl / fl treated or not with 4μ8C , XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs . Bar graphs depict mean ± S . E . M ( n = 3 mice , one representative experiment from 2 independent experiments ) . Tukey ' s ANOVA one - way test , ns : p > 0 . 05 .

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 B-PANEL_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

Figure 4(E) mRNA relative expression level of Il-6, Il-12 and Il-23 from XBP1fl/flIRE1fl/fl, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).(I) Graph depicts the expression (indicated as the Mean Fluorescence Intensity) of H-2Kb by Pru Tomato SAG1‐OVA infected XBP1fl/fl IRE1fl/fl treated or not with 4μ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Tukey's ANOVA one-way test, ns: p>0.05.


Figure 5 ( A , B ) Expression of VenusFP by splenic cDC1 ( CD26 + CD11c + MHCII + XCR1 + CD172a - , upper panels ) and cDC2 ( CD26 + CD11c + MHCII + XCR1 - CD172a + , lower panel ) from WT ( A ) and ERAI ( B ) mice non - infected ( NI ) or infected with Pru ( 6 days post - infection ( dpi ) ) reporting on IRE1α pathway activation . ( E , F ) RT - qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 ( E ) and cDC2 ( F ) sorted by flow cytometry 6 dpi from wild - type mice , non - infected ( NI ) or infected with Pru parasites . Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition . Bar graphs depict mean ± S . E . M . Unpaired Student T - test , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; mean ± S . E . M ( n = 3 - 5 mice pooled from 2 independent experiments ) .

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

Figure 5(A, B) Expression of VenusFP by splenic cDC1 (CD26+ CD11c+ MHCII+ XCR1+ CD172a-, upper panels) and cDC2 (CD26+ CD11c+ MHCII+ XCR1- CD172a+, lower panel) from WT (A) and ERAI (B) mice non-infected (NI) or infected with Pru (6 days post-infection (dpi)) reporting on IRE1α pathway activation.(E, F) RT-qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 (E) and cDC2 (F) sorted by flow cytometry 6 dpi from wild-type mice, non-infected (NI) or infected with Pru parasites. Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition. Bar graphs depict mean ± S.E.M. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).


Figure 6 ( A ) Survival curves of XBP1fl / flIRE1fl / fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites . Log - rank ( Mantel - Cox ) test , p = 0 , 0004 ; n = 10 mice per genotype . ( D , E ) Quantification of the number of total ( left histogram ) and activated ( right histogram ) ( IFNγ - positive ) CD4 + T cells ( D ) and CD8 + T cells ( E ) in the spleen of control mice ( XBP1fl / flIRE1fl / fl ) and XBP1ΔDC IRE1truncDC infected for 6 days . Two - way ANOVA Tukey ' s multiple compare , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 2 independent experiments combined ) .

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Figure 6(A) Survival curves of XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites. Log-rank (Mantel-Cox) test, p=0,0004; n=10 mice per genotype.(D, E) Quantification of the number of total (left histogram) and activated (right histogram) (IFNγ-positive) CD4+ T cells (D) and CD8+ T cells (E) in the spleen of control mice (XBP1fl/flIRE1fl/fl) and XBP1ΔDC IRE1truncDC infected for 6 days. Two-way ANOVA Tukey's multiple compare, ns: p> 0.05; *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; mean ± S.E.M (n=2 independent experiments combined).


Figure 1 ( A ) Raji cells stably expressing HIV - 1 YU - 2 ENV ( Raji - Env ) were stained with bNAbs ( 10 - 1074 or 3BNC117 ) or isotype control ( mGO53 ) and analyzed by flow cytometry . The numbers indicate the % of positive cells . One representative experiment is shown . ( B ) Binding of the 24 indicated antibodies to the surface of Raji - Env cells . Results are expressed as the median fluorescence intensity ( MFI ) of staining . n = 6 independent experiments . Data information : Error bars indicate SEM . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney test . ( C ) Raji - Env cells were incubated with indicated antibodies and either normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 1h , surface levels of C3 were analyzed by flow cytometry . One representative experiment is shown . ( D ) C3 deposition by the 24 indicated antibodies . For each antibody , a normalized MFI is calculated by subtracting the MFI of the " no antibody " condition . ( nd . not done ) . n = 3 donors of serum . Data information : Error bars indicate SEM . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney test . ( E ) Raji - Env cells were incubated with indicated antibodies and either normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 24h , cell death was determined by flow cytometry . The numbers indicate the percentage of dead cells . One representative experiment is shown . ( F ) CDC induction by the 24 indicated antibodies . The % of CDC was calculated as the relative percentage of dead cells compared to the " no antibody " condition . n = 3 donors of serum . Data information : Error bars indicate SEM . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney test . ( G ) Raji - Env cells were incubated for 24h with NHS and indicated concentrations of WT or E430G mutant bNAbs ( 10 - 1074 , 3BNC117 or PGDM1400 ) or isotype control ( mGO53 ) . Cell death was measured by flow cytometry and the % of CDC was calculated as the relative % of dead cells compared to the " no antibody " condition . n = 3 donors of serum .

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

Figure 1(A) Raji cells stably expressing HIV-1 YU-2 ENV (Raji-Env) were stained with bNAbs (10-1074 or 3BNC117) or isotype control (mGO53) and analyzed by flow cytometry. The numbers indicate the % of positive cells. One representative experiment is shown.(B) Binding of the 24 indicated antibodies to the surface of Raji-Env cells. Results are expressed as the median fluorescence intensity (MFI) of staining. n=6 independent experiments. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.(C) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 1h, surface levels of C3 were analyzed by flow cytometry. One representative experiment is shown.(D) C3 deposition by the 24 indicated antibodies. For each antibody, a normalized MFI is calculated by subtracting the MFI of the "no antibody" condition. (nd. not done). n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.(E) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, cell death was determined by flow cytometry. The numbers indicate the percentage of dead cells. One representative experiment is shown.(F) CDC induction by the 24 indicated antibodies. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.(G) Raji-Env cells were incubated for 24h with NHS and indicated concentrations of WT or E430G mutant bNAbs (10-1074, 3BNC117 or PGDM1400) or isotype control (mGO53). Cell death was measured by flow cytometry and the % of CDC was calculated as the relative % of dead cells compared to the "no antibody" condition. n=3 donors of serum.


Figure 2 ( A ) Activated primary CD4 T cells were infected by HIV - 1 ( strain NLAD8 ) and subjected to antibody binding assay . The numbers indicate the % of Gag + cells that are decorated by the indicated antibodies . One representative experiment is shown . ( B ) Antibody binding at the surface of HIV - 1 - infected primary CD4 T cells . Results are expressed as median fluorescence ( MFI ) of staining on infected ( Gag + ) cells . Each dot represents a single donor of CD4 T cells . n = 9 donors of CD4 T cells . Data information : Error bars indicate SEM . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon test . Only significant comparisons are depicted . ( C ) HIV - 1 - infected ( strain NLAD8 ) CD4 T cells were cultured with the indicated antibody and NHS . After 24h , the surface levels of C3 and the frequency of infected cells ( Gag + ) were determined by flow cytometry . One representative experiment is shown . ( D ) C3 surface levels at the surface of HIV - 1 - infected primary CD4 T cells . Results are expressed as MFI of staining on infected ( Gag + ) cells . Each dot represents a single donor of CD4 T cells . n = 7 donors of CD4 T cells . Data information : Error bars indicate SEM . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon test . Only significant comparisons are depicted . ( E ) HIV - 1 - infected CD4 T cells ( strain NLAD8 ) were cultured with the indicated antibody and normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 24h , the surface levels of C5b - 9 ( MAC ) on infected ( Gag + ) cells were determined by flow cytometry . One representative experiment is shown . ( F ) C5b - 9 ( MAC ) surface levels on HIV - 1 - infected cells ( strain NLAD8 ) . Results are expressed as MFI of staining on infected ( Gag + ) cells . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells . Data information : Error bars indicate SEM . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon test . Only significant comparisons are depicted . ( G ) HIV - 1 - infected CD4 T cells ( strain NLAD8 ) were cultured with the indicated antibody and NHS or HIHS to determine complement - mediated cytotoxicity . After 24h , the percentage of infected ( Gag + ) cells in each condition was determined by flow cytometry . One representative experiment is shown . ( H ) Complement - mediated cytotoxicity of HIV - 1 - infected CD4 T cells ( strain NLAD8 ) . For each antibody , the % of CDC was calculated at 24h as the % of disappearance of Gag + cells in NHS compared to HIHS . Each dot represents a single donor of CD4 T cells . n = 7 donors of CD4 T cells . Data information : Error bars indicate SEM . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon test . Only significant comparisons are depicted .

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 B-PANEL_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 B-PANEL_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activated primary CD4 T cells were infected by HIV-1 (strain NLAD8) and subjected to antibody binding assay. The numbers indicate the % of Gag+ cells that are decorated by the indicated antibodies. One representative experiment is shown.(B) Antibody binding at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as median fluorescence (MFI) of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=9 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.(C) HIV-1-infected (strain NLAD8) CD4 T cells were cultured with the indicated antibody and NHS. After 24h, the surface levels of C3 and the frequency of infected cells (Gag+) were determined by flow cytometry. One representative experiment is shown.(D) C3 surface levels at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.(E) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, the surface levels of C5b-9 (MAC) on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown.(F) C5b-9 (MAC) surface levels on HIV-1-infected cells (strain NLAD8). Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.(G) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. One representative experiment is shown.(H) Complement-mediated cytotoxicity of HIV-1-infected CD4 T cells (strain NLAD8). For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


Figure 3 ( A ) Raji - Env cells were subcloned to obtain cell lines expressing increasing amounts of Env . Env surface levels were determined by the binding of 10 - 1074 and measured by flow cytometry . One representative experiment is shown . ( B ) Parental Raji cells and the different Raji - Env clones were cultured with 10 - 1074 or an isotype control ( mGO53 ) and NHS for 24h . The % of CDC was calculated as the relative percentage of dead cells compared to the " no antibody " condition . n = 3 donors of serum . ( C ) Non - infected ( NI ) and HIV - 1 - infected primary CD4 T cells ( strain NLAD8 ) as well as three Raji - Env clones ( D1 , D2 and D4 ) were incubated with 10 - 1074 and antibody surface levels were analyzed by flow cytometry . Results are expressed as median fluorescence ( MFI ) of staining in total cells ( Raji , n = 3 independent experiments ) or in infected ( Gag + ) cells ( CD4 T cells , n = 5 donors ) . ( D ) HIV - or HIV ∆ Vpu - infected CD4 T cells ( strain NLAD8 ) were subjected to antibody binding with indicated antibodies and surface levels were determined by flow cytometry ( left panel ) . The staining obtained with mGO53 was similar to the background signal observed on unstained cells . Infected cells were cultured with the indicated antibodies and NHS for 24h and the C3 surface levels on infected ( Gag + ) cells were determined by flow cytometry ( right panel ) . One representative experiment is shown . ( E ) HIV - or HIV ∆ Vpu - infected CD4 T cells ( strain NLAD8 ) were incubated with NHS and either 10 - 1074 ( left panel ) or 3BNC117 ( right panel ) . Both WT or E430G - mutated antibodies were used . After 24h , C3 surface levels on infected cells were analyzed by flow cytometry . Results are expressed as MFI of staining on infected ( Gag + ) cells . The MFI of C3 staining on Gag - cells was included as a control . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells .

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 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 B-PANEL_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 B-PANEL_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 3(A) Raji-Env cells were subcloned to obtain cell lines expressing increasing amounts of Env. Env surface levels were determined by the binding of 10-1074 and measured by flow cytometry. One representative experiment is shown.(B) Parental Raji cells and the different Raji-Env clones were cultured with 10-1074 or an isotype control (mGO53) and NHS for 24h. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum.(C) Non-infected (NI) and HIV-1-infected primary CD4 T cells (strain NLAD8) as well as three Raji-Env clones (D1, D2 and D4) were incubated with 10-1074 and antibody surface levels were analyzed by flow cytometry. Results are expressed as median fluorescence (MFI) of staining in total cells (Raji, n=3 independent experiments) or in infected (Gag+) cells (CD4 T cells, n=5 donors).(D) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were subjected to antibody binding with indicated antibodies and surface levels were determined by flow cytometry (left panel). The staining obtained with mGO53 was similar to the background signal observed on unstained cells. Infected cells were cultured with the indicated antibodies and NHS for 24h and the C3 surface levels on infected (Gag+) cells were determined by flow cytometry (right panel). One representative experiment is shown.(E) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were incubated with NHS and either 10-1074 (left panel) or 3BNC117 (right panel). Both WT or E430G-mutated antibodies were used. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. The MFI of C3 staining on Gag- cells was included as a control. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


Figure 4 ( A ) Primary CD4 T cells were nucleofected with a control ( CRISPR Control ) or anti - CD59 ( CRISPR CD59 ) guide RNA / Cas9 complex . CD59 surface levels were analyzed by flow cytometry 7 days after nucleofection . Isotype control ( grey ) and CD59 stainings ( blue ) . One representative experiment is shown on the left . Numbers indicate the % of CD59 + cells ( middle ) or the MFI of staining in CD59 - cells ( Right ) . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells . ( B ) NLAD8 ∆ Vpu - infected CD59 KO primary CD4 T cells were cultured with indicated antibodies and NHS or HIHS for 24h . Numbers indicate the percentage of infected ( Gag + ) cells . One representative experiment is shown . ( C ) CD59 - KO ( CRISPR CD59 ) or control ( CRISPR Control ) CD4 T cells infected with HIV WT ( left ) or HIVΔVpu ( right ) ( strain NLAD8 ) were cultured with the indicated antibody , and NHS or HIHS for 24h . The % of CDC was calculated as the % of disappearance of Gag + cells after culture with NHS compared to HIHS . Each dot represents a single donor of CD4 T cells . n = at least 6 donors of CD4 T cells . ( D ) CD59 surface levels on non - infected ( NI ) or HIV - 1 - infected ( strain NLAD8 ) primary CD4 T cells were analyzed by flow cytometry . Results are expressed as the MFI of staining with an isotype control or the anti - CD59 antibody . n = 4 donors of CD4 T cells .

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Figure 4(A) Primary CD4 T cells were nucleofected with a control (CRISPR Control) or anti-CD59 (CRISPR CD59) guide RNA/Cas9 complex. CD59 surface levels were analyzed by flow cytometry 7 days after nucleofection. Isotype control (grey) and CD59 stainings (blue). One representative experiment is shown on the left. Numbers indicate the % of CD59+ cells (middle) or the MFI of staining in CD59- cells (Right). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.(B) NLAD8∆Vpu-infected CD59 KO primary CD4 T cells were cultured with indicated antibodies and NHS or HIHS for 24h. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown.(C) CD59-KO (CRISPR CD59) or control (CRISPR Control) CD4 T cells infected with HIV WT (left) or HIVΔVpu (right) (strain NLAD8) were cultured with the indicated antibody, and NHS or HIHS for 24h. The % of CDC was calculated as the % of disappearance of Gag+ cells after culture with NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n= at least 6 donors of CD4 T cells.(D) CD59 surface levels on non-infected (NI) or HIV-1-infected (strain NLAD8) primary CD4 T cells were analyzed by flow cytometry. Results are expressed as the MFI of staining with an isotype control or the anti-CD59 antibody. n=4 donors of CD4 T cells.


Figure 5 ( B ) HIV - 1 - infected primary CD4 T cells were cultured with the indicated antibodies ( E430G ) and with NHS or HIHS . The frequency of infected cells was assessed by flow cytometry in live cells at days 1 , 2 , 3 and 6 . Numbers indicate the percentage of infected ( Gag + ) cells . One representative experiment is shown . ( C ) Frequency of live HIV - 1 - infected cells ( NLAD8 WT or ΔVpu strain ) 6 days after incubation with the indicated antibodies , and with NHS or HIHS . Each donor of CD4 T cells is represented by a different symbol . n = 8 donors of CD4 T cells . ( D ) Elimination of HIV - 1 - infected cells ( strain NLAD8 WT or ΔVpu ) over culture with the indicated antibody ( E430G ) , and NHS or HIHS . The frequency of % infected ( Gag + ) cells is determinined by flow cytometry . The mean relative percentage of infected cells compared to the HIHS condition is depicted . n = 6 - 8 donors of CD4 T cells .

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Figure 5(B) HIV-1-infected primary CD4 T cells were cultured with the indicated antibodies (E430G) and with NHS or HIHS. The frequency of infected cells was assessed by flow cytometry in live cells at days 1, 2, 3 and 6. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown.(C) Frequency of live HIV-1-infected cells (NLAD8 WT or ΔVpu strain) 6 days after incubation with the indicated antibodies, and with NHS or HIHS. Each donor of CD4 T cells is represented by a different symbol. n=8 donors of CD4 T cells.(D) Elimination of HIV-1-infected cells (strain NLAD8 WT or ΔVpu) over culture with the indicated antibody (E430G), and NHS or HIHS. The frequency of % infected (Gag+) cells is determinined by flow cytometry. The mean relative percentage of infected cells compared to the HIHS condition is depicted. n=6-8 donors of CD4 T cells.


Figure 6 ( A ) Primary CD4 T cells infected with HIV - 1 ( Transmitted / Founder strains CH058 and CH077 , and the primary strain vKB18 ) were incubated with the indicated bNAbs and surface levels were analyzed by flow cytometry . The numbers indicate the % of bNAb + cells among infected ( Gag + ) cells . The percentages of infected cells were 27 % , 30 % and 6 % for CH058 , CH077 and vKB18 , respectively . One representative experiment is shown . ( B ) CH058 - infected CD4 T cells were cultured with the indicated WT or E430G mutant antibody and NHS . After 24h , the C3 surface levels on infected ( Gag + ) cells were determined by flow cytometry . One representative experiment is shown . ( C ) CH058 - , CH077 - and vKB18 - infected primary CD4 T cells were incubated with the indicated antibodies and NHS . After 24h , C3 surface levels on infected cells were analyzed by flow cytometry . Results are expressed as MFI of staining on infected ( Gag + ) cells . Each dot represents a donor of CD4 T cells . n = 6 donors of CD4 T cells . ( D ) Primary CD4 T cells infected with the indicated HIV - 1 strains ( CH058 , CH077 or vKB18 ) were cultured with the indicated WT or E430G mutant antibodies and NHS or HIHS to determine complement - mediated cytotoxicity . After 24h , the percentage of infected ( Gag + ) cells in each condition was determined by flow cytometry . For each antibody , the % of CDC was calculated at 24h as the % of disappearance of Gag + cells in NHS compared to HIHS . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells .

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Figure 6(A) Primary CD4 T cells infected with HIV-1 (Transmitted/Founder strains CH058 and CH077, and the primary strain vKB18) were incubated with the indicated bNAbs and surface levels were analyzed by flow cytometry. The numbers indicate the % of bNAb+ cells among infected (Gag+) cells. The percentages of infected cells were 27%, 30% and 6% for CH058, CH077 and vKB18, respectively. One representative experiment is shown.(B) CH058-infected CD4 T cells were cultured with the indicated WT or E430G mutant antibody and NHS. After 24h, the C3 surface levels on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown.(C) CH058-, CH077- and vKB18-infected primary CD4 T cells were incubated with the indicated antibodies and NHS. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a donor of CD4 T cells. n=6 donors of CD4 T cells.(D) Primary CD4 T cells infected with the indicated HIV-1 strains (CH058, CH077 or vKB18) were cultured with the indicated WT or E430G mutant antibodies and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


Figure 7 ( A ) HIV - 1 - infected primary CD4 T cells ( strain CH058 ) were incubated with sera of HIV negative ( HIV - ; n = 9 ) or ART - treated HIV - 1 - infected ( HIV + ; n = 9 ) individuals ( serum dilution 1 : 1000 ) . The levels of IgG binding on infected ( Gag + ) cells were analyzed by flow cytometry . Results are expressed as the MFI on infected ( Gag + ) cells . Error bars indicate SEM . * * * * P < 0 , 0001 ; Mann - Whitney test . ( B ) CH058 - infected primary CD4 T cells were cultured for 24h with sera of healthy ( HIV - ; n = 9 ) or ART - treated HIV - 1 - infected ( HIV + ; n = 9 ) individuals ( serum dilution 1 : 100 ) and 50 % NHS from a healthy donor . C3 surface levels on infected ( Gag + ) cells were analyzed by flow cytometry . Results are expressed as the MFI on infected ( Gag + ) cells . Error bars indicate SEM . * * * * P < 0 , 0001 ; Mann - Whitney test . ( C ) CD4 T cells not infected ( NI ) or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) were incubated with the serum of HIV - 1 - infected ART - treated individuals ( sKB104 or sKB105 , dilution 1 : 1000 ) . The levels of IgG binding on infected ( Gag + ) cells were analyzed by flow cytometry . One representative experiment is shown on the left . On the right , results are expressed as MFI of staining . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells . Error bars indicate SEM . * P < 0 . 05 , Wilcoxon test . Only significant comparisons are depicted . ( D ) Primary CD4 T cells either not infected ( NI ) or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) were incubated with 50 % NHS from a healthy donor and serum of HIV - 1 - infected ART - treated individuals ( sKB104 or sKB105 , dilution 1 : 100 ) . The levels of C3 deposition on infected ( Gag + ) cells were analyzed by flow cytometry . One representative experiment is shown ( left panel ) . Results are expressed as MFI of staining ( right panel ) . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells . ( E ) Correlation between patients ' sera ( sK104 and sKB105 ) antibody binding and C3 deposition on CD4 T cells either mock - infected or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) . Each dot is the mean of 6 donors of CD4 T cells . Correlation was analyzed by Spearman correlation . Correlation coefficient ( r ) and p - value are indicated .

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Figure 7(A) HIV-1-infected primary CD4 T cells (strain CH058) were incubated with sera of HIV negative (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.(B) CH058-infected primary CD4 T cells were cultured for 24h with sera of healthy (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:100) and 50% NHS from a healthy donor. C3 surface levels on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.(C) CD4 T cells not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with the serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown on the left. On the right, results are expressed as MFI of staining. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Error bars indicate SEM. *P<0.05, Wilcoxon test. Only significant comparisons are depicted.(D) Primary CD4 T cells either not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with 50% NHS from a healthy donor and serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:100). The levels of C3 deposition on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown (left panel). Results are expressed as MFI of staining (right panel). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.(E) Correlation between patients' sera (sK104 and sKB105) antibody binding and C3 deposition on CD4 T cells either mock-infected or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058). Each dot is the mean of 6 donors of CD4 T cells. Correlation was analyzed by Spearman correlation. Correlation coefficient (r) and p-value are indicated.


Figure 1 ( A ) 786 - 0 or 769 - P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times , and then cell viability was assessed . ( B ) 786 - 0 or 769 - P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT ( 3 * WT indicates 3 - fold expression WT ) and then treated with aa starvation for the indicated time . Cell viability was subsequently assessed . ( Data information : The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t - test . ( * * p < 0 . 01 ; * * * p < 0 . 001 . ) ( I ) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed ( left ) . Quantitative analysis of the GDH1 protein levels was performed using unpaired t - test ( right ) . * * * p < 0 . 001 . Scale bars : 50 μm . ( K ) Overall survival of patients in different tumor stages in the TCGA - KIRC cohort . The R package " survminer " - based maximally selected log - rank statistic was used to determine the best cutoff . The log - rank test was used to determine the p value .

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Figure 1(A) 786-0 or 769-P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times, and then cell viability was assessed. (B) 786-0 or 769-P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT (3*WT indicates 3-fold expression WT) and then treated with aa starvation for the indicated time. Cell viability was subsequently assessed. ( Data information: The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t-test. (**p < 0.01; ***p < 0.001.)(I) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed (left). Quantitative analysis of the GDH1 protein levels was performed using unpaired t-test (right). ***p < 0.001. Scale bars: 50 μm.(K) Overall survival of patients in different tumor stages in the TCGA-KIRC cohort. The R package "survminer"-based maximally selected log-rank statistic was used to determine the best cutoff. The log-rank test was used to determine the p value.


Figure 2 ( D ) Immunofluorescence ( IF ) assay was performed to determine the subcellular location of GDH1 with or without aa starvation . GDH1 localization was indicated by GDH1 antibody and MitoTracker ( left ) . Signal intensities and distances were quantified ( right ) . Scale bars : 20 μm . ( E ) Cytoplasm - mitochondrial fractionation assay was performed to determine the subcellular location of GDH1 with or without aa starvation . The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 ( mitochondrial membrane marker ) and GDH1 . Tubulin was used as loading control . ( G ) Immunofluorescence ( IF ) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted ( dMSP ) GDH1 . GDH1 localization was indicated by GDH1 antibody and MitoTracker ( left ) . Signal intensities and distances were quantified ( right ) . Scale bars : 20 μm . ( H ) Cytoplasm - mitochondrial fractionation assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted ( dMSP ) GDH1 . The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 ( mitochondrial membrane marker ) and GDH1 . Tubulin was used as loading control .

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Figure 2(D) Immunofluorescence (IF) assay was performed to determine the subcellular location of GDH1 with or without aa starvation. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.(E) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of GDH1 with or without aa starvation. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.(G) Immunofluorescence (IF) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.(H) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.


Figure 3 ( D ) FLAG - tagged GDH1 ( including WT and mutants with different lysine mutations to arginine ) was co - transfected with HA - tagged ubiquitin for 48 hours , and then cells were subjected to aa starvation for 12 hours before collecting cells . WB and qPCR separately tested the levels of GDH1 protein and mRNA , respectively . Data information : The data are represented as the mean ± SEM of three independent experiments . The one - way ANOVA test ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( H ) 786 - 0 or 769 - P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time . Cell viability was determined . Unpaired two - tailed t - test was used . Data information : The data are represented as the mean ± SEM of three independent experiments . The unpaired t - test were used ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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Figure 3(D) FLAG-tagged GDH1 (including WT and mutants with different lysine mutations to arginine) was co-transfected with HA-tagged ubiquitin for 48 hours, and then cells were subjected to aa starvation for 12 hours before collecting cells. WB and qPCR separately tested the levels of GDH1 protein and mRNA, respectively. Data information: The data are represented as the mean ± SEM of three independent experiments. The one-way ANOVA test (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).(H) 786-0 or 769-P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time. Cell viability was determined. Unpaired two-tailed t-test was used. Data information: The data are represented as the mean ± SEM of three independent experiments. The unpaired t-test were used (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


Figure 4786 - 0 cells were treated with MG132 ( 10 μM ) under aa starvation . Antibodies against GDH1 ( C ) were used to enrich protein complexes in cells with or without GDH1 . IgG was used as a control . WB was performed using the indicated antibodies . ( H ) 786 - 0 or 769 - P cells ( WT or stably expressing shGDH1 ) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times . Cell viability was determined . Unpaired t - test was used with * * * p < 0 . 001 . ( I ) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 ( upper panel ) . The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated ( lower panel ) . The cor . test was used to determine the p - value . Scale bars : 50 μm .

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Figure 4786-0 cells were treated with MG132 (10 μM) under aa starvation. Antibodies against GDH1 (C) were used to enrich protein complexes in cells with or without GDH1. IgG was used as a control. WB was performed using the indicated antibodies.(H) 786-0 or 769-P cells (WT or stably expressing shGDH1) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times. Cell viability was determined. Unpaired t-test was used with ***p < 0.001.(I) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 (upper panel). The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated (lower panel). The cor.test was used to determine the p-value. Scale bars: 50 μm.


Figure 5786 - 0 cells expressing WT or enzymatically dead ( ED ) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours . In the above cells in ( A ) , qPCR was used to measure the mRNA of the indicated RP genes ( C ) . ( J ) KIRC clinical samples were collected for αKG measurement , and we assessed overall survival ( left panel ) with log - rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation ( right panel ) . Unpaired t - test was used in the right panel with * * * p < 0 . 001 .

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Figure 5786-0 cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. In the above cells in (A), qPCR was used to measure the mRNA of the indicated RP genes (C).(J) KIRC clinical samples were collected for αKG measurement, and we assessed overall survival (left panel) with log-rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation (right panel). Unpaired t-test was used in the right panel with ***p < 0.001.


Figure 6 ( G ) An in vitro demethylation assay was performed with recombinant KDM4A ( 0 . 05 mM ) or KDM6A ( 0 . 05 mM ) , and the indicated histone H3 lysine methylation peptides under 50 - μM αKG conditions ( no αKG was used as a blank control ) . Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system . ( H ) 786 - 0 cells were supplemented with or without 2HG ( 500 μM ) for 48 hours , and the cells were fixed with 1 % formaldehyde . ChIP assays were performed using antibodies against H3K9me3 or H3K27me3 . qPCR was used to test the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes .

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Figure 6(G) An in vitro demethylation assay was performed with recombinant KDM4A (0.05 mM) or KDM6A (0.05 mM), and the indicated histone H3 lysine methylation peptides under 50-μM αKG conditions (no αKG was used as a blank control). Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system.(H) 786-0 cells were supplemented with or without 2HG (500 μM) for 48 hours, and the cells were fixed with 1% formaldehyde. ChIP assays were performed using antibodies against H3K9me3 or H3K27me3. qPCR was used to test the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes.


Figure 7Xenogeneic inoculation of 786 - 0 cells lacking GDH1 with restored expression of WT , ED or 3KR mutant GDH1 into the left groin of nude mice . The solid tumor formed by the indicated 786 - 0 cells is shown ( B ) . Xenogeneic inoculation of 786 - 0 cells lacking GDH1 with restored expression of WT , ED or 3KR mutant GDH1 into the left groin of nude mice . The staining of Ki67 ( G , upper panel ) and TUNEL ( G , lower panel ) using tumors from ( B ) are shown and analyzed ( G , right panel ) . We used 6 mice in each group . Scale bars : 50 μm . Data information : All statistical analyses were conducted using unpaired t - test ( n = 6 ) . ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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Figure 7Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The solid tumor formed by the indicated 786-0 cells is shown (B).Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The staining of Ki67 (G, upper panel) and TUNEL (G, lower panel) using tumors from (B) are shown and analyzed (G, right panel). We used 6 mice in each group. Scale bars: 50 μm. Data information: All statistical analyses were conducted using unpaired t-test (n = 6). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


Figure 1 ( B ) The confirmative RT - PCRs were resolved in a 2 % agarose gel . Untreated reverse transcriptase RNAs ( - RT ) and genomic DNA served as negative and positive controls , respectively . ( D ) Left panel : Absorption spectra of dark - adapted XccBphP after illumination with red , far - red , fluorescent white lamp light , sunlight or sunlight filtered through green leaves ( LF ) . Pr / Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel . Right panel : The XccBphP - C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red , far - red and white light or dark adaptation .

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Figure 1(B) The confirmative RT-PCRs were resolved in a 2% agarose gel. Untreated reverse transcriptase RNAs (-RT) and genomic DNA served as negative and positive controls, respectively.(D) Left panel: Absorption spectra of dark-adapted XccBphP after illumination with red, far-red, fluorescent white lamp light, sunlight or sunlight filtered through green leaves (LF). Pr/Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel. Right panel: The XccBphP-C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red, far-red and white light or dark adaptation.


Figure 2 ( A - B ) A . thaliana plants were inoculated with wild - type , XccbphP , pXccBphP or pC13S bacterial strains cultured prior to inoculation under ( A ) normal laboratory illumination or ( B ) under light or dark conditions . After 1 , 2 or 3 d . p . i . , CFU per plant mg were determined ( n = 3 replicates ) . Data presented here derive from more than 4 independent experiments . ( C ) Stomatal closure in the presence of bacterial strains or not ( untreated ) after 1 h under light conditions . ( D ) Stomatal opening after 3 h of incubation with bacterial strains or not ( untreated ) in the dark . A control treatment without bacteria and illuminated was included ( Light ) . ( C - D ) Stomatal apertures were recorded ( n = 80 replicates ) . Data are representative of two independent experiments . ( E ) A . thalianaleaves were inoculated with wild - type , XccbphP , pXccBphP and pC13S strains , stained for callose deposits and observed by fluorescence microscopy . MgCl2 buffer ( untreated ) and flg22 peptide were used as negative and positive controls , respectively . Top panel : representative pictures of three independent experiments . Scale bar represents 200 µm . Bottom panel : the number of callose deposits per field of view ( 0 . 45 mm2 ) were determined ( n = 8 replicates ) . Data are representative of two independent experiments . ( A - E ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a two - tailed Mann - Whitney test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 )

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

Figure 2(A-B) A. thaliana plants were inoculated with wild-type, XccbphP, pXccBphP or pC13S bacterial strains cultured prior to inoculation under (A) normal laboratory illumination or (B) under light or dark conditions. After 1, 2 or 3 d.p.i., CFU per plant mg were determined (n = 3 replicates). Data presented here derive from more than 4 independent experiments.(C) Stomatal closure in the presence of bacterial strains or not (untreated) after 1 h under light conditions. (D) Stomatal opening after 3 h of incubation with bacterial strains or not (untreated) in the dark. A control treatment without bacteria and illuminated was included (Light). (C-D) Stomatal apertures were recorded (n = 80 replicates). Data are representative of two independent experiments.(E) A. thalianaleaves were inoculated with wild-type, XccbphP, pXccBphP and pC13S strains, stained for callose deposits and observed by fluorescence microscopy. MgCl2 buffer (untreated) and flg22 peptide were used as negative and positive controls, respectively. Top panel: representative pictures of three independent experiments. Scale bar represents 200 µm. Bottom panel: the number of callose deposits per field of view (0.45 mm2) were determined (n = 8 replicates). Data are representative of two independent experiments. (A-E) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a two-tailed Mann-Whitney test (*P < 0.05, **P < 0.01, ***P < 0.001)


Figure 3 ( A ) Five µL supernatants from wild - type , XccbphP , pXccBphP and pC13 strains bacterial cultures ( OD600 = 1 ) grown under light or dark conditions were plated onto PYM - carboxymethyl cellulose ( CMC ) agar plates and revealed with Congo redstaining ( n = 2 replicates ) . The extracellular β - 1 , 4 - endoglucanase production levels correlated with CMC degradation halo radiuses ( top panel ) . Halo measurements are presented in the bottom panel . Data derive from 10 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( B ) The extracellular β - 1 , 4 - endoglucanase activity from bacterial cultures supernatants ( OD600 = 1 ) was determined by a colorimetric assay in solution ( n = 2 replicates ) . Wild - type , XccbphP , pXccBphP and pC13 strains were assayed under normal laboratory conditions . Data derive from 3 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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 B-PANEL_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

Figure 3(A) Five µL supernatants from wild-type, XccbphP, pXccBphP and pC13 strains bacterial cultures (OD600 = 1) grown under light or dark conditions were plated onto PYM-carboxymethyl cellulose (CMC) agar plates and revealed with Congo redstaining (n = 2 replicates). The extracellular β-1,4-endoglucanase production levels correlated with CMC degradation halo radiuses (top panel). Halo measurements are presented in the bottom panel. Data derive from 10 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).(B) The extracellular β-1,4-endoglucanase activity from bacterial cultures supernatants (OD600 = 1) was determined by a colorimetric assay in solution (n = 2 replicates). Wild-type, XccbphP, pXccBphP and pC13 strains were assayed under normal laboratory conditions. Data derive from 3 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).