HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . F Measurement of TO - PRO - 3 uptake as in ( B ) . Dead TO - PRO - 3high cells were discarded . Data are means ± SEM of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). F Measurement of TO-PRO-3 uptake as in (B). Dead TO-PRO-3high cells were discarded. Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . G Small EVs fractions were purified from the culture medium by ultracentrifugation . Shown is a representative diagram of particle count obtained with tunable resistive pulse sensing analysis ( TRPS , qNano , IZON ) .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). G Small EVs fractions were purified from the culture medium by ultracentrifugation. Shown is a representative diagram of particle count obtained with tunable resistive pulse sensing analysis (TRPS, qNano, IZON).


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . H Cells were subjected to Western blotting analysis Data information : Arrowhead , MLKL cleaved fragment .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). H Cells were subjected to Western blotting analysis Data information: Arrowhead, MLKL cleaved fragment.


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . I MLKL oligomerization was determined Data information Arrowhead , MLKL cleaved fragment .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). I MLKL oligomerization was determined Data information Arrowhead, MLKL cleaved fragment.


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . J Cell survival by CellTiter - Glo after 24 hours ( mean ± SEM , n = 4 biological replicates , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA ) .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). J Cell survival by CellTiter-Glo after 24 hours (mean ± SEM, n=4 biological replicates, **P<0.01, ***P<0.001, ****P<0.0001, ANOVA).


HT - 29 cells were transfected with a nonspecific ( NS ) siRNA or with the indicated siRNAs for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) . K TO - PRO - 3 uptake Data are means ± SEM , n = 3 biological replicates , * * * * P < 0 . 0001 ( ANOVA ) . The presented data are representative of at least three independent experiments .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). K TO-PRO-3 uptake Data are means ± SEM, n=3 biological replicates, ****P<0.0001 (ANOVA). The presented data are representative of at least three independent experiments.


A HT - 29 cells were transfected with a siRNA for PANX1 ( # 3 ) , or scramble non - specific ( NS ) siRNA for 72 hours . Cells were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , and exposed to 10 ng . mL - 1 of TNFα ( T ) for 6 hours . Shown is a normalized densitometric analysis for the presence of 120 cytokines with an antibody array . The color scale ( 0 - 10 ) represents the means of normalized densitometry values ( n = 2 technical replicates ) .

O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HT-29 cells were transfected with a siRNA for PANX1 (#3), or scramble non-specific (NS) siRNA for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T) for 6 hours. Shown is a normalized densitometric analysis for the presence of 120 cytokines with an antibody array. The color scale (0-10) represents the means of normalized densitometry values (n=2 technical replicates).


B , C HT - 29 transfected as indicated ( B ) or PANX1 knockout HT - 29 cells ( C ) were treated with TQS for 6 hours . The mRNA levels of Cxcl8 relative to untreated NS or NC samples were measured by qPCR ( means ± SEM , n = 3 biological replicates , * * * * P < 0 . 0001 , ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C HT-29 transfected as indicated (B) or PANX1 knockout HT-29 cells (C) were treated with TQS for 6 hours. The mRNA levels of Cxcl8 relative to untreated NS or NC samples were measured by qPCR (means ± SEM, n=3 biological replicates, ****P<0.0001, ANOVA).


D Lysates from cells treated as indicated were prepared and subjected to Western blotting analysis as indicated . Molecular weight markers ( Mr ) are shown . Data information Cleaved MLKL is indicated with an arrowhead .

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

D Lysates from cells treated as indicated were prepared and subjected to Western blotting analysis as indicated. Molecular weight markers (Mr) are shown. Data information Cleaved MLKL is indicated with an arrowhead.


E Abundance of IL - 8 , measured by ELISA , in the supernatants of cells treated as indicated after 6 hours of stimulation ( means ± SEM , n = 4 biological replicates , * * * P < 0 . 001 , ANOVA ) .

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

E Abundance of IL-8, measured by ELISA, in the supernatants of cells treated as indicated after 6 hours of stimulation (means ± SEM, n=4 biological replicates, ***P<0.001, ANOVA).


HT - 29 cells were treated as indicated . Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment ( F ) . Data are means ± SEM of three ( F ) independent experiments ( * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment (F). Data are means ± SEM of three (F) independent experiments (***P<0.001, ****P<0.0001, ANOVA).


HT - 29 cells were treated as indicated . Cell lysates were analyzed by Western blotting ( G ) . Data information : Cleaved MLKL is indicated with an arrowhead .

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

HT-29 cells were treated as indicated. Cell lysates were analyzed by Western blotting (G). Data information: Cleaved MLKL is indicated with an arrowhead.


HT - 29 cells were treated as indicated . The release of IL - 8 in cell culture media was analyzed by ELISA ( H ) . Data are means ± SEM of four ( H ) independent experiments ( * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. The release of IL-8 in cell culture media was analyzed by ELISA (H). Data are means ± SEM of four (H) independent experiments (***P<0.001, ****P<0.0001, ANOVA).


HT - 29 cells were treated as indicated . Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment ( I ) . Data are means ± SEM of three ( I ) independent experiments ( * * P < 0 . 01 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment (I). Data are means ± SEM of three (I) independent experiments (**P<0.01, ****P<0.0001, ANOVA).


HT - 29 cells were treated as indicated . Cell lysates were analyzed by Western blotting ( J ) . Data information : Cleaved MLKL is indicated with an arrowhead .

HT-29 cells were treated as indicated. Cell lysates were analyzed by Western blotting (J). Data information: Cleaved MLKL is indicated with an arrowhead.


HT - 29 cells were treated as indicated . IL - 8 release was analyzed in cell culture media by ELISA ( K ) . Data are means ± SEM of four ( K ) independent experiments ( * * P < 0 . 01 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. IL-8 release was analyzed in cell culture media by ELISA (K). Data are means ± SEM of four (K) independent experiments (**P<0.01, ****P<0.0001, ANOVA).


( B ) Western blotting of WT and Drp1 - KO MEFs with or without FCCP treatment ( 30 min ) using the indicated antibodies .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(B) Western blotting of WT and Drp1-KO MEFs with or without FCCP treatment (30 min) using the indicated antibodies.


( C ) Quantification of band intensity . Values are average ± SD ( n = 3 ) . Significance was calculated using ANOVA with post - hoc Tukey : * * p < 0 . 01 , * * * p < 0 . 001 .

(C) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.


( B ) Western blotting of WT MEFs , Drp1 - KO MEFs , and Drp1 - KO MEFs carrying Drp1 , all of which express L2 - HA , using the indicated antibodies . The expression of L2 - HA was induced for 16 h ( 0 . 1 µg / ml doxycycline ) . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting of WT MEFs, Drp1-KO MEFs, and Drp1-KO MEFs carrying Drp1, all of which express L2-HA, using the indicated antibodies. The expression of L2-HA was induced for 16 h (0.1 µg/ml doxycycline). The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

(C) Quantification of band intensity. Values are average ± SD (n = 3).


( E ) WT and Drp1 - KO MEFs , both of which express L2 - HA , were transduced with lentiviruses carrying either scramble or Oma1 - targeted shRNAs . Whole - cell lysates were analyzed by Western blotting using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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

(E) WT and Drp1-KO MEFs, both of which express L2-HA, were transduced with lentiviruses carrying either scramble or Oma1-targeted shRNAs. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( F ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

(F) Quantification of band intensity. Values are average ± SD (n = 3).


( A ) The mitochondrial membrane potential was measured in WT and Drp1 - KO MEFs using a membrane potential - dependent dye ( MitoLite NIR ) and flow cytometry .

(A) The mitochondrial membrane potential was measured in WT and Drp1-KO MEFs using a membrane potential-dependent dye (MitoLite NIR) and flow cytometry.


( B ) Drp1 - KO MEFs carrying Su9 - GFP were treated with DMSO ( control ) or 10 nM antimycin A or 10 ng / ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10 - s intervals in the presence of TMRE . The arrows indicate mitochondria that showed flickering . Three frames from the time - lapse analysis are shown ( please also see Movie EV1 - EV4 ) . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Drp1-KO MEFs carrying Su9-GFP were treated with DMSO (control) or 10 nM antimycin A or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1-EV4). Scale bar, 10 µm.


The percentage of cells that showed flickering in 30 min . Values are average ± SD ( n = 3 experiments ) . In each experiment , 25 - 89 cells were analyzed .

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

The percentage of cells that showed flickering in 30 min. Values are average ± SD (n = 3 experiments). In each experiment, 25-89 cells were analyzed.


( F ) Drp1 - KO MEFs were treated with DMSO or 10 nM antimycin A or 10 ng / ml oligomycin or 10 µM FCCP for 1 h . The mitochondrial membrane potential was analyzed using MitoLite NIR and flow cytometry .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(F) Drp1-KO MEFs were treated with DMSO or 10 nM antimycin A or 10 ng/ml oligomycin or 10 µM FCCP for 1 h. The mitochondrial membrane potential was analyzed using MitoLite NIR and flow cytometry.


( G ) Drp1 - KO MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 nM antimycin A along with 10 µM FCCP . Western blotting was performed using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( H ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Drp1-KO MEFs expressing L2-HA (induced for 16 h) were treated with 10 nM antimycin A along with 10 µM FCCP. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (H) Quantification of band intensity. Values are average ± SD (n = 3).


( G ) Drp1 - KO MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 nM antimycin A along with 10 µM FCCP . Western blotting was performed using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(G) Drp1-KO MEFs expressing L2-HA (induced for 16 h) were treated with 10 nM antimycin A along with 10 µM FCCP. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( I ) Western blotting of Drp1 - KO MEFs with or without 10 ng / ml oligomycin treatment . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( J ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Western blotting of Drp1-KO MEFs with or without 10 ng/ml oligomycin treatment. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (J) Quantification of band intensity. Values are average ± SD (n = 3).


( I ) Western blotting of Drp1 - KO MEFs with or without 10 ng / ml oligomycin treatment . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(I) Western blotting of Drp1-KO MEFs with or without 10 ng/ml oligomycin treatment. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( K ) WT MEFs carrying Su9 - GFP were treated with DMSO or 10 ng / ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10 - s intervals in the presence of TMRE . The arrows indicate mitochondria that showed flickering . Three frames from the time - lapse analysis are shown ( please also see Movie EV1 and EV5 ) . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) WT MEFs carrying Su9-GFP were treated with DMSO or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1 and EV5). Scale bar, 10 µm.


( L ) WT MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 ng / ml oligomycin . Western blotting was performed using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( M ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) WT MEFs expressing L2-HA (induced for 16 h) were treated with 10 ng/ml oligomycin. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (M) Quantification of band intensity. Values are average ± SD (n = 3).


( L ) WT MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 ng / ml oligomycin . Western blotting was performed using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(L) WT MEFs expressing L2-HA (induced for 16 h) were treated with 10 ng/ml oligomycin. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( A ) Western blotting of Drp1flox / floxOpa1flox / flox and Drp1Opa1 - KO MEFs . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( B ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

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

(A) Western blotting of Drp1flox/floxOpa1flox/flox and Drp1Opa1-KO MEFs. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (B) Quantification of band intensity. Values are average ± SD (n = 3).


( A ) Western blotting of Drp1flox / floxOpa1flox / flox and Drp1Opa1 - KO MEFs . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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

(A) Western blotting of Drp1flox/floxOpa1flox/flox and Drp1Opa1-KO MEFs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C ) Drp1flox / floxOpa1flox / flox MEFs and Drp1Opa1 - KO MEFs were transduced with the indicated Opa1 constructs . The cells were observed using laser scanning confocal microscopy for 30 min with 10 - s intervals in the presence of TMRE . The percentage of cells that exhibited flickering is shown . Values are average ± SD ( n = 3 experiments ) . In each experiment , 48 - 62 cells were analyzed .

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

(C) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs were transduced with the indicated Opa1 constructs. The cells were observed using laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The percentage of cells that exhibited flickering is shown. Values are average ± SD (n = 3 experiments). In each experiment, 48-62 cells were analyzed.


( D ) Drp1flox / floxOpa1flox / flox MEFs and Drp1Opa1 - KO MEFs carrying the indicated Opa1 constructs were subjected to confocal immunofluorescence microscopy using anti - PDH antibodies . Scale bar , 10 µm .

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

(D) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs carrying the indicated Opa1 constructs were subjected to confocal immunofluorescence microscopy using anti-PDH antibodies. Scale bar, 10 µm.


( E ) Quantification of mitochondrial morphology is shown ( n = 3 experiments ) . In each experiment , 30 cells were analyzed .

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

(E) Quantification of mitochondrial morphology is shown (n = 3 experiments). In each experiment, 30 cells were analyzed.


( G ) Western blotting of Drp1flox / floxOpa1flox / flox MEFs and Drp1Opa1 - KO MEFs , both of which carry the indicated Opa1 constructs . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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

(G) Western blotting of Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs, both of which carry the indicated Opa1 constructs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( H ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

(H) Quantification of band intensity. Values are average ± SD (n = 3).


( B ) Three frames from the time - lapse analysis during artificial flickering are shown . The boxed regions are magnified . Scale bar , 5 µm .

O O O O O 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) Three frames from the time-lapse analysis during artificial flickering are shown. The boxed regions are magnified. Scale bar, 5 µm.


( C ) The fluorescence intensity of Su9 - GFP and TMRE in mitochondria was quantified .

(C) The fluorescence intensity of Su9-GFP and TMRE in mitochondria was quantified.


( D ) Western blotting of WT MEFs after the indicated FCCP treatments . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( E ) Quantification of band intensity . Values are average ± SD ( n = 3 ) . Significance was calculated using ANOVA with post - hoc Tukey : * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (E) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.


( D ) Western blotting of WT MEFs after the indicated FCCP treatments . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( A ) Western blotting of WT MEFs and Drp1 - KO MEFs , both of which carry shRNAs ( Oma1 or scramble ) and / or ectopic Mfn1 . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( B ) Quantification of band intensity is shown . Values are average ± SD ( n = 3 ) .

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

(A) Western blotting of WT MEFs and Drp1-KO MEFs, both of which carry shRNAs (Oma1 or scramble) and/or ectopic Mfn1. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (B) Quantification of band intensity is shown. Values are average ± SD (n = 3).


( A ) Western blotting of WT MEFs and Drp1 - KO MEFs , both of which carry shRNAs ( Oma1 or scramble ) and / or ectopic Mfn1 . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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

(A) Western blotting of WT MEFs and Drp1-KO MEFs, both of which carry shRNAs (Oma1 or scramble) and/or ectopic Mfn1. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C - F ) WT MEFs ( C and E ) and Drp1 - KO MEFs ( D and F ) were transduced with Su9 - Eos , along with Oma1 shRNAs and ectopic Mfn1 . Su9 - Eos in the small region of mitochondria ( indicated by the asterisks ) was photoconverted using a 405 - nm laser on confocal microscope . Within 1 s after photoconversion , images were obtained for both photoconverted and unconverted Su9 - Eos signals . Images before the photoconversion were also taken to detect background signals . Scale bar , 10 µm . ( E and F ) To determine the connectivity of mitochondria , the area containing photoconverted Su9 - Eos signals was divided by the area containing the photoconverted and unconverted Su9 - Eos signals ( total mitochondria ) . Values are average ± SD ( n = 30 cells ) .

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

(C-F) WT MEFs (C and E) and Drp1-KO MEFs (D and F) were transduced with Su9-Eos, along with Oma1 shRNAs and ectopic Mfn1. Su9-Eos in the small region of mitochondria (indicated by the asterisks) was photoconverted using a 405-nm laser on confocal microscope. Within 1 s after photoconversion, images were obtained for both photoconverted and unconverted Su9-Eos signals. Images before the photoconversion were also taken to detect background signals. Scale bar, 10 µm. (E and F) To determine the connectivity of mitochondria, the area containing photoconverted Su9-Eos signals was divided by the area containing the photoconverted and unconverted Su9-Eos signals (total mitochondria). Values are average ± SD (n = 30 cells).


WT which carry shRNAs ( Oma1 or scramble ) and ectopic Mfn1 , were stained with TMRE . The percentage of cells that maintained the membrane potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 cells were analyzed in each experiment .

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

WT which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.


Drp1 - KO MEFs which carry shRNAs ( Oma1 or scramble ) and ectopic Mfn1 , were stained with TMRE . The arrows indicate cells that lost the mitochondrial membrane potential in ( C ) . Scale bar , 10 µm . The percentage of cells that maintained the membrane potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 cells were analyzed in each experiment .

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

Drp1-KO MEFs which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The arrows indicate cells that lost the mitochondrial membrane potential in (C). Scale bar, 10 µm. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.


OCRs were analyzed in the same set of MEFs . Quantification of the basal OCRs and respiratory capacity is shown . Values are average ± SD ( n = 3 ) .

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

OCRs were analyzed in the same set of MEFs. Quantification of the basal OCRs and respiratory capacity is shown. Values are average ± SD (n = 3).


( A ) Western blotting of livers isolated from the indicated mice at 3 months of age using the indicated antibodies . ( B ) Quantification of band intensity . Values are average ± SD ( n = 3 mice for each genotype ) . Significance was calculated using ANOVA with post - hoc Tukey : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) Western blotting of livers isolated from the indicated mice at 3 months of age using the indicated antibodies. (B) Quantification of band intensity. Values are average ± SD (n = 3 mice for each genotype). Significance was calculated using ANOVA with post-hoc Tukey: *p<0.05, **p<0.01, ***p<0.001.


( B ) 48h post - transfection of HeLa - CIITA cells with siRNAs targeting NDP52 , T6BP , OPTN , and p62 , AR expression was analyzed using Western Blot . Tubulin was used as control . The results are representative of at least 3 independent experiments and correspond to AR expression levels of the experiment in Figure 1B , left panel .

(B) 48h post-transfection of HeLa-CIITA cells with siRNAs targeting NDP52, T6BP, OPTN, and p62, AR expression was analyzed using Western Blot. Tubulin was used as control. The results are representative of at least 3 independent experiments and correspond to AR expression levels of the experiment in Figure 1B, left panel.


( C ) Monitoring of Gag - specific T cell activation . HeLa - CIITA cells were treated as indicated above and transfected with a plasmid encoding Gag - LC3 . Left panel , a representative experiment is shown ( + / - SD of three technical replicates ) . Right panel , three biological replicates are combined and presented as mean percentage ( + / - SD ) . The y - axis represents the relative percentage of IFNγ spots reported to the secretion of IFNγ by the CD4 + T cell clones incubated with the siCTRL - treated HeLa - CIITA and set to 100 % . The mean T cell activation levels of the three experiments using siCTRL or siT6BP were ( 100 , 195 , 119 ) and ( 35 , 53 , 38 ) IFNγ + spots , respectively . 5000 T cell clones were seeded per well in technical triplicates . Data information : For all ELISPOT experiments , the background secretions of IFNγ by CD4 + T cells co - cultured with mock - treated HeLa - CIITA cells were used as negative controls and subtracted . CTRL : control . Wilcoxon ' s test ; the symbols correspond to * * p < 0 . 01 and # p > 0 . 05 comparing each experimental conditions solely with its internal control ( siCTRL ) .

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

(C) Monitoring of Gag-specific T cell activation. HeLa-CIITA cells were treated as indicated above and transfected with a plasmid encoding Gag-LC3. Left panel, a representative experiment is shown (+/- SD of three technical replicates). Right panel, three biological replicates are combined and presented as mean percentage (+/- SD). The y-axis represents the relative percentage of IFNγ spots reported to the secretion of IFNγ by the CD4+ T cell clones incubated with the siCTRL-treated HeLa-CIITA and set to 100%. The mean T cell activation levels of the three experiments using siCTRL or siT6BP were (100, 195, 119) and (35, 53, 38) IFNγ+ spots, respectively. 5000 T cell clones were seeded per well in technical triplicates. Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols correspond to ** p<0.01 and # p>0.05 comparing each experimental conditions solely with its internal control (siCTRL).


( D ) Left panel , as in Figure 1B right panel , but using DNA encoding Gag - LC3 , Gag - LC3G120A , or Gag . With Gag - LC3 , Gag - LC3 G120A or Gag antigens , the mean T cell activation levels using siCTRL or siT6BP were ( 199 , 435 , 384 ) , ( 91 , 141 , 119 ) and ( 343 , 415 , 385 ) or ( 98 , 273 , 171 ) , ( 27 , 50 , 19 ) and ( 97 , 148 , 131 ) IFNγ + spots , respectively . 5000 T cell clones were seeded per well in technical triplicates . Three biological replicates are combined and presented as mean percentage ( + / - SD ) . Right panel , influence of T6BP silencing on peptide presentation by Hela - CIITA cells . The cognate peptide was added exogenously ( gag2 , 0 , 1µg / mL ) on siRNA - treated cells ( 2h , 37 ° C ) , washed and T cell activation monitored using IFNγ - ELISPOT . The mean T cell activation levels using siCTRL or siT6BP were ( 100 , 45 , 42 ) and ( 71 , 45 , 33 ) IFNγ + spots , respectively . 1000 T cell clones were seeded per well in technical triplicates . Three biological replicates are combined and presented as mean percentage ( + / - SD ) . Data information : For all ELISPOT experiments , the background secretions of IFNγ by CD4 + T cells co - cultured with mock - treated HeLa - CIITA cells were used as negative controls and subtracted . CTRL : control . Wilcoxon ' s test ; the

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

(D) Left panel, as in Figure 1B right panel, but using DNA encoding Gag-LC3, Gag-LC3G120A, or Gag. With Gag-LC3, Gag-LC3 G120A or Gag antigens, the mean T cell activation levels using siCTRL or siT6BP were (199, 435, 384), (91, 141, 119) and (343, 415, 385) or (98, 273, 171), (27, 50, 19) and (97, 148, 131) IFNγ+ spots, respectively. 5000 T cell clones were seeded per well in technical triplicates. Three biological replicates are combined and presented as mean percentage (+/- SD). Right panel, influence of T6BP silencing on peptide presentation by Hela-CIITA cells. The cognate peptide was added exogenously (gag2, 0,1µg/mL) on siRNA-treated cells (2h, 37°C), washed and T cell activation monitored using IFNγ-ELISPOT. The mean T cell activation levels using siCTRL or siT6BP were (100, 45, 42) and (71, 45, 33) IFNγ+ spots, respectively. 1000 T cell clones were seeded per well in technical triplicates. Three biological replicates are combined and presented as mean percentage (+/- SD). Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols


( E ) As in ( D ) but using cDNA encoding HCMV pp65 antigen ( left panel ) or pp65 peptide ( 0 , 5µg / mL ; right panel ) and a pp65 - specific CD4 + T cell line . Results of three independent experiments are represented . The mean T cell activation levels using siCTRL or siT6BP and pp65 DNA were ( 66 , 106 , 99 ) and ( 39 , 50 , 33 ) IFNγ + spots , respectively . For the peptide : siCTRL or siT6BP : ( 185 , 317 , 415 ) and ( 163 , 292 , 381 ) IFNγ + spots , respectively . 1000 or 2000 T cell clones were seeded per well in technical triplicates . Data information : For all ELISPOT experiments , the background secretions of IFNγ by CD4 + T cells co - cultured with mock - treated HeLa - CIITA cells were used as negative controls and subtracted . CTRL : control . Wilcoxon ' s test ; the symbols correspond to * * p < 0 . 01 and # p > 0 . 05 comparing each experimental conditions solely with its internal control ( siCTRL ) .

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

(E) As in (D) but using cDNA encoding HCMV pp65 antigen (left panel) or pp65 peptide (0,5µg/mL; right panel) and a pp65-specific CD4+ T cell line. Results of three independent experiments are represented. The mean T cell activation levels using siCTRL or siT6BP and pp65 DNA were (66, 106, 99) and (39, 50, 33) IFNγ+ spots, respectively. For the peptide: siCTRL or siT6BP: (185, 317, 415) and (163, 292, 381) IFNγ+ spots, respectively. 1000 or 2000 T cell clones were seeded per well in technical triplicates. Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols correspond to ** p<0.01 and # p>0.05 comparing each experimental conditions solely with its internal control (siCTRL).


( D ) T6BP silencing affects the formation of stable MHC - peptide complexes . HeLa - CIITA cells were transfected as above and pulse - labeled with 35S - Met / Cys for 30 min , washed and chased for 4h at 37 ° C . MHC - II molecules were then immunoprecipitated using TÜ36 antibody and analyzed on SDS - PAGE after incubation of the immunoprecipitated protein complexes with SDS at 95 ° C ( B : boiled ) or room temperature ( NB : non - boiled ) to visualize α , β and Ii chains and SDS resistant αβ dimers , respectively . The bands corresponding to α , β and Ii chains are indicated . The arrow indicates the SDS - resistant stable HLA αβ heterodimers . This gel is representative of two independent experiments .

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

(D) T6BP silencing affects the formation of stable MHC-peptide complexes. HeLa-CIITA cells were transfected as above and pulse-labeled with 35S-Met/Cys for 30 min, washed and chased for 4h at 37°C. MHC-II molecules were then immunoprecipitated using TÜ36 antibody and analyzed on SDS-PAGE after incubation of the immunoprecipitated protein complexes with SDS at 95°C (B: boiled) or room temperature (NB: non-boiled) to visualize α, β and Ii chains and SDS resistant αβ dimers, respectively. The bands corresponding to α, β and Ii chains are indicated. The arrow indicates the SDS-resistant stable HLA αβ heterodimers. This gel is representative of two independent experiments.


( A ) MHC - II and T6BP expressions were assessed using confocal microscopy . HeLa - CIITA cells were transfected with control and T6BP - silencing siRNAs . 48h post - treatment , MHC - II and T6BP were detected using L243 and anti - T6BP antibodies , respectively , and revealed with species specific secondary antibodies . Nuclei were stained using DAPI . ( B ) Quantitative analysis using in - house ImageJ script displaying distance of each MHC - II + vesicles to the nucleus and number of vesicles per cell . At least 20 , 000 vesicles from 160 cells corresponding to five biological replicates were analyzed . Right panel , quantification in the siCTRL cells of the colocalization between MHC - II + and T6BP + dots using Pearson ' s coefficient where the dotted lines ( at 0 . 5 ) indicate the limit under which no significant co - localization is measured ( number of cells = 54 ) . Data information : In graphs representing the number of vesicles per cells , each dot displayed corresponds to a single cell . Within the violin plots , continuous and dotted lines correspond to medians and quartiles , respectively . Scale bars : 2μm . CTRL : control . Mann - Whitney ' s test ; * p < 0 . 05 ; * * p < 0 . 002 ; * * * p < 0 . 0003 ; * * * * p < 0 . 0001 ; # p > 0 . 05 .

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

(A) MHC-II and T6BP expressions were assessed using confocal microscopy. HeLa-CIITA cells were transfected with control and T6BP-silencing siRNAs. 48h post-treatment, MHC-II and T6BP were detected using L243 and anti-T6BP antibodies, respectively, and revealed with species specific secondary antibodies. Nuclei were stained using DAPI. (B) Quantitative analysis using in-house ImageJ script displaying distance of each MHC-II+ vesicles to the nucleus and number of vesicles per cell. At least 20,000 vesicles from 160 cells corresponding to five biological replicates were analyzed. Right panel, quantification in the siCTRL cells of the colocalization between MHC-II+ and T6BP+ dots using Pearson's coefficient where the dotted lines (at 0.5) indicate the limit under which no significant co-localization is measured (number of cells = 54). Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.


( C ) As in A , T6BP and LAMP - 1 expressions were analyzed . ( D ) The localization of LAMP - 1 + vesicles and the number of vesicles per cell were quantified as in B . At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed . Data information : In graphs representing the number of vesicles per cells , each dot displayed corresponds to a single cell . Within the violin plots , continuous and dotted lines correspond to medians and quartiles , respectively . Scale bars : 2μm . CTRL : control . Mann - Whitney ' s test ; * p < 0 . 05 ; * * p < 0 . 002 ; * * * p < 0 . 0003 ; * * * * p < 0 . 0001 ; # p > 0 . 05 .

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

(C) As in A, T6BP and LAMP-1 expressions were analyzed. (D) The localization of LAMP-1+ vesicles and the number of vesicles per cell were quantified as in B. At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed. Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.


( E ) As in A , adding Lysotracker staining . ( F ) As in B , quantitative analysis of Lysotracker + vesicles : localization to the nucleus and number of vesicles per cell . Colocalization of Lysotracker + vesicles with MHC - II + puncta was analyzed using JACoP plugin ( scales start at 0 . 5 above which the % of co - localization is considered significant ) , number of vesicles > 2000 from at least three biological replicates were analyzed corresponding to 20 cells . Data information : In graphs representing the number of vesicles per cells , each dot displayed corresponds to a single cell . Within the violin plots , continuous and dotted lines correspond to medians and quartiles , respectively . Scale bars : 2μm . CTRL : control . Mann - Whitney ' s test ; * p < 0 . 05 ; * * p < 0 . 002 ; * * * p < 0 . 0003 ; * * * * p < 0 . 0001 ; # p > 0 . 05 .

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

(E) As in A, adding Lysotracker staining. (F) As in B, quantitative analysis of Lysotracker+ vesicles: localization to the nucleus and number of vesicles per cell. Colocalization of Lysotracker+ vesicles with MHC-II+ puncta was analyzed using JACoP plugin (scales start at 0.5 above which the % of co-localization is considered significant), number of vesicles > 2000 from at least three biological replicates were analyzed corresponding to 20 cells. Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.


( G ) As in E , CD63 and MHC - II expressions were assessed . ( H ) Quantitative analysis as represented in F . At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed . Data information : In graphs representing the number of vesicles per cells , each dot displayed corresponds to a single cell . Within the violin plots , continuous and dotted lines correspond to medians and quartiles , respectively . Scale bars : 2μm . CTRL : control . Mann - Whitney ' s test ; * p < 0 . 05 ; * * p < 0 . 002 ; * * * p < 0 . 0003 ; * * * * p < 0 . 0001 ; # p > 0 . 05 .

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

(G) As in E, CD63 and MHC-II expressions were assessed. (H) Quantitative analysis as represented in F. At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed. Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.


( A ) CD74 expression was assessed using Western Blot . HeLa - CIITA cells were transfected with siCTRL and siT6BP . 48h post treatment , the Iip33 CD74 isoform , a degradation product ( Iip16 ) , T6BP and actin were detected using indicated antibodies . Left panel , a representative Western Blot experiment is shown . Right panel , expressions levels five biological replicates were quantified using ImageJ and are presented as mean of ratios ( + / - SD ) of CD74 to actin used as control housekeeping gene expression .

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

(A) CD74 expression was assessed using Western Blot. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post treatment, the Iip33 CD74 isoform, a degradation product (Iip16), T6BP and actin were detected using indicated antibodies. Left panel, a representative Western Blot experiment is shown. Right panel, expressions levels five biological replicates were quantified using ImageJ and are presented as mean of ratios (+/-SD) of CD74 to actin used as control housekeeping gene expression.


( B ) As in ( A ) assessing HLA - DR and actin expression levels . Top panel , a representative Western Blot experiment is shown . Bottom panel , in three biological replicates , HLA - DR expression was quantified using ImageJ and is presented as a mean of ratios to actin ( + / - SD ) . ( C ) As in ( B ) assessing HLA - DM and actin expression levels . Bottom panel , HLA - DM expression was quantified in three biological replicates and presented as mean of ratios to actin ( + / - SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) As in (A) assessing HLA-DR and actin expression levels. Top panel, a representative Western Blot experiment is shown. Bottom panel, in three biological replicates, HLA-DR expression was quantified using ImageJ and is presented as a mean of ratios to actin (+/-SD). (C) As in (B) assessing HLA-DM and actin expression levels. Bottom panel, HLA-DM expression was quantified in three biological replicates and presented as mean of ratios to actin (+/-SD).


( D ) CD74 expression is partially recovered by blocking lysosomal acidification . As in ( A ) , following siRNA transfection of Hela - CIITA cells , CD74 expression was assessed . The last 16h prior to harvesting , cells were treated with chloroquine ( CQ ) or epoxomicin ( Epo ) . Left panel a representative experiment is shown , membranes were blotted using anti - T6BP , - CD74 and - actin antibodies . The different degradation fragments of CD74 are indicated ( Iip33 , Iip22 and Iip16 ) . Right panel , expression levels of Iip33 ( top ) and Iip16 ( bottom ) were quantified in three biological replicates using ImageJ and are presented as mean of ratios of each fragment to actin ( + / - SD ) . Data information : ( D ) One - way Anova statistical test combined with Bonferronis multiple comparison test was applied . Dotted lines indicate statistically significant differences between conditions .

O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) CD74 expression is partially recovered by blocking lysosomal acidification. As in (A), following siRNA transfection of Hela-CIITA cells, CD74 expression was assessed. The last 16h prior to harvesting, cells were treated with chloroquine (CQ) or epoxomicin (Epo). Left panel a representative experiment is shown, membranes were blotted using anti-T6BP, -CD74 and -actin antibodies. The different degradation fragments of CD74 are indicated (Iip33, Iip22 and Iip16). Right panel, expression levels of Iip33 (top) and Iip16 (bottom) were quantified in three biological replicates using ImageJ and are presented as mean of ratios of each fragment to actin (+/-SD). Data information: (D) One-way Anova statistical test combined with Bonferronis multiple comparison test was applied. Dotted lines indicate statistically significant differences between conditions.


( E Analysis of CD74 proteolysis . HeLa - CIITA cells were transfected with siCTRL and siT6BP . 48h post treatment , cells were pulsed for 30 min with 35S - Met / Cys , washed and chased for 1 , 2 and 4h . HLA - DR / CD74 complexes were first immunoprecipitated using Tü36 antibody ( E ) Samples were boiled and analyzed using SDS - PAGE . The bands corresponding to CD74 isoforms ( Iip41 and Iip33 ) and the cleavage products ( Iip16 ) are indicated . ( E right panel ) Iip16 expression was quantified using ImageJ and presented as a percentage of Iip16 normalized to the highest quantity of Iip16 detected after 1h of chase in the control condition . Results are representative of two biological replicates . Ii : invariant chain ( CD74 ) ; CTRL : control .

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(E Analysis of CD74 proteolysis. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post treatment, cells were pulsed for 30 min with 35S-Met/Cys, washed and chased for 1, 2 and 4h. HLA-DR/CD74 complexes were first immunoprecipitated using Tü36 antibody (E) Samples were boiled and analyzed using SDS-PAGE. The bands corresponding to CD74 isoforms (Iip41 and Iip33) and the cleavage products (Iip16) are indicated. (E right panel) Iip16 expression was quantified using ImageJ and presented as a percentage of Iip16 normalized to the highest quantity of Iip16 detected after 1h of chase in the control condition. Results are representative of two biological replicates. Ii: invariant chain (CD74); CTRL: control.


F ) . Analysis of CD74 proteolysis . HeLa - CIITA cells were transfected with siCTRL and siT6BP . 48h post treatment , cells were pulsed for 30 min with 35S - Met / Cys , washed and chased for 1 , 2 and 4h . HLA - DR / CD74 complexes were immunoprecipitated using VICY1 antibody ( F ) . Samples were boiled and analyzed using SDS - PAGE . The bands corresponding to CD74 isoforms Iip33 ) Results are representative of two biological replicates . Ii : invariant chain ( CD74 ) ; CTRL : control .

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

F). Analysis of CD74 proteolysis. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post treatment, cells were pulsed for 30 min with 35S-Met/Cys, washed and chased for 1, 2 and 4h. HLA-DR/CD74 complexes were immunoprecipitated using VICY1 antibody (F). Samples were boiled and analyzed using SDS-PAGE. The bands corresponding to CD74 isoforms Iip33) Results are representative of two biological replicates. Ii: invariant chain (CD74); CTRL: control.


( A - B ) CANX co - immunoprecipitates with T6BP in model and professional APCs . Endogenous T6BP was immunoprecipitated from HeLa - CIITA , B ( DG - 75 ) and dendritic - like ( KG - 1 , DC ) cells . The input , the flow through ( FT ) and the immunoprecipitation ( IP ) fractions were analyzed by Western blot using the indicated antibodies . Bottom panels , control IPs using the beads without antibody ( IP CTRL ) are presented for each IP . ( B ) As in ( A ) but using anti - CANX Ab for the IP . For A and B , one representative experiment out of three biological replicates is shown .

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(A-B) CANX co-immunoprecipitates with T6BP in model and professional APCs. Endogenous T6BP was immunoprecipitated from HeLa-CIITA, B (DG-75) and dendritic-like (KG-1, DC) cells. The input, the flow through (FT) and the immunoprecipitation (IP) fractions were analyzed by Western blot using the indicated antibodies. Bottom panels, control IPs using the beads without antibody (IP CTRL) are presented for each IP. (B) As in (A) but using anti-CANX Ab for the IP. For A and B, one representative experiment out of three biological replicates is shown.


( C ) CANX interacts with T6BP using proximity ligation assay ( PLA ) . 48h post - transfection with the indicated siRNAs , HeLa - CIITA cells were fixed , stained with anti - T6BP and anti - CANX antibodies and proximity revealed using PLA ( Duolink ) . Nuclei were stained using DAPI . Top panel , two representative fields are shown . Bottom panel , quantitative analysis using ImageJ displaying the number of PLA per cell . The continuous line indicates the mean ( + / - SEM ) . PLA were quantified in at least 130 cells corresponding to three biological replicates . Scale bars : 10μm . CTRL : control ; Mann - Whitney ' s test ; * * * : p < 0 . 0003 .

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(C) CANX interacts with T6BP using proximity ligation assay (PLA). 48h post-transfection with the indicated siRNAs, HeLa-CIITA cells were fixed, stained with anti-T6BP and anti-CANX antibodies and proximity revealed using PLA (Duolink). Nuclei were stained using DAPI. Top panel, two representative fields are shown. Bottom panel, quantitative analysis using ImageJ displaying the number of PLA per cell. The continuous line indicates the mean (+/-SEM). PLA were quantified in at least 130 cells corresponding to three biological replicates. Scale bars: 10μm. CTRL: control; Mann-Whitney's test; ***: p<0.0003.


( D ) Calnexin interacts with T6BP through its cytosolic tail . HeLa - CIITA cells were transfected with a plasmid encoding the GST - tagged transmembrane ( TM ) and cytosolic ( CytoTail ) domains of CANX ( GST - TM - CytoTail - CANX ) or the vector not encoding TM - CytoTail - CANX as control and immunoprecipitated with anti - GST antibodies . The input , FT , the wash and the IP fractions were analyzed by Western blot and revealed using the indicated antibodies . One representative experiment out of three biological replicates is shown .

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

(D) Calnexin interacts with T6BP through its cytosolic tail. HeLa-CIITA cells were transfected with a plasmid encoding the GST-tagged transmembrane (TM) and cytosolic (CytoTail) domains of CANX (GST-TM-CytoTail-CANX) or the vector not encoding TM-CytoTail-CANX as control and immunoprecipitated with anti-GST antibodies. The input, FT, the wash and the IP fractions were analyzed by Western blot and revealed using the indicated antibodies. One representative experiment out of three biological replicates is shown.


( E ) Silencing of T6BP does not influence CANX expression levels while silencing of CANX reduces the level of CD74 expression . HeLa - CIITA cells transfected with the indicated siRNAs and samples analyzed , 48h post transfection , by Western blot with the indicated antibodies . These western blot results are representative of at least three biological replicates .

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

(E) Silencing of T6BP does not influence CANX expression levels while silencing of CANX reduces the level of CD74 expression. HeLa-CIITA cells transfected with the indicated siRNAs and samples analyzed, 48h post transfection, by Western blot with the indicated antibodies. These western blot results are representative of at least three biological replicates.


( a ) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody ( Atg16L1 ) or no antibody ( No Ab ) in control , and subjected to SDS - PAGE ; proteins were stained with SimplyBlue safe stain ( Invitrogen ) in accordance with the manufacturer ' s protocol . Bands indicated in the box were cut out and digested with trypsin , and identified by MALDI - TOF MS . HC , heavy chain .

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(a) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody (Atg16L1) or no antibody (No Ab) in control, and subjected to SDS-PAGE; proteins were stained with SimplyBlue safe stain (Invitrogen) in accordance with the manufacturer's protocol. Bands indicated in the box were cut out and digested with trypsin, and identified by MALDI-TOF MS. HC, heavy chain.


( b ) HeLa cells transfected for 24 h with control vector , or wild - type or different deletion mutants ( residues 2 - 77 , 2 - 275 and 232 - 607 ) of Flag - Atg16L1 were immunoprecipitated with anti - Flag antibody ( Atg16L1 ) and immunoblotted with anti - clathrin , anti - Flag and anti - Atg5 antibodies . Clathrin interacts with the N terminus of Atg16L1 , in a similar manner to Atg12 - Atg5 . Total lysates were run alongside as controls for protein input . FL , full - length .

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(b) HeLa cells transfected for 24 h with control vector, or wild-type or different deletion mutants (residues 2-77, 2-275 and 232-607) of Flag-Atg16L1 were immunoprecipitated with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Clathrin interacts with the N terminus of Atg16L1, in a similar manner to Atg12-Atg5. Total lysates were run alongside as controls for protein input. FL, full-length.


( c ) HeLa cell lysates were immunoprecipitated ( IP ) with Atg16L1 antibody ( + Ab ) or no antibody ( no Ab ) and immunoblotted with anti - clathrin , anti - AP2 and anti - Atg16L1 antibodies . Total cell lysates ( TL ) were also blotted for input controls . Endogenous Atg16L1 interacts with clathrin heavy chain and AP2 .

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

(c) HeLa cell lysates were immunoprecipitated (IP) with Atg16L1 antibody (+Ab) or no antibody (no Ab) and immunoblotted with anti-clathrin, anti-AP2 and anti-Atg16L1 antibodies. Total cell lysates (TL) were also blotted for input controls. Endogenous Atg16L1 interacts with clathrin heavy chain and AP2.


( d ) HeLa cells transfected for 24 h with Flag - Atg16L1 and increasing concentrations of Atg5 were immunoprecipitated ( IP ) with anti - Flag antibody ( Atg16L1 ) and immunoblotted with anti - clathrin , anti - Flag and anti - Atg5 antibodies . Atg5 overexpression neither interferes with nor increases clathrin - Atg16L1 binding . Uncropped images of blots are shown in Supplementary Information , Fig . S9 .

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

(d) HeLa cells transfected for 24 h with Flag-Atg16L1 and increasing concentrations of Atg5 were immunoprecipitated (IP) with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Atg5 overexpression neither interferes with nor increases clathrin-Atg16L1 binding. Uncropped images of blots are shown in Supplementary Information, Fig. S9.


( a ) HeLa cells transfected for 5 days with two rounds of control , epsin 1 ( eps . ) , clathrin heavy chain ( cla . ) , AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 ( + / - BafA1 ) , after which they were lysed for western blot analysis with anti - LC3 and anti - tubulin antibodies . Note that LC3 - I is often very faint compared with LC3 - II in HeLa cells under the protein extraction conditions we use . However , this is not a problem because it is advisable to relate LC3 - II to tubulin / actin .

O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) HeLa cells transfected for 5 days with two rounds of control, epsin 1 (eps.), clathrin heavy chain (cla.), AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 (+/- BafA1), after which they were lysed for western blot analysis with anti-LC3 and anti-tubulin antibodies. Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we use. However, this is not a problem because it is advisable to relate LC3-II to tubulin/actin.


( b ) Ratio of LC3 - II to tubulin on knockdown ( KD ) of control , epsin , clathrin heavy chain , AP2 or AP1 , with or without bafilomycin A1 , quantified from three independent experiments ( two for epsin ) and represented in the graph . Asterisk , P 0 . 01 ; two asterisks , P 0 . 001 ; three asterisks , P 0 . 0001 .

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(b) Ratio of LC3-II to tubulin on knockdown (KD) of control, epsin, clathrin heavy chain, AP2 or AP1, with or without bafilomycin A1, quantified from three independent experiments (two for epsin) and represented in the graph. Asterisk, P 0.01; two asterisks, P 0.001; three asterisks, P 0.0001.


( c ) HeLa cells stably expressing GFP - mRFP - LC3 ( ref . 17 ) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA , during which they were either left untreated ( UT ) or treated with bafilomycin A1 ( BafA1 ) for the last 15 h . Cells were then fixed and analysed on a Cellomics ArrayScan system . Quantification of autophagic vacuoles ( AV ) or autolysosomes ( AL ) per cell in the different conditions is shown in the graph . Asterisk , P 0 . 01 ; n . s . , not significant . n = 2 , 000 cells .

O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) HeLa cells stably expressing GFP-mRFP-LC3 (ref. 17) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA, during which they were either left untreated (UT) or treated with bafilomycin A1 (BafA1) for the last 15 h. Cells were then fixed and analysed on a Cellomics ArrayScan system. Quantification of autophagic vacuoles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph. Asterisk, P 0.01; n.s., not significant. n = 2,000 cells.


( d ) HeLa cells transfected for 4 days with control , clathrin heavy chain , AP1 or AP2 siRNA were grown in Hanks balanced salt solution ( HBSS ) for 6 h , after which the cells were fixed and immunostained for endogenous LC3 . The percentages of HeLa cells with more than 50 LC3 vesicles ( marked with arrows ) are quantified in the graph . End . , endogenous . Three asterisks , P 0 . 0001 . n = 500 cells . All error bars represent s . e . m . Scale bars , 10 μm . Uncropped images of blots are shown in Supplementary Information , Fig . S9 .

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(d) HeLa cells transfected for 4 days with control, clathrin heavy chain, AP1 or AP2 siRNA were grown in Hanks balanced salt solution (HBSS) for 6 h, after which the cells were fixed and immunostained for endogenous LC3. The percentages of HeLa cells with more than 50 LC3 vesicles (marked with arrows) are quantified in the graph. End., endogenous. Three asterisks, P 0.0001. n = 500 cells. All error bars represent s.e.m. Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S9.


( a - d ) HeLa cells transfected for 72 h with control , epsin 1 , clathrin heavy chain , AP2 or AP1 siRNA were treated with HBSS ( to induce autophagy ) for 6 h , after which they were fixed and immunostained for endogenous Atg16L1 . Representative cells for the control and epsin 1 siRNA are in a , and percentages of HeLa cells with Atg16L1vesicles are quantified in b ( for epsin 1 ) , c ( for clathrin ) and d ( for AP1 and AP2 ) . Three asterisks , P 0 . 0001 . Scale bars , 10 μm . ( Note that we used single siRNAs for all experiments and the effects were confirmed with two independent sequences for clathrin heavy chain ) . n = 600 cells .

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(a-d) HeLa cells transfected for 72 h with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA were treated with HBSS (to induce autophagy) for 6 h, after which they were fixed and immunostained for endogenous Atg16L1. Representative cells for the control and epsin 1 siRNA are in a, and percentages of HeLa cells with Atg16L1vesicles are quantified in b (for epsin 1), c (for clathrin) and d (for AP1 and AP2). Three asterisks, P 0.0001. Scale bars, 10 μm. (Note that we used single siRNAs for all experiments and the effects were confirmed with two independent sequences for clathrin heavy chain). n = 600 cells.


( e , f ) HeLa cells transfected for 24 h with Flag - tagged wild - type Atg16L1 or the Atg16L1 deletion mutants 2 - 275 Atg16L1 or 232 - 607 Atg16L1 were fixed and immunostained with anti - Flag antibody ( e ) ; the proportions of cells with Flag - Atg16L1 vesicles are represented in f . Representative images of Atg16L1 vesicles with the different mutant constructs are shown in e . The large boxes in each panel are higher magnifications of regions in the smaller boxes . Three asterisks , P 0 . 0001 ; n . s . , not significant . n = 100 cells .

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(e, f) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2-275 Atg16L1 or 232-607 Atg16L1 were fixed and immunostained with anti-Flag antibody (e); the proportions of cells with Flag-Atg16L1 vesicles are represented in f. Representative images of Atg16L1 vesicles with the different mutant constructs are shown in e. The large boxes in each panel are higher magnifications of regions in the smaller boxes. Three asterisks, P 0.0001; n.s., not significant. n = 100 cells.


( g - i ) HeLa cells transfected with control , epsin 1 , clathrin heavy chain , AP2 or AP1 siRNA , as above , were transfected with GFP - Atg16L1 and tomato LC3 for a further 24 h , after which the cells were fixed . g shows representative cells for control , epsin 1 and clathrin siRNA . The percentage of GFP - Atg16L1 ( green ) that co - localized ( yellow , marked by arrows ) with tomato LC3 vesicles ( red ) is quantified as shown in the graph in h ( control , epsin 1 and clathrin siRNA ) and i ( control , AP2 and AP1 siRNA ) . Three asterisks , P 0 . 0001 . n = 21 cells . Scale bars , 10 μm . All error bars represent s . e . m .

(g-i) HeLa cells transfected with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA, as above, were transfected with GFP-Atg16L1 and tomato LC3 for a further 24 h, after which the cells were fixed. g shows representative cells for control, epsin 1 and clathrin siRNA. The percentage of GFP-Atg16L1 (green) that co-localized (yellow, marked by arrows) with tomato LC3 vesicles (red) is quantified as shown in the graph in h (control, epsin 1 and clathrin siRNA) and i (control, AP2 and AP1 siRNA). Three asterisks, P 0.0001. n = 21 cells. Scale bars, 10 μm. All error bars represent s.e.m.


( a ) HeLa cells transfected for 24 h with GFP - Atg16L1 were fixed to reveal the Atg16L1 vesicles ( green ) . Scale bar , 10 μm .

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(a) HeLa cells transfected for 24 h with GFP-Atg16L1 were fixed to reveal the Atg16L1 vesicles (green). Scale bar, 10 μm.


( b ) HeLa cells transiently transfected for 20 h with GFP - Atg16L1 and mRFP - GPi were cultured in starvation medium for a further 2 h . TIRF image with GFP - Atg16L1 ( green ) and mRFP - GPi ( red ) is shown . Arrows indicate co - localization between Atg16L1 and GPi . The percentage co - localization of GFP - Atg16L1 with mRFP - GPi is shown in the graph . n = 2 cells . Scale bar , 5 μm .

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(b) HeLa cells transiently transfected for 20 h with GFP-Atg16L1 and mRFP-GPi were cultured in starvation medium for a further 2 h. TIRF image with GFP-Atg16L1 (green) and mRFP-GPi (red) is shown. Arrows indicate co-localization between Atg16L1 and GPi. The percentage co-localization of GFP-Atg16L1 with mRFP-GPi is shown in the graph. n = 2 cells. Scale bar, 5 μm.


( c ) HeLa cells transfected for 24 h with GFP - Atg16L1 were either left untreated or treated with 50 μM dynasore ( Sigma ) for 4 h and processed for immunogold electron microscopy with anti - GFP antibodies ( 15 - nm gold particles ) and anti - clathrin antibodies ( 10 - nm gold particles ) . Co - localization can be seen in the boxed areas . Quantification of clathrin - coated structures ( CCS ) that were associated with Atg16L1 per 1 , 000 μm2 is shown in the graph . Three asterisks , P 0 . 0001 . Scale bar , 100 nm . All error bars represent s . e . m .

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(c) HeLa cells transfected for 24 h with GFP-Atg16L1 were either left untreated or treated with 50 μM dynasore (Sigma) for 4 h and processed for immunogold electron microscopy with anti-GFP antibodies (15-nm gold particles) and anti-clathrin antibodies (10-nm gold particles). Co-localization can be seen in the boxed areas. Quantification of clathrin-coated structures (CCS) that were associated with Atg16L1 per 1,000 μm2 is shown in the graph. Three asterisks, P 0.0001. Scale bar, 100 nm. All error bars represent s.e.m.


( a ) HeLa cells transfected for 24 h with GFP - Atg16L1 ( green ) and CFP - LC3 ( blue ) were incubated with Alexa Fluor - 555 - conjugated cholera toxin subunit B ( red ) for 15 min at 4 ° C ( allowing toxin to bind to the plasma membrane ) . Then cells were incubated at 37 ° C ( allowing internalization of cholera toxin ) for 10 min and fixed for confocal analysis . Vesicles positive for both Atg16L1 and cholera toxin are yellow ( also see high - magnification images at the right ) . Note that the small Atg16L1 vesicles co - localizing with cholera toxin are negative for LC3 ( as marked with yellow arrows ) , and the Atg16L1 vesicles co - localizing with both cholera toxin and LC3 ( marked with blue arrows ) are shown in the magnified panels at the right . Scale bar , 10 μm .

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

(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) and CFP-LC3 (blue) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) for 15 min at 4 °C (allowing toxin to bind to the plasma membrane). Then cells were incubated at 37 °C (allowing internalization of cholera toxin) for 10 min and fixed for confocal analysis. Vesicles positive for both Atg16L1 and cholera toxin are yellow (also see high-magnification images at the right). Note that the small Atg16L1 vesicles co-localizing with cholera toxin are negative for LC3 (as marked with yellow arrows), and the Atg16L1 vesicles co-localizing with both cholera toxin and LC3 (marked with blue arrows) are shown in the magnified panels at the right. Scale bar, 10 μm.


( b ) HeLa cells transfected for 24 h with GFP - Atg16L1 ( green ) were incubated with Alexa Fluor - 555 - conjugated cholera toxin subunit B ( red ) as in a , after which they were fixed , immunostained for endogenous ( end . ) EEA1 ( blue ) and analysed by confocal microscopy . Vesicles positive for Atg16L1 and cholera toxin B are yellow ( white arrows ) and vesicles positive for both EEA1 and cholera toxin are purple ( blue arrows ) and are shown in the high - magnification images in the middle . The graph shows percentage co - localization . Cho . tox . , cholera toxin . n = 20 cells . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) as in a, after which they were fixed, immunostained for endogenous (end.) EEA1 (blue) and analysed by confocal microscopy. Vesicles positive for Atg16L1 and cholera toxin B are yellow (white arrows) and vesicles positive for both EEA1 and cholera toxin are purple (blue arrows) and are shown in the high-magnification images in the middle. The graph shows percentage co-localization. Cho. tox., cholera toxin. n = 20 cells. Scale bar, 10 μm.


( c ) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor - 555 - labelled cholera toxin subunit B ( red ) as in a . Cells were then fixed , and immunostained for endogenous Atg16L1 ( green ) and endogenous EEA1 ( blue ) . Cells were analysed as in b ; the graph shows quantification of the results . Scale bar , 10 μm . Arrows indicate Atg16L1 - cholera toxin co - localization ; arrowheads indicate EEA1 - cholera toxin co - localization . n = 20 cells .

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

(c) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed, and immunostained for endogenous Atg16L1 (green) and endogenous EEA1 (blue). Cells were analysed as in b; the graph shows quantification of the results. Scale bar, 10 μm. Arrows indicate Atg16L1-cholera toxin co-localization; arrowheads indicate EEA1-cholera toxin co-localization. n = 20 cells.


( d ) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor - 555 - labelled cholera toxin subunit B ( red ) as in a . Cells were then fixed and immunostained for endogenous Atg5 ( green ) or endogenous Atg12 ( green ) . Co - localization of the Atg5 ( n = 18 cells ) or Atg12 vesicles ( n = 39 cells ) with cholera toxin is shown in yellow ( arrows ) and is quantified in the graph . Scale bar , 10 μm . All error bars represent s . e . m .

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

(d) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed and immunostained for endogenous Atg5 (green) or endogenous Atg12 (green). Co-localization of the Atg5 (n = 18 cells) or Atg12 vesicles (n = 39 cells) with cholera toxin is shown in yellow (arrows) and is quantified in the graph. Scale bar, 10 μm. All error bars represent s.e.m.


( a ) HeLa cells transfected for 24 h with Flag - tagged wild - type Atg16L1 or the Atg16L1 deletion mutants 2 - 275 or 232 - 607 Atg16L1 , together with GFP - Atg5 and CFP - LC3 , were fixed and immunostained with anti - Flag antibody before confocal analysis . Co - localization of wild - type or 2 - 275 Atg16L1 ( red ) with GFP - Atg5 ( green ) is marked by arrows in the insets . Scale bars , 10 μm .

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

(a) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2-275 or 232-607 Atg16L1, together with GFP-Atg5 and CFP-LC3, were fixed and immunostained with anti-Flag antibody before confocal analysis. Co-localization of wild-type or 2-275 Atg16L1 (red) with GFP-Atg5 (green) is marked by arrows in the insets. Scale bars, 10 μm.


( b ) HeLa cells transfected for 24 h with GFP - Atg16L1 were incubated for 15 min with horseradish peroxidase ( HRP ) - conjugated cholera toxin subunit B at 4 ° C . The cells were then incubated for 10 min at 37 ° C , after which they were processed for double immunogold labelling with anti - HRP antibodies ( 15 - nm gold particles ) and anti - GFP antibodies ( 10 - nm gold particles ) for electron microscopy . PM , plasma membrane . Scale bar , 100 nm . Co - localization can be seen in boxed areas . n = 49 fields . All error bars represent s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells transfected for 24 h with GFP-Atg16L1 were incubated for 15 min with horseradish peroxidase (HRP)-conjugated cholera toxin subunit B at 4 °C. The cells were then incubated for 10 min at 37 °C, after which they were processed for double immunogold labelling with anti-HRP antibodies (15-nm gold particles) and anti-GFP antibodies (10-nm gold particles) for electron microscopy. PM, plasma membrane. Scale bar, 100 nm. Co-localization can be seen in boxed areas. n = 49 fields. All error bars represent s.e.m.


( a ) HeLa cells transfected for 24 h with GFP - Atg16L1 ( green ) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 ° C , after which they were imaged immediately ( in an incubated chamber at 37 ° C ) . A time series after fusion of a CellMask Orange vesicle ( red ) with a GFP - Atg16L1 vesicle ( green ) is shown . Scale bar , 5 μm . A higher - magnification image showing co - localization ( yellow ) of GFP - Atg16L1 vesicle ( green ) with CellMask Orange - positive vesicle ( red ) , indicated by arrows , is also shown in the top and bottom panels .

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

(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 °C, after which they were imaged immediately (in an incubated chamber at 37 °C). A time series after fusion of a CellMask Orange vesicle (red) with a GFP-Atg16L1 vesicle (green) is shown. Scale bar, 5 μm. A higher-magnification image showing co-localization (yellow) of GFP-Atg16L1 vesicle (green) with CellMask Orange-positive vesicle (red), indicated by arrows, is also shown in the top and bottom panels.


( b , c ) HeLa cells transfected with control , clathrin heavy chain , AP1 or AP2 siRNA for 72 h were subsequently transfected with GFP - Atg16L1 ( green ) together with the siRNA for the next 24 h . The cells were then incubated for 15 min with Alexa Fluor - 555 - conjugated cholera toxin subunit B ( red ) at 4 ° C , followed by further incubation for 10 min at 37 ° C , after which the cells were fixed for confocal analysis ( b ) . The co - localization ( yellow ) of GFP - Atg16L1 ( green ) with cholera toxin ( red ) in control or clathrin heavy - chain knockdown is shown and is quantified in the graphs in c . n = 25 cells . Three asterisks , P = 0 . 0002 for clathrin heavy - chain knockdown , and P 0 . 0001 for AP2 knockdown . All error bars represent s . e . m . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, c) HeLa cells transfected with control, clathrin heavy chain, AP1 or AP2 siRNA for 72 h were subsequently transfected with GFP-Atg16L1 (green) together with the siRNA for the next 24 h. The cells were then incubated for 15 min with Alexa Fluor-555-conjugated cholera toxin subunit B (red) at 4 °C, followed by further incubation for 10 min at 37 °C, after which the cells were fixed for confocal analysis (b). The co-localization (yellow) of GFP-Atg16L1 (green) with cholera toxin (red) in control or clathrin heavy-chain knockdown is shown and is quantified in the graphs in c. n = 25 cells. Three asterisks, P = 0.0002 for clathrin heavy-chain knockdown, and P 0.0001 for AP2 knockdown. All error bars represent s.e.m. Scale bar, 5 μm.


( a ) HeLa cells transfected with two rounds of control , clathrin heavy chain or AP2 siRNA for 4 days were collected for immunoprecipitation ( IP ) with anti - Atg16L1 antibody . Western blot for total lysate ( bottom ) and IP ( top ) were performed with anti - Atg16L1 and anti - clathrin antibodies . Note the decrease in Atg16L1 - clathrin interaction with AP2 knockdown . The graph shows quantification of the Atg16L1 - clathrin or Atg16L1 - AP2 with knockdown of control , clathrin heavy chain or AP2 from two independent experiments .

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

(a) HeLa cells transfected with two rounds of control, clathrin heavy chain or AP2 siRNA for 4 days were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot for total lysate (bottom) and IP (top) were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the decrease in Atg16L1-clathrin interaction with AP2 knockdown. The graph shows quantification of the Atg16L1-clathrin or Atg16L1-AP2 with knockdown of control, clathrin heavy chain or AP2 from two independent experiments.


( b ) HeLa cells transfected for 24 h with Flag - tagged wild - type Atg16L1 , GFP - PLC ( PH ) together with empty vector ( control ) or dominant - negative dynamin II mutant ( K44A dynamin ; DN - Dyn ) were fixed and analysed by confocal microscopy . Note the co - localization of Atg16L1 with PLC ( PH ) at the plasma membrane ; this is quantified in the graph at the right . Asterisk , P 0 . 01 . n = 20 cells . Scale bars , 5 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole .

(b) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1, GFP-PLC(PH) together with empty vector (control) or dominant-negative dynamin II mutant (K44A dynamin; DN-Dyn) were fixed and analysed by confocal microscopy. Note the co-localization of Atg16L1 with PLC(PH) at the plasma membrane; this is quantified in the graph at the right. Asterisk, P 0.01. n = 20 cells. Scale bars, 5 μm. DAPI, 4′,6-diamidino-2-phenylindole.


( c ) HeLa cells treated for 5 h with HBSS alone or with HBSS containing 80 μM dynasore were collected for immunoprecipitation ( IP ) with anti - Atg16L1 antibody . Western blot analyses for total lysate ( TL ) and IP were performed with anti - Atg16L1 and anti - clathrin antibodies . Note the strong Atg16L1 - clathrin interaction with dynasore treatment . The graph shows quantification from two independent experiments . All error bars represent s . e . m .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(c) HeLa cells treated for 5 h with HBSS alone or with HBSS containing 80 μM dynasore were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot analyses for total lysate (TL) and IP were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the strong Atg16L1-clathrin interaction with dynasore treatment. The graph shows quantification from two independent experiments. All error bars represent s.e.m.


( a ) Confocal micrographs of HeLa cells expressing yellow fluorescent protein ( YFP ) fused to Nap1 , Sintbad or Tank and infected with S . Typhimurium , stained for ubiquitin ( Ub ) 2 h after infection . At least 100 cells were analyzed . DAPI , 4 , 6 - diamidino - 2 - phenylindole . Scale bar , 10 μm ; arrowheads , bacteria shown in insets .

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

(a) Confocal micrographs of HeLa cells expressing yellow fluorescent protein (YFP) fused to Nap1, Sintbad or Tank and infected with S. Typhimurium, stained for ubiquitin (Ub) 2 h after infection. At least 100 cells were analyzed. DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 10 μm; arrowheads, bacteria shown in insets.


( b - e ) Normalized ratio between luciferase activity bound to beads and present in lysates . The indicated purified GST fusion proteins coupled to beads were incubated with lysates of 293ET cells expressing Nap1 , Sintbad or Tank , each fused to luciferase ( b )

O O O O O O O O O O O O O O O O O O O O O O O O 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-e) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with lysates of 293ET cells expressing Nap1, Sintbad or Tank, each fused to luciferase (b)


( c ) , lysates of 293ET cells expressing the indicated Nap1 mutants fused to

(c), lysates of 293ET cells expressing the indicated Nap1 mutants fused to luciferase


( d ) , lysates of E . coli expressing Nap1 ( 1 - 85 ) fused to

(d), lysates of E. coli expressing Nap1(1-85) fused to luciferase


( e ) lysates of 293ET cells expressing Nap1 , NEMO or Optineurin fused to

(e) lysates of 293ET cells expressing Nap1, NEMO or Optineurin fused to luciferase


( a ) Flowthrough from nickel - agarose column , separated by SDS - PAGE and stained with Coomassie blue .

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

(a) Flowthrough from nickel-agarose column, separated by SDS-PAGE and stained with Coomassie blue.


( c , d ) Normalized ratio between luciferase activity bound to beads and present in lysates . The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1 ( 1 - 85 ) fused to luciferase . Reactions were complemented with lysates from 293ET cells that had been depleted with the indicated ( α - ) antibodies ( c , top ) . Immunoblot of lysates after depletion ( c , bottom ) .

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

(c,d) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1(1-85) fused to luciferase. Reactions were complemented with lysates from 293ET cells that had been depleted with the indicated (α-) antibodies (c, top). Immunoblot of lysates after depletion (c, bottom).


( c , d ) Normalized ratio between luciferase activity bound to beads and present in lysates . The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1 ( 1 - 85 ) fused to luciferase . Reactions were complemented with the indicated amounts of lysates from E . coli cultures expressing myelin basic protein ( MBP ) fused to NDP52 ( d ) .

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

(c,d) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1(1-85) fused to luciferase.Reactions were complemented with the indicated amounts of lysates from E. coli cultures expressing myelin basic protein (MBP) fused to NDP52 (d).


( e ) Lysates of 293ET cells were immunoprecipitated ( IP ) using the indicated antibodies or an isotype control . Lysates and precipitates were blotted ( IB ) for the presence of TBK1 , IKKε and IKKβ .

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

(e) Lysates of 293ET cells were immunoprecipitated (IP) using the indicated antibodies or an isotype control. Lysates and precipitates were blotted (IB) for the presence of TBK1, IKKε and IKKβ.


( f ) Lysates of 293ET cells were immunoprecipitated using the indicated antibodies or an isotype control . Precipitates were blotted for the presence of TBK1 and NDP52 . Fusion proteins used in Figure 2 are further characterized in Supplementary Figure 1 . Data in panels c - f are representative of at least two independent experiments . Ub , ubiquitin ; PCNA , proliferating cell nuclear antigen .

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

(f) Lysates of 293ET cells were immunoprecipitated using the indicated antibodies or an isotype control. Precipitates were blotted for the presence of TBK1 and NDP52. Fusion proteins used in Figure 2 are further characterized in Supplementary Figure 1. Data in panels c-f are representative of at least two independent experiments. Ub, ubiquitin; PCNA, proliferating cell nuclear antigen.


( a ) Confocal micrographs of HeLa cells 2 h after infection with S . Typhimurium , stained with antibodies to Cocoa , NDP52 and ubiquitin ( Ub ) . ( b ) Colocalization , at the indicated time points after infection , of ubiquitin - positive S . Typhimurium with NDP52 or Cocoa .

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

(a) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to Cocoa, NDP52 and ubiquitin (Ub). (b) Colocalization, at the indicated time points after infection, of ubiquitin-positive S. Typhimurium with NDP52 or Cocoa.


( c ) Confocal micrographs of HeLa cells 2 h after infection with S . Typhimurium , stained with antibodies to NDP52 and LAMP1 . ( d ) Colocalization , at the indicated time points after infection , of NDP52 - positive S . Typhimurium with LAMP1 . DAPI , 4 , 6 - diamidino - 2 - phenylindole . All data are representative of at least two independent experiments . Error bars , s . d . ; three coverslips per experiment , 50 marker - positive cells evaluated per coverslip . Scale bars , 10 μm ; arrowheads , bacteria shown in insets .

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

(c) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to NDP52 and LAMP1. (d) Colocalization, at the indicated time points after infection, of NDP52-positive S. Typhimurium with LAMP1. DAPI, 4,6-diamidino-2-phenylindole. All data are representative of at least two independent experiments. Error bars, s.d.; three coverslips per experiment, 50 marker-positive cells evaluated per coverslip. Scale bars, 10 μm; arrowheads, bacteria shown in insets.