figf1 ( a ) vBcl - 2 binding proteins . After transfection , 293T cells were used for GST - pulldown and the purified proteins were detected by silver staining . ( c ) UVRAG binds Beclin1 through its CCD . After transfection with Beclin1 - V5 , together with full - length or fragments of Flag - UVRAG , 293T whole cell lysates ( WCLs ) were used for immunoprecipitation ( IP ) with anti - Flag followed by immunoblotting ( IB ) with anti - V5 . ( d ) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI ( 3 ) KC3 . 293T cell lysates were used for immunoprecipitation with preimmune sera or anti - Beclin1 , followed by immunoblotting with anti - UVRAG or anti - Beclin1 , or for immunoprecipitation with preimmune sera or anti - UVRAG , followed by immunoblotting with anti - PI ( 3 ) KC3 . ( e ) UVRAGCCD is required for Beclin1 interaction . After transfection with Beclin1 - V5 ( lanes 2 and 4 ) together with Flag - UVRAGΔCCD ( lanes 1 and 2 ) or Flag - UVRAGΔC2 ( lanes 3 and 4 ) , WCLs were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - V5 . ( f ) Beclin1 binds UVRAG through its CCD . After transfection with Flag - UVRAG together with full - length or fragments of Beclin1 - V5 , WCLs were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - Flag . ( g ) An increase in UVRAG stability induced by Beclin1 . After transfection with Flag - UVRAG and empty vector or Flag - UVRAG and Beclin1 - V5 , WCLs were treated with cycloheximide ( CHX , 20 μg ml − 1 ) for the indicated time , followed by immunoblotting with anti - Flag . ( h ) UVRAG directly interacts with Beclin1 . MBP - UVRAGCCD ( lane 1 ) , His6 - Beclin1CCD ( lane 2 ) or mixtures of both proteins in a concentration dependent manner ( lanes 3 - 8 ) , were subjected to native gel electrophoresis . The numbers indicate the concentration ( nmol ) of protein . Arrows indicate the purified MBP - UVRAGCCD , His6 - Beclin1CCD and complex between these two . ( i ) Beclin1CCD and UVRAGCCD associate into a α - helical heterodimer . After removing the N - terminal His6 and MBP tags , the Beclin1CCD and the preformed Beclin1CCD - UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis . The raw data for this experiment are presented in the Supplementary Information , Fig . S5 .

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figf1(a) vBcl-2 binding proteins. After transfection, 293T cells were used for GST-pulldown and the purified proteins were detected by silver staining.(c) UVRAG binds Beclin1 through its CCD. After transfection with Beclin1-V5, together with full-length or fragments of Flag-UVRAG, 293T whole cell lysates (WCLs) were used for immunoprecipitation (IP) with anti-Flag followed by immunoblotting (IB) with anti-V5.(d) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI(3)KC3. 293T cell lysates were used for immunoprecipitation with preimmune sera or anti-Beclin1, followed by immunoblotting with anti-UVRAG or anti-Beclin1, or for immunoprecipitation with preimmune sera or anti-UVRAG, followed by immunoblotting with anti-PI(3)KC3.(e) UVRAGCCD is required for Beclin1 interaction. After transfection with Beclin1-V5 (lanes 2 and 4) together with Flag-UVRAGΔCCD (lanes 1 and 2) or Flag-UVRAGΔC2 (lanes 3 and 4), WCLs were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5.(f) Beclin1 binds UVRAG through its CCD. After transfection with Flag-UVRAG together with full-length or fragments of Beclin1-V5, WCLs were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag.(g) An increase in UVRAG stability induced by Beclin1. After transfection with Flag-UVRAG and empty vector or Flag-UVRAG and Beclin1-V5, WCLs were treated with cycloheximide (CHX, 20 μg ml−1) for the indicated time, followed by immunoblotting with anti-Flag.(h) UVRAG directly interacts with Beclin1. MBP-UVRAGCCD (lane 1), His6-Beclin1CCD (lane 2) or mixtures of both proteins in a concentration dependent manner (lanes 3-8), were subjected to native gel electrophoresis. The numbers indicate the concentration (nmol) of protein. Arrows indicate the purified MBP-UVRAGCCD, His6-Beclin1CCD and complex between these two.(i) Beclin1CCD and UVRAGCCD associate into a α-helical heterodimer. After removing the N-terminal His6 and MBP tags, the Beclin1CCD and the preformed Beclin1CCD-UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.


figf2 ( a ) The Bcl - 2 binding domain of Beclin1 is sufficient to bind vBcl - 2 . At 48 h posttransfection with wild - type and mutant Beclin1 - V5 , together with HA - vBcl - 2 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - HA . ( b ) The loss of Beclin1 interaction of vBcl - 2AAA mutant . At 48 h posttransfection with wild - type HA - vBcl - 2 or vBcl - 2AAA mutant , together with Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 , followed by immunoblotting with anti - HA . ( c ) vBcl - 2 interaction with wild - type UVRAG in the presence of Beclin1 . At 48 h posttransfection with Flag - UVRAG together with Beclin1 - V5 and HA - vBcl - 2 or vBcl - 2AAA mutant , WCLs of 293T cells were used for immunoprecipitation with anti - HA followed by immunoblotting with anti - Flag . ( d ) vBcl - 2 interaction with PI ( 3 ) KC3 in the presence of Beclin1 . At 48 h posttransfection with HA - vBcl - 2 together with Flag - PI ( 3 ) KC3 and Beclin1 - V5 , WCLs of 293T cells were used for immunprecipitation with anti - HA followed by immunoblotting with anti - Flag . ( e ) PI ( 3 ) KC3 N - terminal C2 domain binds Beclin1 . At 48 h posttransfection with Beclin1 - V5 together with full - length or fragments of Flag - PI ( 3 ) KC3 , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - V5 . CAT , catalytic domain . ( f ) Beclin1CCD and Beclin1ECD are sufficient for binding PI ( 3 ) KC3 . At 48 h posttransfection with Flag - PI ( 3 ) KC3 together with full - length or fragments of Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - Flag . CT , C terminus ( aa 337 - 448 ) . ( g ) PI ( 3 ) KC3 interaction with UVRAG in the presence of Beclin1 . At 48 h posttransfection with Flag - UVRAGΔCCD or UVRAGCCD together with HA - PI ( 3 ) KC3 and Beclin1 - V5 , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - HA .

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

figf2(a) The Bcl-2 binding domain of Beclin1 is sufficient to bind vBcl-2. At 48 h posttransfection with wild-type and mutant Beclin1-V5, together with HA-vBcl-2, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-HA.(b) The loss of Beclin1 interaction of vBcl-2AAA mutant. At 48 h posttransfection with wild-type HA-vBcl-2 or vBcl-2AAA mutant, together with Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5, followed by immunoblotting with anti-HA.(c) vBcl-2 interaction with wild-type UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag-UVRAG together with Beclin1-V5 and HA-vBcl-2 or vBcl-2AAA mutant, WCLs of 293T cells were used for immunoprecipitation with anti-HA followed by immunoblotting with anti-Flag.(d) vBcl-2 interaction with PI(3)KC3 in the presence of Beclin1. At 48 h posttransfection with HA-vBcl-2 together with Flag-PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunprecipitation with anti-HA followed by immunoblotting with anti-Flag.(e) PI(3)KC3 N-terminal C2 domain binds Beclin1. At 48 h posttransfection with Beclin1-V5 together with full-length or fragments of Flag-PI(3)KC3, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5. CAT, catalytic domain.(f) Beclin1CCD and Beclin1ECD are sufficient for binding PI(3)KC3. At 48 h posttransfection with Flag-PI(3)KC3 together with full-length or fragments of Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag. CT, C terminus (aa 337-448).(g) PI(3)KC3 interaction with UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag-UVRAGΔCCD or UVRAGCCD together with HA- PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-HA.


figf3 ( a ) UVRAG expression and LC3 process in HCT116 cells . The same protein amounts of 293T , NIH3T3 , HCT116 and MCF7 cells were used for immunoblotting with anti - UVRAG and anti - tubulin . WCLs of 293T cells transfected with Flag - tagged UVRAG were included as controls . WCLs of HCT116 . vector and HCT116 . UVRAG were incubated with or without starvation conditions and used for immunoblotting with anti - LC3 and anti - tubulin . The raw data for this experiment is presented in the Supplementary information , Fig . S5 . ( b ) Electron microscopy of autophagy in HCT116 cells . HCT116 . vector and HCT116 . UVRAG cells were incubated under normal or starvation conditions for 4 h and subjected to scanning electron microscopy . Scale bars ( from left to right panels ) represent 2 nm , 0 . 5 nm , 0 . 2 nm and 0 . 2 nm . The arrows indicate autophagosome or autolysosome . ( c ) UVRAG induces autophagosome formation in HCT116 cells . After transfection with GFP - LC3 vector , HCT116 . vector and HCT116 . UVRAG cells were maintained under normal conditions or starved for 3 h . GFP - LC3 was detected using an inverted fluorescence microscope . Arrows indicate autophagosomes . The scale bars represent 10 μm . ( d ) After transfection with GFP - LC3 vector , HCT116 . vector and HCT116 . UVRAG cells were maintained under normal conditions , starved for 3 h in the absence or presence of 10 mM 3 - methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3 - methyladenine . Autophagome was quantified as the mean ± s . d . of combined results from three independent experiments .

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figf3(a) UVRAG expression and LC3 process in HCT116 cells. The same protein amounts of 293T, NIH3T3, HCT116 and MCF7 cells were used for immunoblotting with anti-UVRAG and anti-tubulin. WCLs of 293T cells transfected with Flag-tagged UVRAG were included as controls. WCLs of HCT116.vector and HCT116.UVRAG were incubated with or without starvation conditions and used for immunoblotting with anti-LC3 and anti-tubulin. The raw data for this experiment is presented in the Supplementary information, Fig. S5.(b) Electron microscopy of autophagy in HCT116 cells. HCT116.vector and HCT116.UVRAG cells were incubated under normal or starvation conditions for 4 h and subjected to scanning electron microscopy. Scale bars (from left to right panels) represent 2 nm, 0.5 nm, 0.2 nm and 0.2 nm. The arrows indicate autophagosome or autolysosome.(c) UVRAG induces autophagosome formation in HCT116 cells. After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions or starved for 3 h. GFP-LC3 was detected using an inverted fluorescence microscope. Arrows indicate autophagosomes. The scale bars represent 10 μm.(d) After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Autophagome was quantified as the mean ± s.d. of combined results from three independent experiments.


figf4 ( a ) HCT116 . vector and HCT116 . UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis . HCT116 . vector , HCT116 . UVRAG , and HCT116 . UVRAG ΔCCD cells were treated with or without wortmannin ( 1 μM ) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity . Data represents mean ± s . d . of three experiments . ( b ) UVRAG induces autophagosome formation in NIH3T3 cells . After transfection with GFP - LC3 vector , NIH3T3 . vector and NIH3T3 . UVRAG cells were maintained under normal conditions , starved for 3 h in the absence or presence of 10 mM 3 - methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3 - methyladenine . Data represents mean ± s . d . of three experiments .

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figf4(a) HCT116.vector and HCT116.UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis. HCT116.vector, HCT116.UVRAG, and HCT116.UVRAG ΔCCD cells were treated with or without wortmannin (1 μM) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity. Data represents mean ± s.d. of three experiments.(b) UVRAG induces autophagosome formation in NIH3T3 cells. After transfection with GFP-LC3 vector, NIH3T3.vector and NIH3T3.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Data represents mean ± s.d. of three experiments.


figf5 ( a ) Autophagic activity of UVRAG mutants . HCT116 cells expressing wild - type or mutant UVRAG were transfected with GFP - LC3 expression vector and incubated under normal or starvation conditions for 3 h . Autophagy was quantified as described in Fig . 3 . ( b ) Effect of wild - type and mutant UVRAG on Beclin1 - mediated autophagy . MCF7 . vector , MCF7 . Beclin1 and MCF7 . Beclin1ΔECD cells were cotransfected with wild - type UVRAG or its mutant expression vector together with GFP - LC3 expression vector . At 16 - 18 h posttransfection , cells were incubated under normal or starvation conditions for 4 h . Autophagy was quantified as described in Fig . 3 . The scale bars represent 20 μm . ( c , d ) Interdependence between UVRAG and Beclin1 for autophagosome induction . HCT116 . vector and HCT116 . UVRAG cells were transfected with control siRNA , Beclin1 siRNA or UVRAG siRNA together with GFP - LC3 expression vector as indicated . Autophagy was quantified as described in Fig . 3 . WCLs of these cells were used for immunoblotting with anti - UVRAG , anti - Beclin1 or anti - tubulin . The scale bars represent 20 μm . In all figures , data represents mean ± s . d . of three experiments . The raw data for c and d are presented in the Supplementary Information , Fig S5 . ( c , d ) Interdependence between UVRAG and Beclin1 for autophagosome induction . MCF7 . vector , MCF7 . Beclin1 and MCF7 . Beclin1ΔECD cells ( d ) were transfected with control siRNA , Beclin1 siRNA or UVRAG siRNA together with GFP - LC3 expression vector as indicated . Autophagy was quantified as described in Fig . 3 . WCLs of these cells were used for immunoblotting with anti - UVRAG , anti - Beclin1 or anti - tubulin . The scale bars represent 20 μm . In all figures , data represents mean ± s . d . of three experiments . The raw data for c and d are presented in the Supplementary Information , Fig S5 .

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

figf5(a) Autophagic activity of UVRAG mutants. HCT116 cells expressing wild-type or mutant UVRAG were transfected with GFP-LC3 expression vector and incubated under normal or starvation conditions for 3 h. Autophagy was quantified as described in Fig. 3.(b) Effect of wild-type and mutant UVRAG on Beclin1-mediated autophagy. MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells were cotransfected with wild-type UVRAG or its mutant expression vector together with GFP-LC3 expression vector. At 16-18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as described in Fig. 3. The scale bars represent 20 μm.(c, d) Interdependence between UVRAG and Beclin1 for autophagosome induction. HCT116.vector and HCT116.UVRAG cells were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP-LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.(c, d) Interdependence between UVRAG and Beclin1 for autophagosome induction.MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells (d) were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP-LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.


figf6 ( a ) Increased Beclin1 - PI ( 3 ) KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions . HCT116 . vector , HCT116 . UVRAG and HCT116 . UVRAGΔCCD cells were used for immunoprecipitation with anti - PI ( 3 ) KC3 followed by immunoblotting with anti - Beclin1 . WCLs were used for immunoblotting with anti - Beclin1 and anti - PI ( 3 ) KC3 . HCT116 . UVRAG cells expressing Flag - tagged UVRAG were incubated under nutrient - rich conditions ( lanes 1 and 2 ) , starvation for 4 h ( lane 3 ) , or 2 μM rapamycin treatment for 2 h ( lane 4 ) . WCLs were then used for immunoprecipitation with anti - Beclin1 ( lanes 2 , 3 and 4 ) or preimmune antibody ( lane 1 ) followed by immunoblotting with anti - Flag . The raw data for this experiment are presented in the Supplementary Information , Fig . S5 . ( b ) Increased PI ( 3 ) KC3 activity by UVRAG . At 48 h posttransfection with Flag - tagged PI ( 3 ) KC3 with Beclin1 ( lane 1 ) , with Beclin1 + UVRAG ( lane 2 ) or with Beclin1 + UVRAGΔCCD vector ( lane 3 ) , WCLs of 293T cells were used for immunoprecipitation with anti - Flag followed by an assay for PI ( 3 ) KC3 lipid kinase activity , as described in the Methods . At 16 h posttransfection with p40 ( phox ) PX - EGFP fusion , HCT116 . vector and HCT116 . UVRAG cells were detected using an inverted fluorescence microscope . p40 ( phox ) PX - EGFP - positive vesicles were quantified as the mean ± s . d . of combined results from three independent experiments . The scale bars represent 10 μm . ( c ) Suppression of autophagy by γHV68 vBcl - 2 . At 18 h posttransfection with GFP - LC3 vector , NIH3T3 . vector , NIH3T3 . vBcl - 2 and NIH3T3 . vBcl - 2AAA , cells were incubated under normal or starvation conditions for 4 h . Subsequently , GFP - LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s . d . of combined results from three independent experiments .

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

figf6(a) Increased Beclin1-PI(3)KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells were used for immunoprecipitation with anti-PI(3)KC3 followed by immunoblotting with anti-Beclin1. WCLs were used for immunoblotting with anti-Beclin1 and anti-PI(3)KC3. HCT116.UVRAG cells expressing Flag-tagged UVRAG were incubated under nutrient-rich conditions (lanes 1 and 2), starvation for 4 h (lane 3), or 2 μM rapamycin treatment for 2 h (lane 4). WCLs were then used for immunoprecipitation with anti-Beclin1 (lanes 2, 3 and 4) or preimmune antibody (lane 1) followed by immunoblotting with anti-Flag. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.(b) Increased PI(3)KC3 activity by UVRAG. At 48 h posttransfection with Flag-tagged PI(3)KC3 with Beclin1 (lane 1), with Beclin1 + UVRAG (lane 2) or with Beclin1 + UVRAGΔCCD vector (lane 3), WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by an assay for PI(3)KC3 lipid kinase activity, as described in the Methods. At 16 h posttransfection with p40(phox) PX-EGFP fusion, HCT116.vector and HCT116.UVRAG cells were detected using an inverted fluorescence microscope. p40(phox) PX-EGFP-positive vesicles were quantified as the mean ± s.d. of combined results from three independent experiments. The scale bars represent 10 μm.(c) Suppression of autophagy by γHV68 vBcl-2. At 18 h posttransfection with GFP-LC3 vector, NIH3T3.vector, NIH3T3.vBcl-2 and NIH3T3.vBcl-2AAA, cells were incubated under normal or starvation conditions for 4 h. Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s.d. of combined results from three independent experiments.


figf7 ( a ) Proliferation rates . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAGΔC2 , HCT116 . UVRAGΔCCD and HCT116 . UVRAGCCD cells ( 1 × 105 per well ) were seeded into 6 - well microtitre plates . Cell numbers were counted on the days indicated . The results represent means of three independent assays . ( b ) Anchorage independence . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAGΔC2 , HCT116 . UVRAGΔCCD and HCT116 . UVRAGCCD cells ( 1 × 104 ) were incubated in soft agar as described in the Methods . After ten days , colonies were photographed and counted . The scale bars represent 50 μm . ( c ) Tumorigenicity . HCT116 . vector , HCT116 . UVRAG and HCT116 . UVRAGΔCCD cells ( 1 × 107 ) were subcutaneously injected into 5 nude mice , separately . At 16 days postinoculation , mice and tumours were photographed and tumour weights were measured . Arrows indicate location of tumours .

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figf7(a) Proliferation rates. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 105 per well) were seeded into 6-well microtitre plates. Cell numbers were counted on the days indicated. The results represent means of three independent assays.(b) Anchorage independence. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 104) were incubated in soft agar as described in the Methods. After ten days, colonies were photographed and counted. The scale bars represent 50 μm.(c) Tumorigenicity. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells (1 × 107) were subcutaneously injected into 5 nude mice, separately. At 16 days postinoculation, mice and tumours were photographed and tumour weights were measured. Arrows indicate location of tumours.


Figure 1C . qRT - PCR data showing induction of each gene in the iniBAC operon in WT Mtb cultured in low iron and in the virR mutant strain cultured in high iron , expressed as fold change relative to WT Mtb cultured in iron sufficient conditions . RNA extracted from biological triplicates was analyzed . The data are presented as means + / - SEM . * * P ≤ 0 . 01 * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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Figure 1C. qRT-PCR data showing induction of each gene in the iniBAC operon in WT Mtb cultured in low iron and in the virR mutant strain cultured in high iron, expressed as fold change relative to WT Mtb cultured in iron sufficient conditions. RNA extracted from biological triplicates was analyzed. The data are presented as means +/- SEM. **P≤ 0.01 ***P ≤ 0.001 (Student's t-test).


Figure 2A . MEV quantitation based on protein and lipid content in vesicle preparations isolated from the same number of Mtb cells treated with INH 50 ng . mlL - 1 for 24 and 48 h relative to untreated cells . Three independent cultures were analyzed . Data are presented as mean ± SEM * * * P ≤ 0 . 001 ( Student ' s t - test ) . B . Immunodot blot assay detecting MEVs obtained from three independent cultures ( 1 , 2 , 3 ) and derived from the same number of Mtb exposed to 50 ng . mlL - 1 INH for 24 and 48 h . A polyclonal antiserum raised against purified MEV ( Prados - Rosales et al . , 2014a ) was used as a primary antibody . E . Protein and lipid content in vesicle preparations isolated from the same number of WT and Msm overexpressing iniACMtb ( Msm iniACMtb ) . Three independent cultures were analyzed . Data are presented as mean ± SEM * P ≤ 0 . 05 ; * * * P ≤ 0 . 001 ( Student ' s t - test ) . H . The graph shows the avarage of nanoparticle size distribution in three independent vesicle preparations derived from WT ( black ) and Msm iniACMtb ( grey ) .

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Figure 2A. MEV quantitation based on protein and lipid content in vesicle preparations isolated from the same number of Mtb cells treated with INH 50 ng.mlL-1 for 24 and 48 h relative to untreated cells. Three independent cultures were analyzed. Data are presented as mean ± SEM ***P ≤ 0.001 (Student's t-test).B. Immunodot blot assay detecting MEVs obtained from three independent cultures (1, 2, 3) and derived from the same number of Mtb exposed to 50 ng.mlL-1 INH for 24 and 48 h. A polyclonal antiserum raised against purified MEV (Prados-Rosales et al., 2014a) was used as a primary antibody.E. Protein and lipid content in vesicle preparations isolated from the same number of WT and Msm overexpressing iniACMtb (Msm iniACMtb). Three independent cultures were analyzed. Data are presented as mean ± SEM *P≤ 0.05; *** P≤ 0.001 (Student's t-test).H. The graph shows the avarage of nanoparticle size distribution in three independent vesicle preparations derived from WT (black) and Msm iniACMtb (grey).


Figure 3A . Quantitation of MEVs released by Mtb strains in low iron cultures based on vesicle protein and lipid content normalized to CFUs . Three biological replicates were processed . Data are presented as mean ± SD * P ≤ 0 . 05 , * * P ≤ 0 . 005 ( Student ' s t - test ) . C . Protein in vesicle isolates derived from three independent cultures of Mtb WT and iniA : : hyg mutant grown in low ( LI ) or high iron ( HI ) MM with or without 50 ng · mL - 1 INH . Data are presented as mean ± SEM * P ≤ 0 . 05 . * * * P ≤ 0 . 001 ( Student ' s t - test ) D . Lipid in vesicle isolates derived from three independent cultures of Mtb WT and iniA : : hyg mutant cultured in low ( LI ) or high iron ( HI ) MM with or without 50 ng · mL - 1 INH . Data are presented as mean ± SD * P ≤ 0 . 05 ( Student ' s t - test )

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Figure 3A. Quantitation of MEVs released by Mtb strains in low iron cultures based on vesicle protein and lipid content normalized to CFUs. Three biological replicates were processed. Data are presented as mean ± SD *P ≤0.05, **P≤ 0.005 (Student's t-test).C. Protein in vesicle isolates derived from three independent cultures of Mtb WT and iniA::hyg mutant grown in low (LI) or high iron (HI) MM with or without 50 ng·mL-1 INH. Data are presented as mean ± SEM *P ≤ 0.05. ***P≤ 0.001 (Student's t-test)D. Lipid in vesicle isolates derived from three independent cultures of Mtb WT and iniA::hyg mutant cultured in low (LI) or high iron (HI) MM with or without 50 ng·mL-1 INH. Data are presented as mean ± SD *P ≤ 0.05 (Student's t-test)


Figure 4B . Mycobactin synthesized by Mtb WT and iniA : : hyg grown in low iron conditions . Data are presented as the mean of mycobactin extracted from two independent cultures . C . The Mtb siderophore synthesis mutant ( ST142 ) was cultured in low iron MM ( medium ) plain or supplemented with MEV preparations derived from equal number of Mtb WT , iniA : : hyg and complemented strains grown in low iron MM . ST142 growth was determined based on the number of viable cells detected by the resazurine cell viability assay . MEV obtained from three independent cultures were tested .

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Figure 4B. Mycobactin synthesized by Mtb WT and iniA::hyg grown in low iron conditions. Data are presented as the mean of mycobactin extracted from two independent cultures.C. The Mtb siderophore synthesis mutant (ST142) was cultured in low iron MM (medium) plain or supplemented with MEV preparations derived from equal number of Mtb WT, iniA::hyg and complemented strains grown in low iron MM. ST142 growth was determined based on the number of viable cells detected by the resazurine cell viability assay. MEV obtained from three independent cultures were tested.


Figure 5A . Representative Immunodot - blot of bacterial antigens associated with EVs isolated from the culture supernatants of THP - 1 cells infected with Mtb WT , iniA : hyg and complemented strains versus uninfected cells ( UI ) . Anti - MEV specific antiserum was used for detection . The decreasing black bar indicates that increasing dilutions were spotted onto the membrane . The experiment was repeated three times with independent cultures . B . Number of nanoparticles per mL determined by Zeta View NTA in three EV preparations derived from the culture supernatans of THP - 1 cells infected with Mtb WT , iniA : hyg and iniA : : hyg complemented strains . C . Representative Western blot of total EVs isolated from the CSF of THP - 1 cells infected with Mtb WT , iniA : : hyg and complemented strains versus uninfected cells ( UI ) detecting the exosome marker CD9 . The experiment was repeated twice with similar results .

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Figure 5A. Representative Immunodot-blot of bacterial antigens associated with EVs isolated from the culture supernatants of THP-1 cells infected with Mtb WT, iniA:hyg and complemented strains versus uninfected cells (UI). Anti-MEV specific antiserum was used for detection. The decreasing black bar indicates that increasing dilutions were spotted onto the membrane. The experiment was repeated three times with independent cultures.B. Number of nanoparticles per mL determined by Zeta View NTA in three EV preparations derived from the culture supernatans of THP-1 cells infected with Mtb WT, iniA:hyg and iniA::hyg complemented strains.C. Representative Western blot of total EVs isolated from the CSF of THP-1 cells infected with Mtb WT, iniA::hyg and complemented strains versus uninfected cells (UI) detecting the exosome marker CD9. The experiment was repeated twice with similar results.


Figure 1B . Changes in the expression of cytokines in THP1 macrophages ( left ) and PBMCs ( right ) upon treatment with full - length S1 subunit at 1 µg / ml for 24 hr . Data information , E . Effect of an anti - RBD antibody on S1 subunit stimulated changes in the expression of cytokines in PBMCs . Left , a schematic showing the domain structure of S1 spike protein and anti - RBD antibody . Right , changes in cytokine gene expression in response to S1 spike protein in the presence or abscene of anti - RBD antibody . Gene expression was measured by qPCR . Cytokine release in the conditioned media was measured by Luminex . Full - length S1 and S1 subunits are purified from HEK293 cells . Data information , data are shown as the mean of three technical replicates ; in ( D ) , data are shown as the mean of 20 individual donors . Error bars denote SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Multiple t - test .

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Figure 1B. Changes in the expression of cytokines in THP1 macrophages (left) and PBMCs (right) upon treatment with full-length S1 subunit at 1 µg/ml for 24 hr. Data information,E. Effect of an anti-RBD antibody on S1 subunit stimulated changes in the expression of cytokines in PBMCs. Left, a schematic showing the domain structure of S1 spike protein and anti-RBD antibody. Right, changes in cytokine gene expression in response to S1 spike protein in the presence or abscene of anti-RBD antibody. Gene expression was measured by qPCR. Cytokine release in the conditioned media was measured by Luminex. Full-length S1 and S1 subunits are purified from HEK293 cells. Data information, data are shown as the mean of three technical replicates; in (D), data are shown as the mean of 20 individual donors. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Multiple t-test.


Figure 2C . Validation of predicted kinases as drivers of cytokine release by siRNAs in THP1 cells . Cytokine release was measured by Luminex and normalized to scrambled siRNA .

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Figure 2C. Validation of predicted kinases as drivers of cytokine release by siRNAs in THP1 cells. Cytokine release was measured by Luminex and normalized to scrambled siRNA.


Figure 3C . Comparison of kinase inhibition profile of Ponatinib and Baricitinib .

Figure 3C. Comparison of kinase inhibition profile of Ponatinib and Baricitinib.


Figure 4B . Dose - response curves of ponatinib treatment on SARS - Cov2 NTD ( Wuhan ) - mediated cytokine release in PBMCs . Right , a table showing EC50 ( nM ) values of ponatinib - mediated inhibition of cytokine release in response to indicated SARS - Cov2 variant NTD treatments in PBMCs .

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Figure 4B. Dose-response curves of ponatinib treatment on SARS-Cov2 NTD (Wuhan)-mediated cytokine release in PBMCs. Right, a table showing EC50 (nM) values of ponatinib-mediated inhibition of cytokine release in response to indicated SARS-Cov2 variant NTD treatments in PBMCs.


Figure 5A . Comparison of LPS and full length S1 spike protein - mediated changes in cytokines in THP1 macrophages . Data information , , data are shown as the mean of three technical replicates . Error bars denote SEM . D . Representative H & E images showing Ponatinib alleviates LPS - induced inflammatory cell infiltration , septal thickening , alveolar edema in mouse lungs . Scale bars 100 µm ( upper ) and 50 µm ( lower ) . E . Plots showing pre - treatment ( 60 min ) with Ponatinib ( 35 mg / Kg ) inhibits LPS - induced cytokine release in BALF . Cytokine levels were measured using Luminex . Data information , data are mean of five biological replicates from two independent studies , error bars denote SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Welch ' s t - test .

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Figure 5A. Comparison of LPS and full length S1 spike protein-mediated changes in cytokines in THP1 macrophages. Data information, , data are shown as the mean of three technical replicates. Error bars denote SEM.D. Representative H&E images showing Ponatinib alleviates LPS-induced inflammatory cell infiltration, septal thickening, alveolar edema in mouse lungs. Scale bars 100 µm (upper) and 50 µm (lower).E. Plots showing pre-treatment (60 min) with Ponatinib (35 mg/Kg) inhibits LPS-induced cytokine release in BALF. Cytokine levels were measured using Luminex. Data information, data are mean of five biological replicates from two independent studies, error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Welch's t-test.


Figure 1 ( A ) 293T cells were transfected with Flag - ULK1 or control empty vector and subjected to immunoprecipitation with anti - Flag antibody , followed by immunoblot analysis with the indicated antibodies . ( B ) 293T cells were co - transfected with Flag - AMPKα2 and HA - ULK1 . Cell lysates were subjected to immunoprecipitation using anti - Flag antibody followed by SDS - PAGE / immunoblot analysis with anti - HA and anti - Flag antibodies . ( C ) 293T cells were transiently transfected with Flag - AMPKα2 or Flag - AMPKβ1 . After 24 h , immunoprecipitation was performed using anti - Flag or control anti - HA antibodies and analyzed by immunoblotting with anti - ULK1 and anti - Flag polyclonal antibodies . ( D ) 293T cell lysates were subjected to immunoprecipitation with anti - ULK1 polyclonal antibody or control preimmune rabbit serum ( NRS ) , followed by immunoblot analysis with anti - ULK1 and anti - AMPKα antibodies . ( E ) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti - AMPKα antibody followed by immunoblotting with anti - ULK1 and anti - AMPKα antibodies . ( F ) Purified His6 - AMPKα1 / β1 / γ1 fusion proteins ( 250 ng ) were mixed with purified His6 - tagged ULK1 ( 1 µg ) or Bcl - XL ( 200 ng ) proteins in 1 % Triton X - 100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti - ULK1 antibody . The resulting protein complexes and 10 % of the input proteins were analyzed by immunoblotting with anti - His - Tag polyclonal antibody .

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Figure 1(A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies.(B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies.(C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies.(D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies.(E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies.(F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.


Figure 2 ( B , C ) 293T cells were transfected with empty vector or HA - tagged wild type ( WT ) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti - HA antibody . The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR , raptor , AMPKα or HA - tag .

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Figure 2(B, C) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti-HA antibody. The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR, raptor, AMPKα or HA-tag.


Figure 3 ( A ) 293T cells were transfected with empty vector or HA - tagged wild type ( WT ) or deletion mutant ( Δ654 - 828 ) ULK1 and subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with the indicated antibodies . ( B ) U - 2OS cells stably expressing GFP - LC3 were infected with lentivirus expressing human Ulk1 shRNA ( shhUlk1 ) or scrambled shRNA ( shScr ) and subjected to selection with 1 µg / ml puromycin for 5 days . The cells were then transfected with wild type mouse Ulk1 ( mUlk1 ) , mUlk1 Δ654 - 828 or control empty vector for 24 h , incubated in serum - free DMEM overnight , and cultured in complete medium with or without 10 mM metformin for 20 h . The images were obtained using a fluorescence microscope . The scale bars represent 10 µm . ( C ) The number of GFP - LC3 dots per GFP - positive cell in ( B ) was quantified ( mean ± s . d . ; n = 81 ) . ( D ) U - 2OS cells stably expressing GFP - LC3 were transfected with 0 , 2 or 4 µg of pcDNA3 encoding HA - tagged ULK1 deletion mutant ( 554 - 828 ) . The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector . After 24 h transfection , the cells were starved in nutrient - free medium for 1 . 5 h , fixed in 4 % paraformaldehyde , and analyzed by fluorescence microscopy . The number of GFP - LC3 dots per GFP - positive cell was quantified ( mean ± s . d . ; n = 100 ) . Statistical significance was determined by Student ' s t - test and the asterisks indicate P < 0 . 0001 .

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Figure 3(A) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutant (Δ654-828) ULK1 and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies.(B) U-2OS cells stably expressing GFP-LC3 were infected with lentivirus expressing human Ulk1 shRNA (shhUlk1) or scrambled shRNA (shScr) and subjected to selection with 1 µg/ml puromycin for 5 days. The cells were then transfected with wild type mouse Ulk1 (mUlk1), mUlk1 Δ654-828 or control empty vector for 24 h, incubated in serum-free DMEM overnight, and cultured in complete medium with or without 10 mM metformin for 20 h. The images were obtained using a fluorescence microscope. The scale bars represent 10 µm.(C) The number of GFP-LC3 dots per GFP-positive cell in (B) was quantified (mean ± s.d.; n = 81).(D) U-2OS cells stably expressing GFP-LC3 were transfected with 0, 2 or 4 µg of pcDNA3 encoding HA-tagged ULK1 deletion mutant (554-828). The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector. After 24 h transfection, the cells were starved in nutrient-free medium for 1.5 h, fixed in 4% paraformaldehyde, and analyzed by fluorescence microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 100). Statistical significance was determined by Student's t-test and the asterisks indicate P<0.0001.


Figure 4 293 cells stably expressing HA - ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti - HA or control anti - FLAG antibodies . The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies .

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Figure 4  293 cells stably expressing HA-ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti-HA or control anti-FLAG antibodies. The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies.


Figure 5 ( A ) TSC2 − / − , p53 − / − MEFs stably reconstituted with wild - type raptor or S722A / S792A mutant raptor were transfected with GFP - LC3 , treated with 2 mM AICAR or control vehicle for 4 h , and then analyzed by fluorescent microscopy . The number of GFP - LC3 dots per GFP - positive cell was counted ( mean ± s . d . ; n = 60 ) . The scale bars represent 10 µm . ( B ) TSC2 − / − , p53 − / − MEFs stably reconstituted with wild - type raptor or S722A / S792A mutant raptor were treated with 2 mM AICAR for the indicated times . Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies . The levels of p62 are listed relative to those of untreated WT raptor cells , which were set as 1 .

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Figure 5(A) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were transfected with GFP-LC3, treated with 2 mM AICAR or control vehicle for 4 h, and then analyzed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 60). The scale bars represent 10 µm.(B) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were treated with 2 mM AICAR for the indicated times. Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies. The levels of p62 are listed relative to those of untreated WT raptor cells, which were set as 1.


Figure 1 ( C ) Clonal cell lines expressing p53 - Venus were engineered to monitor p53 expression dynamics ( " input " ) and two canonically regulated p53 target promoters expressing mCherry ( " output " ) in response to Nutlin - 3 treatment . ( D ) Representative phase contrast and yellow fluorescent ( indicating p53 - Venus levels ) images at the indicated time points for a cell exposed to the " natural dynamics " Nutlin - 3 dosing regimen . ( E - F ) p53 - Venus expression in response to the " natural dynamics " Nutlin - 3 dosing regimen . Single cell traces ( gray ; E ) and the mean ( red ; E ) are shown for p53 - Venus expression in response to each Nutlin - 3 regimen . Heat map ( F ) shows an alternative representation of all traces as shown in ( E ) . N = 51 cells .

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Figure 1(C) Clonal cell lines expressing p53-Venus were engineered to monitor p53 expression dynamics ("input") and two canonically regulated p53 target promoters expressing mCherry ("output") in response to Nutlin-3 treatment.(D) Representative phase contrast and yellow fluorescent (indicating p53-Venus levels) images at the indicated time points for a cell exposed to the "natural dynamics" Nutlin-3 dosing regimen.(E-F) p53-Venus expression in response to the "natural dynamics" Nutlin-3 dosing regimen. Single cell traces (gray; E) and the mean (red; E) are shown for p53-Venus expression in response to each Nutlin-3 regimen. Heat map (F) shows an alternative representation of all traces as shown in (E). N = 51 cells.


Figure 2 ( A ) Representative phase contrast , yellow fluorescent ( indicating p53 - Venus levels ) , and red fluorescent ( indicating reporter mCherry levels ) images at the indicated time points for MDM2 promoter reporter cells exposed to the " natural dynamics " Nutlin - 3 dosing regimen . ( B - G ) Single cell traces of mCherry expression for the " responding " ( light gray ) or " not responding " ( dark gray ) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics ( B ) , low amplitude ( D ) , high amplitude ( D ) , high frequency ( E ) , low frequency / short duration ( F ) , or long duration ( G ) Nutlin - 3 dosing regimens . The average trace for responding and not responding cells is shown in red and blue , respectively . Heat maps show alternative representation of all single cell traces below each associated time course plot .

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Figure 2(A) Representative phase contrast, yellow fluorescent (indicating p53-Venus levels), and red fluorescent (indicating reporter mCherry levels) images at the indicated time points for MDM2 promoter reporter cells exposed to the "natural dynamics" Nutlin-3 dosing regimen.(B-G) Single cell traces of mCherry expression for the "responding" (light gray) or "not responding" (dark gray) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics (B), low amplitude (D), high amplitude (D), high frequency (E), low frequency/short duration (F), or long duration (G) Nutlin-3 dosing regimens. The average trace for responding and not responding cells is shown in red and blue, respectively. Heat maps show alternative representation of all single cell traces below each associated time course plot.


Figure 3Dose - response curves representing the rate of mCherry expression from the MDM2 ( pink dots ) and CDKN1A ( purple dots ) promoters as a function of total p53 levels . Values of expression levels were averaged across all cells in the first 15 - h response to the long duration Nutlin - 3 dosing regimen . Solid black lines represent the best fit to a Hill function model of promoter activation . Dashed lines indicate the half - maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter .

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Figure 3Dose-response curves representing the rate of mCherry expression from the MDM2 (pink dots) and CDKN1A (purple dots) promoters as a function of total p53 levels. Values of expression levels were averaged across all cells in the first 15-h response to the long duration Nutlin-3 dosing regimen. Solid black lines represent the best fit to a Hill function model of promoter activation. Dashed lines indicate the half-maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter.


Figure 4 ( B - G ) Effects of p53 duration modulation on target promoter activation in terms of the mean timing ( B , E ) , magnitude ( C , F ) , and rate ( D , G ) for the MDM2 ( B - D ) and CDKN1A ( E - G ) promoters in response to short ( light green ) and long ( dark green ) p53 durations . N = at least 45 cells per condition , error bars = SEM . ( H ) Mean nuclear MDM2 - YFP levels in single cells at times corresponding to the first pulse ( 5 . 5 h ) or second pulse ( 11 h ) of p53 expression in response to natural and long duration Nutlin - 3 dosing regimens . N = at least 40 cells per condition , error bars = SEM .

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Figure 4(B-G) Effects of p53 duration modulation on target promoter activation in terms of the mean timing (B, E), magnitude (C, F), and rate (D, G) for the MDM2 (B-D) and CDKN1A (E-G) promoters in response to short (light green) and long (dark green) p53 durations. N = at least 45 cells per condition, error bars = SEM.(H) Mean nuclear MDM2-YFP levels in single cells at times corresponding to the first pulse (5.5 h) or second pulse (11 h) of p53 expression in response to natural and long duration Nutlin-3 dosing regimens. N = at least 40 cells per condition, error bars = SEM.


Figure 5 ( A ) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing ( A , D ) , magnitude ( B , E ) , and rate ( C , F ) for the MDM2 ( A - C ) and CDKN1A ( D - F ) promoters in response to low ( light blue ) , natural ( medium blue ) , high ( dark blue ) p53 frequencies . N = at least 45 cells per condition , error bars = SEM . ( H ) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low , natural , and high frequency p53 pulses .

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Figure 5(A) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing (A, D), magnitude (B, E), and rate (C, F) for the MDM2 (A-C) and CDKN1A (D-F) promoters in response to low (light blue), natural (medium blue), high (dark blue) p53 frequencies. N = at least 45 cells per condition, error bars = SEM.(H) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low, natural, and high frequency p53 pulses.


Figure 6 ( A - B ) Representative phase contrast , red fluorescence ( indicating geminin - mCherry levels ) , and yellow fluorescence ( indicating p53 - Venus levels ) images of cells in response to a low ( A ) or high ( B ) frequency Nutlin - 3 dosing regimen over 40 h . ( C ) Geminin - mCherry traces in single cells treated with the low , natural , or high frequency Nutlin - 3 dosing regimens . N = at least 50 cells per condition . Percentage of cells undergoing cell division ( D ) within the 40 h imaging in response to the low , natural , and high frequency Nutlin - 3 dosing regimensPercentage of cells positive for geminin - mCherry expression ( E ) within the 40 h imaging in response to the low , natural , and high frequency Nutlin - 3 dosing

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Figure 6(A-B) Representative phase contrast, red fluorescence (indicating geminin-mCherry levels), and yellow fluorescence (indicating p53-Venus levels) images of cells in response to a low (A) or high (B) frequency Nutlin-3 dosing regimen over 40 h.(C) Geminin-mCherry traces in single cells treated with the low, natural, or high frequency Nutlin-3 dosing regimens. N = at least 50 cells per condition.Percentage of cells undergoing cell division (D) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimensPercentage of cells positive for geminin-mCherry expression (E) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens


Figure 1B HeLa cells expressing the indicated GFP - tagged Nups were semi - permeabilized and incubated with mitotic HeLa cell extract at 30ºC . NPC disassembly was followed by monitoring the fluorescence intensity of the respective Nups at the nuclear rim and influx of TRITC - dextran . Scale bar , 30 μm . C Quantification of dextran - positive nuclei over time of the experiments in ( B ) . N = 3 biological replicates , n > 100 cells . Error bars , SEM . E Quantification of 3GFP - Nup133 , GFP - Nup107 and 2GFP - Nup58 intensity at the NE over time of the experiments in ( B ) . Error bars , SEM .

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Figure 1B HeLa cells expressing the indicated GFP-tagged Nups were semi-permeabilized and incubated with mitotic HeLa cell extract at 30ºC. NPC disassembly was followed by monitoring the fluorescence intensity of the respective Nups at the nuclear rim and influx of TRITC-dextran. Scale bar, 30 μm.C Quantification of dextran-positive nuclei over time of the experiments in (B). N = 3 biological replicates, n > 100 cells. Error bars, SEM.E Quantification of 3GFP-Nup133, GFP-Nup107 and 2GFP-Nup58 intensity at the NE over time of the experiments in (B). Error bars, SEM.


Figure 2B Input , light and dense fractions were analyzed by immunoblotting using the indicated antibodies . The samples were derived from the same experiment but processed using several gels and blots . C Quantification of the percentage of Nups in the light fractions relative to the total amount of Nups over all fractions . N = 4 biological replicates , except for Nup214 ( N = 2 biological replicates ) . Error bars , SEM .

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Figure 2B Input, light and dense fractions were analyzed by immunoblotting using the indicated antibodies. The samples were derived from the same experiment but processed using several gels and blots.C Quantification of the percentage of Nups in the light fractions relative to the total amount of Nups over all fractions. N = 4 biological replicates, except for Nup214 (N = 2 biological replicates). Error bars, SEM.


Figure 3B Confocal images of metaphase - arrested HeLa cells expressing 3GFP - Nup133 , GFP - Nup107 , GFP - Nup53 or 2GFP - Nup58 that were either directly fixed ( untreated ) or first semi - permeabilized and then fixed . The ER was visualized by immunostaining with an antibody targeting calnexin ; DNA was visualized with Hoechst ( visible only in the merged channels ) . Representative images of 3 biological replicates . Scale bar , 10 μm and inset scale bar , 1 μm .

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Figure 3B Confocal images of metaphase-arrested HeLa cells expressing 3GFP-Nup133, GFP-Nup107, GFP-Nup53 or 2GFP-Nup58 that were either directly fixed (untreated) or first semi-permeabilized and then fixed. The ER was visualized by immunostaining with an antibody targeting calnexin; DNA was visualized with Hoechst (visible only in the merged channels). Representative images of 3 biological replicates. Scale bar, 10 μm and inset scale bar, 1 μm.


Figure 4A , B 2D dSTORM images of interphase ( A ) or metaphase ( B ) U2OS cells expressing SNAP - tagged Nup96 . Cells were semi - permeabilized with digitonin and Nup96 was labelled with the BG - AF647 SNAP dye . The SMLM data was reconstructed using Thunderstorm . Left panels were rendered to black and white and enhanced for viewing purposes . Insets ( yellow dashed boxes ) are displayed to the right . Insets in ( A ) show ring - shaped structures built by Y - complexes in interphase cells . Insets in ( B ) represents mitotic clusters outside the spindle area ( class 1 , inset 1 ) and at kinetochores ( class 2 ) . Scale bar ( left panels ) , 10 μm . Representative images of 4 biological replicates . Inset scale bars , 200 nm .

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Figure 4A, B 2D dSTORM images of interphase (A) or metaphase (B) U2OS cells expressing SNAP-tagged Nup96. Cells were semi-permeabilized with digitonin and Nup96 was labelled with the BG-AF647 SNAP dye. The SMLM data was reconstructed using Thunderstorm. Left panels were rendered to black and white and enhanced for viewing purposes. Insets (yellow dashed boxes) are displayed to the right. Insets in (A) show ring-shaped structures built by Y-complexes in interphase cells. Insets in (B) represents mitotic clusters outside the spindle area (class 1, inset 1) and at kinetochores (class 2). Scale bar (left panels), 10 μm. Representative images of 4 biological replicates. Inset scale bars, 200 nm.


Figure 5A 2D dSTORM images of a metaphase - arrested U2OS cell expressing Nup96 with a SNAP - tag , labelled with BG - Alexa Fluor 647 ( orange ) , in combination with confocal imaging of the ER immunostained for Climp63 ( red ) and phospho - histone H3 ( cyan ) to label DNA . Inset 1 ( mitotic clusters ) and inset 2 ( kinetochores ) are indicated with white dashed boxes . SMLM data was reconstructed using Thunderstorm . Representative images of N = 4 biological replicates . Scale bars , 10 μm . Inset scale bars , 200 nm . C Quantification of the number of localizations in interphase NPCs ( NPC ) , mitotic clusters and kinetochores of the experiments in ( A ) using DBSCAN . Error bars , SD . D Quantification of the number of protein copies in interphase NPCs ( NPC ) , mitotic clusters and kinetochores of the experiments in ( A ) using DBSCAN . Error bars , SD . E Quantification of the cluster width of interphase NPCs ( NPC ) , mitotic clusters and kinetochores of the experiments in ( A ) using DBSCAN . Error bars , SD .

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Figure 5A 2D dSTORM images of a metaphase-arrested U2OS cell expressing Nup96 with a SNAP-tag, labelled with BG-Alexa Fluor 647 (orange), in combination with confocal imaging of the ER immunostained for Climp63 (red) and phospho-histone H3 (cyan) to label DNA. Inset 1 (mitotic clusters) and inset 2 (kinetochores) are indicated with white dashed boxes. SMLM data was reconstructed using Thunderstorm. Representative images of N = 4 biological replicates. Scale bars, 10 μm. Inset scale bars, 200 nm.C Quantification of the number of localizations in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. D Quantification of the number of protein copies in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. E Quantification of the cluster width of interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD.


Figure 1A Generation of myeloid cells genetically inactivated for Elp3 . Elp3 mRNA and protein levels were assessed by Real - Time PCR with extracts isolated from peritoneal macrophages of Elp3Control and Elp3 ∆ Mye mice . mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels ( n = 3 mice ; mean + / - SD , Student T test , * * * = p < 0 . 001 ) . An anti - Elp3 western blot ( WB ) is also illustrated . The anti - Gapdh blot is shown for normalization purposes . C and D Defective mTORC2 signaling upon Elp3 deficiency in myeloid cells . Peritoneal macrophages from Elp3Control and Elp3 ∆ Mye mice were isolated and cultured ex - vivo . They were untreated or stimulated with a combination of LPS ( 100 ng / ml ) and IFNγ ( 50 ng / ml ) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses . F mTORC2 activation by M1 polarization signals relies on Ctu2 . Control and Ctu2 - depleted bone marrow - derived macrophages ( BMDMs ) ( SiRNA - mediated depletions ) were treated or not with IFNγ ( 50 ng / ml ) and LPS ( 100 ng / ml ) and the resulting cell extracts were subjected to WB analyses .

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Figure 1A Generation of myeloid cells genetically inactivated for Elp3. Elp3 mRNA and protein levels were assessed by Real-Time PCR with extracts isolated from peritoneal macrophages of Elp3Control and Elp3∆Mye mice. mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice; mean +/- SD, Student T test, ***= p< 0.001). An anti-Elp3 western blot (WB) is also illustrated. The anti-Gapdh blot is shown for normalization purposes.C and D Defective mTORC2 signaling upon Elp3 deficiency in myeloid cells. Peritoneal macrophages from Elp3Control and Elp3∆Mye mice were isolated and cultured ex-vivo. They were untreated or stimulated with a combination of LPS (100 ng/ml) and IFNγ (50 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.F mTORC2 activation by M1 polarization signals relies on Ctu2. Control and Ctu2-depleted bone marrow-derived macrophages (BMDMs) (SiRNA-mediated depletions) were treated or not with IFNγ (50 ng/ml) and LPS (100 ng/ml) and the resulting cell extracts were subjected to WB analyses.


Figure 2C Elp3 deficiency in myeloid cells exacerbates the decrease of colon length upon DSS administration ( n = 7 mice ; mean + / - SD , * * = p < 0 . 01 ) . E More severe histological score in mice lacking Elp3 in myeloid cells upon DSS administration . Representative intestinal crypts from the indicated genotypes are illustrated . The histological score was established as described in methods ( n = 5 mice per genotype ; mean + / - SD , Student T test , * = p < 0 . 05 ) . F Increased infiltration of iNOS + / CD68 + cells upon DSS administration in Elp3 ∆ Mye mice . The percentage of iNOS + in CD68 + cells was established in both genotypes treated with DSS ( n = 7 mice per genotype ; mean + / - SD , Student T test , * * = p < 0 . 01 ) . Anti - iNOS and - CD68 immunofluorescence analyses carried out in intestinal crypts from both genotypes are illustrated . G Patients suffering from ulcerative colitis ( UC ) show reduced levels of both Elp1 and Elp3 . The central band represents the mean value of relative expression in the investigated cohort . Boxes represent 75th and 25th percentile . Whiskers represent maximum and minimum values before the upper and lower fence , respectively ( * * = p < 0 . 01 , * * * = p < 0 . 001 ) ( GSE9452 - GEO DataSets , Definition of an ulcerative colitis pre - inflammatory state ) .

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Figure 2C Elp3 deficiency in myeloid cells exacerbates the decrease of colon length upon DSS administration (n = 7 mice; mean +/- SD, **= p< 0.01).E More severe histological score in mice lacking Elp3 in myeloid cells upon DSS administration. Representative intestinal crypts from the indicated genotypes are illustrated. The histological score was established as described in methods (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05).F Increased infiltration of iNOS+/CD68+ cells upon DSS administration in Elp3∆Mye mice. The percentage of iNOS+ in CD68+ cells was established in both genotypes treated with DSS (n = 7 mice per genotype; mean +/- SD, Student T test, **= p< 0.01). Anti-iNOS and -CD68 immunofluorescence analyses carried out in intestinal crypts from both genotypes are illustrated.G Patients suffering from ulcerative colitis (UC) show reduced levels of both Elp1 and Elp3. The central band represents the mean value of relative expression in the investigated cohort. Boxes represent 75th and 25th percentile. Whiskers represent maximum and minimum values before the upper and lower fence, respectively (**= p< 0.01, ***= p<0.001) (GSE9452-GEO DataSets, Definition of an ulcerative colitis pre-inflammatory state).


Figure 3A IL - 4 and IL - 13 induce the expression of tRNA - modifying enzymes in macrophages . Peritoneal macrophages were untreated ( « Control » ) or stimulated with IL - 4 ( 10 ng / ml ) , IL - 13 ( 10 ng / ml ) or with both cytokines for 24 hours and cell extracts from the resulting cells were subjected to WB analyses . B The pool of thiolated tRNAs in macrophages is enhanced by IL - 4 / IL - 13 but decreased by LPS / IFNγ . On the left , Northern blot analysis assessing t : Q ( UUG ) tRNA thiolation in Thioglycollate - elicited peritoneal macrophages subjected to the indicated treatments for 24 hours . On the right , quantification of t : Q ( UUG ) tRNA thiolation , calculated as the ration of thiolated over nonthiolated t : Q ( UUG ) , in the indicated experimental conditions . C Elp3 induction by IL - 4 / IL - 13 occurs through a PI3K - dependent pathway . Peritoneal macrophages were pretreated with DMSO ( control vehicle ) or with Wortmannin ( 200 nM ) for one hour and subsequently left untreated or stimulated with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for the indicated periods of time . Cell extracts from the resulting cells were subjected to WB analyses . E Rictor but not Raptor deficiency impairs Elp3 expression . BMDMs were transfected with the indicated siRNAs and the resulting cells were treated or not with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for 24 hours . Cell extracts were subjected to WB analyses . F IL - 4 / IL - 13 rely on Stat6 to induce Elp3 expression . Peritoneal macrophages were pretreated with the control vehicle ( DMSO ) or with the Stat6 pharmacological inhibitor AS1517499 ( 5 μM ) for one hour and subsequently left unstimulated or treated with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for the indicated periods of time . Cell extracts from the resulting cells were subjected to WB analyses . Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL - 4 ( H , , IL - 13 ( H ) or by both cytokines ( H ) . Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL - 4 ( 10 ng / ml ) ( H , IL - 13 ( 10 ng / ml ) ( H ) or with both cytokines ( H ) for 8 hours ( H ) and the resulting cell extracts were subjected to WB analyses . J Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL - 4 I and J ) Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL - 4 ( 10 ng / ml ) , I and J ) , or for the indicated periods of time ( I and J ) and the resulting cell extracts were subjected to WB analyses . L Elp3 promotes M2 macrophage polarization . Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL - 4 / IL - 13 ( 10 ng / ml ) for 24 hours and the resulting cell extracts were subjected to WB analyses .

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

Figure 3A IL-4 and IL-13 induce the expression of tRNA-modifying enzymes in macrophages. Peritoneal macrophages were untreated (« Control ») or stimulated with IL-4 (10 ng/ml), IL-13 (10 ng/ml) or with both cytokines for 24 hours and cell extracts from the resulting cells were subjected to WB analyses.B The pool of thiolated tRNAs in macrophages is enhanced by IL-4/IL-13 but decreased by LPS/IFNγ. On the left, Northern blot analysis assessing t:Q(UUG) tRNA thiolation in Thioglycollate-elicited peritoneal macrophages subjected to the indicated treatments for 24 hours. On the right, quantification of t:Q(UUG) tRNA thiolation, calculated as the ration of thiolated over nonthiolated t:Q(UUG), in the indicated experimental conditions.C Elp3 induction by IL-4/IL-13 occurs through a PI3K-dependent pathway. Peritoneal macrophages were pretreated with DMSO (control vehicle) or with Wortmannin (200 nM) for one hour and subsequently left untreated or stimulated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.E Rictor but not Raptor deficiency impairs Elp3 expression. BMDMs were transfected with the indicated siRNAs and the resulting cells were treated or not with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for 24 hours. Cell extracts were subjected to WB analyses.F IL-4/IL-13 rely on Stat6 to induce Elp3 expression. Peritoneal macrophages were pretreated with the control vehicle (DMSO) or with the Stat6 pharmacological inhibitor AS1517499 (5 μM) for one hour and subsequently left unstimulated or treated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 (H, , IL-13 (H) or by both cytokines (H). Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) (H, IL-13 (10 ng/ml) (H) or with both cytokines (H) for 8 hours (H) and the resulting cell extracts were subjected to WB analyses.J Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 I and J) Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) , I and J), or for the indicated periods of time (I and J) and the resulting cell extracts were subjected to WB analyses.L Elp3 promotes M2 macrophage polarization. Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4/IL-13 (10 ng/ml) for 24 hours and the resulting cell extracts were subjected to WB analyses.


Figure 4B Defective induction of Pparγ by IL - 4 / IL - 13 upon Elp3 deficiency . Cell extracts from peritoneal macrophages of Elp3Control and Elp3 ∆ Mye mice treated or not with IL - 4 / IL - 13 were subjected to WB analyses .

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Figure 4B Defective induction of Pparγ by IL-4/IL-13 upon Elp3 deficiency. Cell extracts from peritoneal macrophages of Elp3Control and Elp3∆Mye mice treated or not with IL-4/IL-13 were subjected to WB analyses.


Figure 7A The induction of Ric8b expression by IL - 4 relies on Elp3 . BMDMs from the indicated genotypes were treated or not with IL - 4 ( 10 ng / ml ) up to 24 hours and the resulting cell extracts were subjected to WB analyses . B Elp3 promotes Ric8B mRNA translation . BMDMs from Elp3Control and Elp3 ∆ Mye mice were treated by IL - 4 for 24 hours , followed by a treatment with 10 μg / ml puromycin for 5 minutes . To detect newly synthesized Ric8b proteins , a Puro - PLA assay was conducted . Representative images detecting Ric8b ( Red ) and α - Tubulin + microtubules ( green ) in BMDMs are illustrated . On the right , the graph shows a quantification of signals for Ric8b in α - Tubulin + areas . A random of six different areas were counted ( 6 technical replicates , mean + / - SD , Student ' s t - test , * = p < 0 . 05 ) . C Ric8b is a direct target of Elp3 . BMDMs from Elp3Control and Elp3 ∆ Mye mice were infected with a lentiviral GFP construct ( negative control ) , Flag - Ric8b WT or with Flag - Ricb MUT , as indicated and the resulting protein extracts were subjected to WB analyses to assess Ric8b and Elp3 protein levels . Gapdh was used as a loading control . D and E Ric8b promotes IL - 4 - dependent mTORC2 activation . Control or Ric8b - depleted BMDMs were treated or not with IL - 4 ( 10 ng / ml ) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses .

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Figure 7A The induction of Ric8b expression by IL-4 relies on Elp3. BMDMs from the indicated genotypes were treated or not with IL-4 (10 ng/ml) up to 24 hours and the resulting cell extracts were subjected to WB analyses.B Elp3 promotes Ric8B mRNA translation. BMDMs from Elp3Control and Elp3∆Mye mice were treated by IL-4 for 24 hours, followed by a treatment with 10 μg/ml puromycin for 5 minutes. To detect newly synthesized Ric8b proteins, a Puro-PLA assay was conducted. Representative images detecting Ric8b (Red) and α-Tubulin+ microtubules (green) in BMDMs are illustrated. On the right, the graph shows a quantification of signals for Ric8b in α-Tubulin+ areas. A random of six different areas were counted (6 technical replicates, mean +/- SD, Student's t-test, *= p< 0.05).C Ric8b is a direct target of Elp3. BMDMs from Elp3Control and Elp3∆Mye mice were infected with a lentiviral GFP construct (negative control), Flag-Ric8b WT or with Flag-Ricb MUT, as indicated and the resulting protein extracts were subjected to WB analyses to assess Ric8b and Elp3 protein levels. Gapdh was used as a loading control.D and E Ric8b promotes IL-4-dependent mTORC2 activation. Control or Ric8b-depleted BMDMs were treated or not with IL-4 (10 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.


Figure 8C Elp3 promotes mitochondrial translation . BMDMs from the indicated genotypes were treated or not with IL - 4 / IL - 13 ( 10 ng / ml ) for the indicated hours and the resulting cell extracts were subjected to WB analyses .

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Figure 8C Elp3 promotes mitochondrial translation. BMDMs from the indicated genotypes were treated or not with IL-4/IL-13 (10 ng/ml) for the indicated hours and the resulting cell extracts were subjected to WB analyses.


Figure 9D Elp3 expression in myeloid cells maintains the pool of tumor - associated macrophages ( TAMs ) . The number of TAMs , defined as CD301 + / F4 / 80 + cells among F4 / 80 + cells was quantified in intestinal crypts from both Apc + / Min Elp3Control and Apc + / Min Elp3 ∆ Mye mice ( n = 5 mice per genotype ; mean + / - SD , Student T test , * = p < 0 . 05 ) . Representative immunofluorescence analyses are illustrated .

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Figure 9D Elp3 expression in myeloid cells maintains the pool of tumor-associated macrophages (TAMs). The number of TAMs, defined as CD301+/F4/80+ cells among F4/80+ cells was quantified in intestinal crypts from both Apc+/Min Elp3Control and Apc+/Min Elp3∆Mye mice (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05). Representative immunofluorescence analyses are illustrated.


Figure 1B . Typical examples of stage 1 ( day 0 ) , stage 2 ( day 7 ) and stage 3 ( day 12 ) human iPSC - derived NSCs / neurons immunostained for γ - Tubulin and Centrin , marked by arrows . Cells were co - immunostained with Ki67 and DAPI ( day 0 ) or Trim46 and MAP2 ( day 7 and 12 ) to define their neuronal stages . Arrowheads mark the AIS . Scale bar = 10µm in overview , 2µm in zooms . D . 3D STED imaging of αTubulin in stage 1 ( day 0 ) , stage 2 ( day 7 ) and stage 3 ( day 13 ) hiPSC - derived neurons . Scale bar = 5µm in overview , 1µm in zoom . F . Typical examples of centrosomes of human iPSC - derived NSCs ( day 0 ) with STED imaging of Trim46 and Centrin immunostaining , and confocal imaging of γ - Tubulin immunostaining . Scale bar = 1µm . G . Typical examples of stage 2 and stage 3 human iPSC - derived neurons immunostained for Trim46 and MAP2 . Inserts represent centrosomes . Zooms on the right represent a non - polarized neurite or a developing axon in stage 2 or stage 3 neurons , respectively . Scale bar = 20µm in overview , 1µm in insert , 10µm in zoom .

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Figure 1B. Typical examples of stage 1 (day 0), stage 2 (day 7) and stage 3 (day 12) human iPSC-derived NSCs/neurons immunostained for γ-Tubulin and Centrin, marked by arrows. Cells were co-immunostained with Ki67 and DAPI (day 0) or Trim46 and MAP2 (day 7 and 12) to define their neuronal stages. Arrowheads mark the AIS. Scale bar = 10µm in overview, 2µm in zooms.D. 3D STED imaging of αTubulin in stage 1 (day 0), stage 2 (day 7) and stage 3 (day 13) hiPSC-derived neurons. Scale bar = 5µm in overview, 1µm in zoom.F. Typical examples of centrosomes of human iPSC-derived NSCs (day 0) with STED imaging of Trim46 and Centrin immunostaining, and confocal imaging of γ-Tubulin immunostaining. Scale bar = 1µm.G. Typical examples of stage 2 and stage 3 human iPSC-derived neurons immunostained for Trim46 and MAP2. Inserts represent centrosomes. Zooms on the right represent a non-polarized neurite or a developing axon in stage 2 or stage 3 neurons, respectively. Scale bar = 20µm in overview, 1µm in insert, 10µm in zoom.


Figure 2A . Typical examples of Centrinone - B treated or control human iPSC - derived NSCs immunostained for Pericentrin and Centrin . Inserts represent centriole ( s ) . Scale bar = 5µm in overview , 2µm in inserts . B . Quantifications of the percentage of cells with 0 , 1 or 2 centrioles per cell after 0 ( control ) , 2 or 5 days Centrinone - B treatment and prior to neuronal induction . n = 48 - 51 cells in 2 independent experiments . C Data information : Data represents mean ± SEM . Chi - square - test including post - hoc analysis with Bonferroni correction * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns p > 0 . 05C . Typical examples of Centrinone - B treated or control human iPSC - derived neurons ( 12 - 15 days ) immunostained for AnkG , Trim46 , MAP2 and Centrin . Arrowheads mark AIS structures , arrows mark centrosomes . Inserts represent centrosomes , zooms on the right represent AIS structures . Scale bar = 20µm in overview , 2µm in insert , 10µm in zooms . M . AP threshold , amplitude and after - hyperpolarization recorded in Centrinone - B treated ( n = 43 cells in 3 independent experiments ) and control human iPSC - derived neuron cultures ( n = 59 cells in 4 independent experiments ) . Data information : Data represents mean ± SEM . Chi - square - test including post - hoc analysis with Mann Whitney test after - hyperpolarization Student ' s t test ( M : threshold , AP amplitude ) . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns p > 0 .

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Figure 2A. Typical examples of Centrinone-B treated or control human iPSC-derived NSCs immunostained for Pericentrin and Centrin. Inserts represent centriole(s). Scale bar = 5µm in overview, 2µm in inserts. B. Quantifications of the percentage of cells with 0, 1 or 2 centrioles per cell after 0 (control), 2 or 5 days Centrinone-B treatment and prior to neuronal induction. n = 48-51 cells in 2 independent experiments. C Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Bonferroni correction *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05C. Typical examples of Centrinone-B treated or control human iPSC-derived neurons (12-15 days) immunostained for AnkG, Trim46, MAP2 and Centrin. Arrowheads mark AIS structures, arrows mark centrosomes. Inserts represent centrosomes, zooms on the right represent AIS structures. Scale bar = 20µm in overview, 2µm in insert, 10µm in zooms.M. AP threshold, amplitude and after-hyperpolarization recorded in Centrinone-B treated (n = 43 cells in 3 independent experiments) and control human iPSC-derived neuron cultures (n = 59 cells in 4 independent experiments). Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Mann Whitney test after-hyperpolarization Student's t test (M: threshold, AP amplitude). *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05


Figure 3D . Representative images of fan - like growth cones at day 5 of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions . Growth cone visualized by immunostaining for phalloidin . Scale bar = 5µm . E . Quantifications of the average area ( µm² ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . n = 25 - 83 growth cones in 3 independent experiments . F Data information : Data represent mean ± SEM . One - way ANOVA including Tukey ' s post - hoc analysis * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 , ns p ≥ 0 . 05F . Representative images of different growth cone morphological categories : fan - like , torpedo - like and bulb - like . Scale bar = 5µm . G . Quantifications of the ratios of different subtypes ( fan - like , torpedo - like , bulb - like ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . H . Quantifications of the average area ( µm² ) of different subtypes ( fan - like , torpedo - like , bulb - like ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . n = 5 - 55 growth cones in 3 independent experiments . Da Data information : Chi - square - test including post - hoc analysis with Bonferroni correction ( G ) , One - way ANOVA including Sidak ' s post - hoc analysis ( H ) . * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 , ns p ≥ 0 .

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Figure 3D. Representative images of fan-like growth cones at day 5 of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions. Growth cone visualized by immunostaining for phalloidin. Scale bar = 5µm. E. Quantifications of the average area (µm²) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 25-83 growth cones in 3 independent experiments. F Data information: Data represent mean ± SEM. One-way ANOVA including Tukey's post-hoc analysis *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05F. Representative images of different growth cone morphological categories: fan-like, torpedo-like and bulb-like. Scale bar = 5µm. G. Quantifications of the ratios of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. H. Quantifications of the average area (µm²) of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 5-55 growth cones in 3 independent experiments. Da Data information: Chi-square-test including post-hoc analysis with Bonferroni correction (G), One-way ANOVA including Sidak's post-hoc analysis (H). *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05