following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : Spinning disc confocal live imaging of Alexa 555 - EGF ( 555 - EGF ) shown as vesicle tracking with time represented as a colour spectrum . Tracking started 5 min after addition of 20 ng / mL 555 - EGF for indicated durations . Scale bar : 10 μm .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O O O B-MEASURED_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 B-CONTROLLED_VAR O O O O O O O O O O

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Spinning disc confocal live cell imaging of Alexa 555-EGF (555-EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/mL 555-EGF for the indicated durations. Scale bar: 10 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . B : Immunofluorescence of EGFR following 5 or 15 min stimulation with 20 ng / mL EGF . Scale bar : 20 μm .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). B: Immunofluorescence staining of EGFR following 5 or 15 min stimulation with 20 ng/mL EGF. Scale bar: 20 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : of EGFR vesicles in a perinuclear region ( within 30 μm diameter of centre of nucleus ) Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Quantification of EGFR vesicles in a perinuclear region (within 30 μm diameter of the centre of the nucleus) Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . D : were stimulated with 20 ng / mL 555 - EGF for 15 or 30 min before immunofluorescence against EGFR . Scale bar : 10 μm .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Cells were stimulated with 20 ng/mL 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : of of total EGFR vesicles that colocalise with 555 - EGF Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Quantification of percentage of total EGFR vesicles that colocalise with 555-EGF Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . F : stably expressing mCherry - Rab5 were stimulated with 20 ng / mL EGF for indicated times before immunofluorescence against EGFR . Scale bar : 10 μm .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). F: Cells stably expressing mCherry-Rab5 were stimulated with 20 ng/mL EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . G : Pearson ' s colocalisation coefficient between mCherry - Rab5 EGFR Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). G: Pearson's colocalisation coefficient between mCherry-Rab5 and EGFR Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . A : Control , sgAtg7 , or sgAtg16l1 were treated with 2 ng / mL EGF for 15 min . were then processed for using anti - EEA1 antibodies PI ( 3 ) P probe ( Alexa 488 - labelled 2XFYVE domains ) . To ensure specificity of probe , control were pre - treated with 5 mM 3 ' MA for 30 min . Scale bar : 10 μm . B : of PI ( 3 ) P + label per ( in A ) . C : Pearson ' s colocalisation coefficient between PI ( 3 ) P EEA1 ( in A ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. A: Control, sgAtg7, or sgAtg16l1 cells were treated with 2 ng/mL EGF for 15 min. Cells were then processed for staining using anti-EEA1 antibodies and PI(3)P probe (Alexa 488-labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm. B: Quantification of PI(3)P+ label intensity per cell (in A). C: Pearson's colocalisation coefficient between PI(3)P and EEA1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . D : Control sgAtg7 were stimulated 20 ng / mL EGF for 5 or 15 min before immunofluorescence against EEA1 EGFR . Scale bar : 10 μm . E : Pearson ' s colocalisation coefficient between EEA1 EGFR ( in D ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. D: Control and sgAtg7 cells were stimulated 20 ng/mL EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR. Scale bar: 10 μm. E: Pearson's colocalisation coefficient between EEA1 and EGFR (in D). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . F : Endogenous VPS34 was immunoprecipitated from control sgAtg7 that were treated with 2 ng / mL EGF for 15 min then lysed in CHAPS - containing detergent buffer binding partners detected by western blotting . G : Densitometry analyses of coimmunoprecipitated with endogenous VPS34 ( in F ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. F: Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng/mL EGF for 15 min then lysed in CHAPS-containing detergent buffer and binding partners detected by western blotting. G: Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : transiently expressing YFP - Galectins ( YFP - Gal3 , - Gal8 , or - Gal9 ) were stimulated with 2 ng / mL EGF for 15 min before fixation immunofluorescence against EEA1 . Scale bar : 10 μm . B : of of total EEA1 vesicles that colocalise with YFP - Galectins ( in A ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells transiently expressing YFP-Galectins (YFP-Gal3, -Gal8, or -Gal9) were stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Galectins (in A).Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : transiently expressing YFP - Gal8 were stimulated with 20 ng / mL Alexa 555 - EGF ( 555 - EGF ) for 15 min . Scale bar : 10 μm . D : of of YFP - Gal8 - labelled vesicles that colocalise with 555 - EGF in control or ATG7 - deficient ( in C ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells transiently expressing YFP-Gal8 were stimulated with 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min. Scale bar: 10 μm. D: Quantification of the percentage of YFP-Gal8-labelled vesicles that colocalise with 555-EGF in control or ATG7-deficient cells (in C). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : transiently expressing YFP - Gal8 were treated 100 μM monensin for 1 hr stimulated with 2 ng / mL EGF for 15 min before fixation immunofluorescence against EEA1 . Scale bar : 10 μm . F : of of total EEA1 vesicles that colocalise with YFP - Gal8 upon monensin treatment ( in E ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Cells transiently expressing YFP-Gal8 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. F: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Gal8 upon monensin treatment (in E). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : stably expressing GFP - LC3 were treated with 100 μM monensin in presence or absence of bafilomycin A1 ( 20 nM ) for 1 hr stimulated with 2 ng / mL EGF for 15 min before fixation immunofluorescence against EEA1 . arrows indicate colocalisation . Scale bar : 10 μm . B : of of total EEA1 vesicles that colocalise with GFP - LC3 ( in A ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells stably expressing GFP-LC3 were treated with 100 μM monensin in the presence or absence of bafilomycin A1 (20 nM) for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. White arrows indicate colocalisation. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with GFP-LC3 (in A). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : were treated for 1 hr with 100 μM monensin then stimulated with 2 ng / mL EGF for 15 min . LAMP2 EEA1 were then detected by immunofluorescence . arrows indicate colocalisation . Scale bar : 10 μm . D : of of total EEA1 vesicles that colocalise with LAMP2 ( in C ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells were treated for 1 hr with 100 μM monensin then stimulated with 2 ng/mL EGF for 15 min. LAMP2 and EEA1 were then detected by immunofluorescence staining. White arrows indicate colocalisation. Scale bar: 10 μm. D: Quantification of the percentage of total EEA1 vesicles that colocalise with LAMP2 (in C). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : were treated with 2 ng / mL EGF for 15 min before immunofluorescence against endogenous ATG16L1 EEA1 . Scale bar : 10 μm . F : of of total EEA1 that colocalise with ATG16L1 ( in E ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Cells were treated with 2 ng/mL EGF for 15 min before immunofluorescence against endogenous ATG16L1 and EEA1. Scale bar: 10 μm. F: Quantification of percentage of total EEA1 puncta that colocalise with ATG16L1 (in E). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . G : stably expressing Flag - S - ATG16L1 were treated for 1 hr with either 100 μM monensin or 30 μM Dynasore , then stimulated with 2 ng / mL EGF for 15 min . were then stained by immunofluorescence against EEA1 Flag tag . Scale bar : 10 μm . H : of of total EEA1 vesicles that colocalise with Flag - S - ATG16L1 ( in G ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). G: Cells stably expressing Flag-S-ATG16L1 were treated for 1 hr with either 100 μM monensin or 30 μM Dynasore, then stimulated with 2 ng/mL EGF for 15 min. Cells were then stained by immunofluorescence against EEA1 and Flag tag. Scale bar: 10 μm. H: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 (in G). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . I : Untreated control or sgAtg7 , or control pre - treated with 100 μM monensin 1 hr , stably expressing Flag - S - ATG16L1 were stimulated 20 ng / mL Alexa 555 - EGF ( 555 - EGF ) for 15 min before fixation immunofluorescence against Flag tag EEA1 . were then imaged by structured illumination microscopy ( SIM ) were reconstructed in Nikon Elements software . Scale bar : 10 μm . J : of of EGF - EEA1 colocalised vesicles that stained triple - positive with ATG16L1 by SIM ( in I ) . Due to low throughput nature of this , following numbers were counted : control untreated ( 9 ) , sgAtg7 # 1 ( 10 ) , sgAtg7 # 2 ( 9 ) , control + monensin : ( 11 ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O B-MEASURED_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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). I: Untreated control or sgAtg7 cells, or control cells pre-treated with 100 μM monensin 1 hr, stably expressing Flag-S-ATG16L1 were stimulated 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min before fixation and immunofluorescence staining against Flag tag and EEA1. Cells were then imaged by structured illumination microscopy (SIM) and images were reconstructed in Nikon Elements software. Scale bar: 10 μm. J: Quantification of the percentage of EGF-EEA1 colocalised vesicles that stained triple-positive with ATG16L1 by SIM (in I). Due to the low throughput nature of this assay, the following cell numbers were counted: control untreated (9 cells), sgAtg7#1 (10 cells), sgAtg7#2 (9 cells), and control + monensin: (11 cells). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . K : were treated with 2 ng / mL EGF for 15 min before immunofluorescence against endogenous WIPI2 EEA1 . Scale bar : 10 μm . L : of of total EEA1 that colocalise with WIPI2 ( in K ) . Data information : arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). K: Cells were treated with 2 ng/mL EGF for 15 min before immunofluorescence against endogenous WIPI2 and EEA1. Scale bar: 10 μm. L: Quantification of percentage of total EEA1 puncta that colocalise with WIPI2 (in K). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : were treated for 15 min with 2 ng / mL EGF before fixation immunofluorescence against EEA1 p - TBK1 . arrows indicate colocalisation . Scale bar : 10 μm . B : of of total EEA1 vesicles that colocalise with p - TBK1 ( in A ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells were treated for 15 min with 2 ng/mL EGF before fixation and immunofluorescence staining against EEA1 and p-TBK1. White arrows indicate colocalisation. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with p-TBK1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : stably expressing Flag - S - ATG16L1 were pre - treated for 1 hr with TBK1 inhibitors ( 100 μM MRT68601 or 5 μM momelotinib ) . Control were also treated 100 μM monensin for 1 hr as indicated . were stimulated for 15 min with 2 ng / mL EGF followed by fixation immunofluorescence against Flag tag EEA1 . Scale bar : 10 μm .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells stably expressing Flag-S-ATG16L1 were pre-treated for 1 hr with TBK1 inhibitors (100 μM MRT68601 or 5 μM momelotinib). Control cells were also treated 100 μM monensin for 1 hr as indicated. All cells were stimulated for 15 min with 2 ng/mL EGF followed by fixation and immunofluorescence staining against Flag tag and EEA1. Scale bar: 10 μm.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . D : of total EEA1 vesicles that colocalise with Flag - S - ATG16L1 Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Quantification of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : Western blotting of shNf - 1 / shTp53 glial expressing gRNA sequences targeting Gal8 .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Western blotting of shNf-1/shTp53 glial cells expressing gRNA sequences targeting Gal8.


following experiments were performed in glial shNf - 1 / shTp53 glial serum starved 4 hr before assaying . were expressing either Cas9 alone ( control ) or Cas9 sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . F : Control sgGal8 stably expressing Flag - S - ATG16L1 were treated 100 μM monensin for 1 hr stimulated with 2 ng / mL EGF for 15 min before fixation immunofluorescence against EEA1 Flag tag . arrows indicate colocalisation . Scale bar : 10 μm . G : of of total EEA1 vesicles that colocalise with Flag - S - ATG16L1 in sgGal8 ( in F ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). F: Control and sgGal8 cells stably expressing Flag-S-ATG16L1 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 and Flag tag. White arrows indicate colocalisation. Scale bar: 10 μm. G: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 in sgGal8 cells (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


A : Glial shNf - 1 / shTp53 control sgAtg7 were serum starved 4 hr then stimulated with 20 ng / mL TexasRed - EGF ( TxRed - EGF ) for 30 min . Scale bar : 10 μm . B : of TxRed - EGF per , relative to number taken up by control ( in A ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_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

A: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 20 ng/mL TexasRed-EGF (TxRed-EGF) for 30 min. Scale bar: 10 μm. B: Quantification of TxRed-EGF puncta per cell, relative to the number taken up by control cells (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.


C : Endocytosis rate of wild type ( WT ) EGFR was assayed in control , sgAtg7 , or sgAtg16l1 glial shNf - 1 / shTp53 or EGFRvIII - expressing by surface biotinylation one application of 2 ng / mL EGF for 15 min . Monensin treatment was added as indicated . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Endocytosis rate of wild type (WT) EGFR was assayed in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells by cell surface biotinylation and one application of 2 ng/mL EGF for 15 min. Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.


D : Plasma recycling rate of WT EGFR in control , sgAtg7 , or sgAtg16l1 glial shNf - 1 / shTp53 or EGFRvIII - expressing was assayed by surface biotinylation successive applications of 2 ng / mL EGF treatments for 15 min Monensin treatment was added as indicated . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Plasma membrane recycling rate of WT EGFR in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells was assayed by cell surface biotinylation and successive applications of 2 ng/mL EGF treatments for 15 min Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.


E : Glial shNf - 1 / shTp53 control sgAtg7 stably overexpressing mCherry - Rab11 were serum starved 4 hr then stimulated with 20 ng / mL EGF for 15 min then fixed stained by immunofluorescence against EGFR . arrows indicate colocalisation . Scale bar : 10 μm . F : of Pearson ' s colocalisation coefficient between mCherry - Rab11 EGFR ( in E ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 .

O O O O B-CONTROLLED_VAR I-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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_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

E: Glial shNf-1/shTp53 control and sgAtg7 cells stably overexpressing mCherry-Rab11 were serum starved 4 hr then stimulated with 20 ng/mL EGF for 15 min then fixed and stained by immunofluorescence against EGFR. White arrows indicate colocalisation. Scale bar: 10 μm. F: Quantification of Pearson's colocalisation coefficient between mCherry-Rab11 and EGFR (in E). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.


Glial shNf - 1 / shTp53 sgAtg7 ( A ) were either left untreated ( + serum ) or serum starved ( - serum ) 4 hr then stimulated 2 ng / mL EGF for 15 min before analyses by western blotting .

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Glial shNf-1/shTp53 sgAtg7 (A) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.


Glial shNf - 1 / shTp53 sgAtg16l1 ( B ) were either left untreated ( + serum ) or serum starved ( - serum ) 4 hr then stimulated 2 ng / mL EGF for 15 min before analyses by western blotting .

Glial shNf-1/shTp53 sgAtg16l1 (B) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.


Glial shNf - 1 / shTp53 sgGal8 ( C ) were either left untreated ( + serum ) or serum starved ( - serum ) 4 hr then stimulated 2 ng / mL EGF for 15 min before analyses by western blotting .

Glial shNf-1/shTp53 sgGal8 (C) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.


D : Glial shNf - 1 / shTp53 control sgAtg7 were serum starved 4 hr then stimulated with 2 ng / mL EGF for indicated times before of EGFR signalling pathway activity by western blotting . E : Phosphorylated : total ratios for EGFR AKT calculated from densitometry analyses of western blots were made relative to their respective points of maximal stimulation ( 100 % ) ( for EGFR : t = 5 min in control for AKT : t = 7 min in control ) Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_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

D: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 2 ng/mL EGF for the indicated times before analysis of EGFR signalling pathway activity by western blotting. E: Phosphorylated:total protein ratios for EGFR and AKT calculated from densitometry analyses of western blots were made relative to their respective points of maximal stimulation (100%) (for EGFR: t=5 min in control cells and for AKT: t=7 min in control cells) Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.


F : Glial shNf - 1 / shTp53 control sgAtg7 were serum starved 4 hr with or without 20 ng / mL EGF 30 min stimulation before subcellular fractionation by western blotting .

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F: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr with or without 20 ng/mL EGF 30 min stimulation before subcellular fractionation and analysis by western blotting.


G : Radar chart indicating relative of pAKT ( pink ) , pEGFR ( blue ) , or total EGFR ( blue ) , in autophagy - deficient lines relative to their control counterparts . following knockouts were introduced : glial ( sgAtg7 ) , HT29 ( crATG7 ) , MCF - 7 ( sgATG5 ) , A549 ( sgATG5 ) , U2OS ( sgATG5 ) , MEFs ( Atg16l1 - / - ) .

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G: Radar chart indicating the relative levels of pAKT (pink line), pEGFR (light blue line), or total EGFR levels (dark blue line), in autophagy-deficient cell lines relative to their control counterparts. The following knockouts were introduced: glial cells (sgAtg7), HT29 (crATG7), MCF-7 (sgATG5), A549 (sgATG5), U2OS (sgATG5), and MEFs (Atg16l1-/-).


H : Control or sgAtg7 glial shNf - 1 / shTp53 were serum starved for 24 hr in presence or absence of 20 ng / mL EGF . of of propidium iodide ( PI ) - positive is shown . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent p values represent a two - Student ' s t : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

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H: Control or sgAtg7 glial shNf-1/shTp53 cells were serum starved for 24 hr in the presence or absence of 20 ng/mL EGF. Quantification of the percentage of propidium iodide (PI)-positive cells is shown. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.


( B ) Expression of CH25H in heathy donors COVID - 19 . box plot shows expression of CH25H in macrophages of bronchoalveolar lavage fluids from three healthy donors , three moderate COVID - 19 six severe COVID - 19 by scRNA - seq ( Liao et al . , 2020 ) .

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(B) Expression of CH25H in heathy donors and COVID-19 patients. The box plot shows the expression of CH25H in macrophages of bronchoalveolar lavage fluids from three healthy donors, three moderate COVID-19 patients and six severe COVID-19 patients by scRNA-seq analysis (Liao et al., 2020).


Characterization of SARS - CoV - 2 pseudovirus . ( C ) SARS - CoV - 2 , SARS - CoV VSV pseudovirus ( VSVG ) were concentrated by ultracentrifuge . Incorporation of spike on viral was analyzed by western blot using an antibody recognizing S2 subunit of SARS - CoV SARS - CoV - 2 . VSV Matrix served as a loading control for viral particles . Black arrows indicate full S spike ( S2 subunit ) , respectively .

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Characterization of SARS-CoV-2 pseudovirus. (C) SARS-CoV-2, SARS-CoV and VSV pseudovirus (VSVG) were concentrated by ultracentrifuge. Incorporation of spike protein on viral membrane was analyzed by western blot using an antibody recognizing S2 subunit of SARS-CoV and SARS-CoV-2. VSV Matrix protein served as a loading control for viral particles. Black arrows indicate full length S and cleaved spike (S2 subunit), respectively.


25HC inhibits fusion mediated by SARS - CoV - 2 S ( F ) 293FT or Vero were treated with EtOH or 25HC ( 5 µM ) for 16h prior to transfection with pLVX plasmids encoding SARS - CoV - 2 S ( F ) in absence of EtOH or 25HC . At 24h post - transfection , syncytium formation was visualized by microscopy . arrows indicate syncytia . of fusion was performed by calculating percentages of nuclei involved in syncytia formation from nuclei in GFP positive .

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25HC inhibits membrane fusion mediated by SARS-CoV-2 S (F) 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding SARS-CoV-2 S (F) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.


25HC inhibits fusion mediated by , SARS - CoV S ( G , left ) , MERS - CoV S ( G , right ) . 293FT or Vero were treated with EtOH or 25HC ( 5 µM ) for 16h prior to transfection with pLVX plasmids encoding , SARS - CoV S ( G , left ) , MERS - CoV S ( G , right ) in absence of EtOH or 25HC . At 24h post - transfection , syncytium formation was visualized by microscopy . Scale bar , 50 μm . arrows indicate syncytia . of fusion was performed by calculating percentages of nuclei involved in syncytia formation from nuclei in GFP positive .

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25HC inhibits membrane fusion mediated by , SARS-CoV S (G, left), and MERS-CoV S (G, right). 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding , SARS-CoV S (G, left), and MERS-CoV S (G, right) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. Scale bar, 50 μm. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.


( B - C ) 25HC depletes accessible cholesterol on plasma . ( B ) Calu - 3 were treated with indicated concentrations of 25HC for 1h , subsequently incubated with ALOD4 for 30 min . were washed twice with PBS , lysed , amounts of bound ALOD4 were examined by western blotting . ( C ) Calu - 3 were treated with 5 µM 25HC for 1h , subsequently incubated with AF488 labeled ALOD4 for 30 min . were washed twice examined by microscopy .

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(B-C) 25HC depletes accessible cholesterol on the plasma membrane. (B) Calu-3 cells were treated with indicated concentrations of 25HC for 1h, and subsequently incubated with ALOD4 for 30 min. Cell were washed twice with PBS, lysed, and the amounts of bound ALOD4 were examined by western blotting. (C) Calu-3 cells were treated with 5 µM 25HC for 1h, and subsequently incubated with AF488 labeled ALOD4 for 30 min. Cells were washed twice and examined by fluorescence microscopy.


( D ) Cholesterol rescues level of accessible cholesterol on plasma of 25HC - treated . Calu - 3 were pretreated with ethanol or 5 µM 25HC for 16h . were washed incubated with PBS or 20 µM cholesterol for 1 h . After further incubation with ALOD4 , were washed with PBS twice lysed . amount of bound ALOD4 was examined by western blotting .

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(D) Cholesterol rescues the level of accessible cholesterol on the plasma membrane of 25HC-treated cells. Calu-3 cells were pretreated with ethanol or 5 µM 25HC for 16h. Cells were washed and incubated with PBS or 20 µM cholesterol for 1 h. After further incubation with ALOD4, cells were washed with PBS twice and lysed. The amount of bound ALOD4 was examined by western blotting.


Addition of cholesterol rescues entry of SARS - CoV - 2 pseudovirus , SARS - CoV pseudovirus into 25HC - treated Calu - 3 . were pretreated with ethanol or 5 µM 25HC for 16h , washed incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS - CoV - 2 pseudovirus . Entry of SARS - CoV - 2 pseudovirus ( E F ) was assessed by luciferase or by microscopy at 24h post - infection .

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Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. Entry of SARS-CoV-2 pseudovirus (E and F) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.


Addition of cholesterol rescues entry of SARS - CoV - 2 pseudovirus , SARS - CoV pseudovirus , MERS - CoV pseudovirus into 25HC - treated Calu - 3 . were pretreated with ethanol or 5 µM 25HC for 16h , washed incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS - CoV - 2 pseudovirus . , SARS - CoV pseudovirus ( G ) , MERS - CoV pseudovirus ( H ) was assessed by luciferase or by microscopy at 24h post - infection .

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Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus, and MERS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. , SARS-CoV pseudovirus (G), and MERS-CoV pseudovirus (H) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.


( C ) SZ58 - 035 rescues surface accessible cholesterol in 25HC - treated . Calu - 3 were treated with vehicle or 40 µM SZ58 - 035 for 16 h , prior to treatment with ethanol or 2 . 5 µM 25HC for 1h . were then incubated with ALOD4 for 30 min . amount of bound ALOD4 was examined by western blotting .

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(C) SZ58-035 rescues surface accessible cholesterol in 25HC-treated cells. Calu-3 cells were treated with vehicle or 40 µM SZ58-035 for 16 h, prior to treatment with ethanol or 2.5 µM 25HC for 1h. Cells were then incubated with ALOD4 for 30 min. The amount of bound ALOD4 was examined by western blotting.


A , of Nox4 mRNA ( A ) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum - replete ( 15 % serum ) . n = 4 preparations / group .

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A, Quantification of Nox4 mRNA levels (A) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum-replete cells (15% serum). n=4 cell preparations/group.


B of Nox4 ( B ) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum - replete ( 15 % serum ) . n = 4 preparations / group . A representative immunoblot for Nox4 tubulin as a loading control is shown in panel B .

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B Quantification of Nox4 protein levels (B) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum-replete cells (15% serum). n=4 cell preparations/group. A representative immunoblot for Nox4 and tubulin as a loading control is shown in panel B.


C after 48 h serum starvation in cardiomyocytes in which Nox4 was depleted by an adenoviral shRNA ( Ad . shNox4 ) as compared to infected with a control vector ( Ad . Ctl ) . n = 6 / group . representative immunoblot shows reduction in Nox4 after shRNA - mediated knockdown .

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C Cell death after 48 h serum starvation in cardiomyocytes in which Nox4 was depleted by an adenoviral shRNA (Ad.shNox4) as compared to cells infected with a control vector (Ad.Ctl). n=6/group. The representative immunoblot shows reduction in Nox4 protein levels after shRNA-mediated knockdown.


D Lactate dehydrogenase ( LDH ) activity in supernatants . Staurosporine ( Stauro , 1 μmol / L ) was used to induce by apoptosis . n = 4 preparations / group .

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D Lactate dehydrogenase (LDH) activity in cell supernatants. Staurosporine (Stauro, 1 μmol/L) was used to induce cell death by apoptosis. n=4 cell preparations/group.


E of high mobility group box1 ( HMGB1 ) in supernatants . n = 3 / group . A representative immunoblot is shown at top .

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E Levels of high mobility group box1 protein (HMGB1) in cell supernatants. n=3/group. A representative immunoblot is shown at the top.


F of cytochrome c in fractions of cardiomyocytes . n = 4 / group . A representative immunoblot is shown at top . fraction ( Mem ) containing mitochondria was used as a positive control .

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F Protein levels of cytochrome c in the cytoplasmic fractions of cardiomyocytes. n=4/group. A representative immunoblot is shown at the top. The cellular membrane fraction (Mem) containing mitochondria was used as a positive control.


G of Nox4 - depleted control with depolarized mitochondria , quantified by flow cytometry . n = 3 preparations / group ( 100 , 000 per experiment ) .

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G Percentage of Nox4-depleted and control cells with depolarized mitochondria, quantified by flow cytometry. n=3 cell preparations/group (100,000 cells per experiment).


H Effect of cyclosporin A ( CsA , 1 μmol / L ) on of with depolarized mitochondria . n = 6 - 8 / group .

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H Effect of cyclosporin A (CsA, 1 μmol/L) on the percentage of cells with depolarized mitochondria. n=6-8/group.


I Effect of cyclosporin A ( CsA , 1 μmol / L ) on in serum - starved cardiomyocytes . n = 4 / group .

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I Effect of cyclosporin A (CsA, 1 μmol/L) on cell death in serum-starved cardiomyocytes. n=4/group.


J in serum starved Nox4 knockout MEFs ( Nox4KO ) wild - type MEFs ( WT ) . Cat = PEG - catalase ( 500 U / mL ) . Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant , Nox4P437H ( Mut ) . n = 6 - 12 / group .

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J Cell death in serum starved Nox4 knockout MEFs (Nox4KO) and wild-type MEFs (WT). Cat = PEG-catalase (500 U/mL). Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant, Nox4P437H (Mut). n=6-12/group.


K HMGB1 in supernatants of serum - starved MEFs in similar experiments to those in ( J ) . n = 4 / group . A representative immunoblot is shown to top .

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K HMGB1 levels in the supernatants of serum-starved MEFs in similar experiments to those in (J). n=4/group. A representative immunoblot is shown to the top.


L of MEFs with depolarized mitochondria . n = 6 / group .

L Percentage of MEFs with depolarized mitochondria. n=6/group.


A Basal mitochondrial calcium assessed in serum - starved rat cardiomyocytes using a mitochondrial - targeted cameleon CFP / YFP FRET probe . Nox4 was depleted with silencing RNAs ( siRNAs ) or were transfected with a control scrambled siRNA ( siScr ) . Representative photomicrographs are shown at top . spectrum color scale represents of emitted ( YFP / CFP ) . mean changes in Nox4 - depleted as compared to scrambled control ( dotted ) at bottom . representative immunoblot shows depletion of Nox4 . Scale bars : 10 µm . n = 3 / group ( with > 50 per experiment ) .

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A Basal mitochondrial calcium levels assessed in serum-starved rat cardiomyocytes using a mitochondrial-targeted cameleon CFP/YFP FRET probe. Nox4 was depleted with silencing RNAs (siRNAs) or cells were transfected with a control scrambled siRNA (siScr). Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). The mean changes in Nox4-depleted cells as compared to scrambled control (dotted line) at the bottom. The representative immunoblot shows depletion of Nox4 protein levels. Scale bars: 10 µm. n=3/group (with >50 cells per individual experiment).


B Basal mitochondrial calcium in WT Nox4KO MEFs after serum starvation . Representative photomicrographs are shown at top . spectrum color scale represents of emitted ( YFP / CFP ) . Mean data at bottom . Scale bars : 10 µm . n = 3 / group ( with > 100 per experiment ) .

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B Basal mitochondrial calcium levels in WT and Nox4KO MEFs after serum starvation. Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). Mean data at the bottom. Scale bars: 10 µm. n=3/group (with >100 cells per individual experiment).


Changes in mitochondrial calcium in serum - starved WT Nox4KO MEFs after addition of histamine ( 100 μmol / L , C ) n = 3 / group ( with > 30 per experiment ) .

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Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, C) n=3/group (with >30 cells per individual experiment).


D Changes in mitochondrial calcium in serum - starved WT Nox4KO MEFs after addition of ATP ( 100 μmol / L , D ) . n = 3 / group ( with > 30 per experiment ) .

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D Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, D). n=3/group (with >30 cells per individual experiment).


E , Changes in ER calcium measured with an ER - targeted cameleon probe in serum - starved WT Nox4KO MEFs after addition of histamine ( 100 μmol / L , E ) n = 3 / group ( with > 30 per experiment ) .

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E, Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, E) n=3/group (with >30 cells per individual experiment).


F Changes in ER calcium measured with an ER - targeted cameleon probe in serum - starved WT Nox4KO MEFs after addition of ATP ( 100 μmol / L , F ) . n = 3 / group ( with > 30 per experiment ) .

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F Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, F). n=3/group (with >30 cells per individual experiment).


G Peak increase in mitochondrial calcium response in response to histamine ( 100 μmol / L ) in WT MEFs with or without treatment with PEG - catalase ( Cat ) , in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4P437H mutant ( Mut ) . n = 3 / group ( with > 30 per experiment ) .

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G Peak increase in mitochondrial calcium levels response in response to histamine (100 μmol/L) in WT MEFs with or without treatment with PEG-catalase (Cat), and in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4P437H mutant (Mut). n=3/group (with >30 cells per individual experiment).


H Representative time - course of histamine - induced changes in mitochondrial calcium for experiments shown in ( I ) .

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H Representative time-course of histamine-induced changes in mitochondrial calcium for the experiments shown in (I).


A Immunoblotting of subcellular fractions from serum - starved rat cardiomyoblasts ( H9c2 ) . Input : homogenate before fractionation ; Cyto : cytosol ; ER : endoplasmic reticulum ; Crude M : crude mitochondrial fraction , also containing MAM ; Pure Mito : mitochondria ; MAM : mitochondria associated - ER membranes . Cytochrome c ( Cyt c ) was used as a mitochondrial marker , VDAC as an outer mitochondrial marker , calnexin as an ER marker , Sigma1R FACL4 as MAM markers , tubulin as a marker . B Immunoblotting of subcellular fractions from rat heart . C Immunoblotting of subcellular fractions from rat kidney .

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A Immunoblotting of subcellular fractions from serum-starved rat cardiomyoblasts (H9c2 cells). Input: homogenate before fractionation; Cyto: cytosol; ER: endoplasmic reticulum; Crude M: crude mitochondrial fraction, also containing MAM; Pure Mito: mitochondria; MAM: mitochondria associated-ER membranes. Cytochrome c (Cyt c) was used as a mitochondrial marker, VDAC as an outer mitochondrial membrane marker, calnexin as an ER marker, Sigma1R and FACL4 as MAM markers, and tubulin as a cytosolic marker. B Immunoblotting of subcellular fractions from rat heart. C Immunoblotting of subcellular fractions from rat kidney.


D Electron showing subcellular localization of Nox4 in wild - type ( WT ) Nox4KO mouse hearts , human induced pluripotent - derived cardiomyocytes ( hiPSC - CM ) . hiPSC - CM were depleted of Nox4 using a lentiviral shRNA ( Nox4KD ) or infected with a control lentivirus . FACL4 was used as a MAM marker . Arrow heads show peri - mitochondrial localization of Nox4 FACL4 , in proximity of ER cisternae ribosomes . M = mitochondria , ER = endoplasmic reticulum , T = T - tubule . Scale bars : 100 nm .

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D Electron micrographs showing subcellular localization of Nox4 in wild-type (WT) and Nox4KO mouse hearts, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). hiPSC-CM were depleted of Nox4 using a lentiviral shRNA (Nox4KD) or infected with a control lentivirus. FACL4 was used as a MAM marker. Arrow heads show peri-mitochondrial localization of Nox4 and FACL4, in the proximity of ER cisternae and ribosomes. M=mitochondria, ER=endoplasmic reticulum, T=T-tubule. Scale bars: 100 nm.


A Simplified photomicrographs of proximity ligation studies in WT Nox4KO MEFs , showing borders , nuclei ( blue ) yellow dots corresponding to co - localization of . of number of dots / in each condition is shown to right . Proximity was tested for following couples : FACL4 / Nox4 , InsP3R / Nox4 , FACL4 / InsP3R . Scale bars : 10 µm . n = 3 / group ( with > 30 / experiment ) .

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A Simplified photomicrographs of proximity ligation studies in WT and Nox4KO MEFs, showing cell borders, nuclei (blue) and yellow dots corresponding to co-localization of proteins. Quantification of the number of dots/cell in each condition is shown to the right. Proximity was tested for the following protein couples: FACL4/Nox4, InsP3R/Nox4, and FACL4/InsP3R. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


B Simplified photomicrographs of proximity ligation in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control ( siScr ) , with as in ( A ) . Proximity was tested for FACL4 / Nox4 couple . Scale bars : 10 µm . n = 3 / group ( with > 30 / experiment ) .

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B Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control (siScr), with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


C Simplified photomicrographs of proximity ligation in hiPSC - CM ( human cardiomyocytes ) transduced with Nox4 shRNA or scrambled control , with as in ( A ) . Proximity was tested for FACL4 / Nox4 couple . Scale bars : 10 µm . n = 3 / group ( with > 30 / experiment ) .

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C Simplified photomicrographs of proximity ligation staining in hiPSC-CM (human cardiomyocytes) transduced with Nox4 shRNA or scrambled control, with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


A Immunoblotting of subcellular fractions from serum - starved cardiomyoblasts for InsP3R , Akt PP2a , as in Fig 3A .

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A Immunoblotting of subcellular fractions from serum-starved cardiomyoblasts for InsP3R, Akt and PP2a, as in Fig 3A.


B Fractionation of crude mitochondrial fractions ( containing mitochondria MAM ) from WT MEFs on a sucrose gradient ( 5 - 60 % ) immunoblotting for relevant . Fraction : 200 µL . Representative of 3 independent experiments .

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B Fractionation of crude mitochondrial fractions (containing mitochondria and MAM) from WT MEFs on a sucrose gradient (5-60%) and immunoblotting for relevant proteins. Fraction size: 200 µL. Representative of 3 independent experiments.


C Immunoblotting for phosphorylated total Akt or InsP3R in crude mitochondrial fractions from serum - starved WT Nox4KO MEFs . FACL4 was used as a loading control for MAM . InsP3R was first immunopreciptated then precipitate was immunoblotted for total InsP3R for phosphorylated Akt - substrate motif RXRXX ( pS / T ) ( p - InsP3R ) . Representative immunoblots are shown at top mean data at bottom . Cat = PEG - catalase . Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant ( Mut ) . n = 3 / group .

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C Immunoblotting for phosphorylated and total Akt or InsP3R protein in crude mitochondrial fractions from serum-starved WT and Nox4KO MEFs. FACL4 was used as a loading control for the MAM. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Representative immunoblots are shown at the top and mean data at the bottom. Cat = PEG-catalase. Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant (Mut). n=3/group.


D ROS indexed in MEFs using H2O2 specific HyPer probes targeted to MAM ( FACL4 HyPer ) or cytosol ( Cyto HyPer ) . Imaging was performed after serum starvation for 12h . Representative photomicrographs of are shown to right mean data for changes in presented to left . signal obtained using corresponding ROS - insensitive SypHer probes was used to correct for any pH - induced changes in ; change in between HyPer corresponding SypHer probe ( ΔR ) for each condition is reported . n = 5 independent preparations / group , with at least 20 imaged / preparation . Scale bars : 10 µm .

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D ROS levels indexed in MEFs using H2O2 specific HyPer probes targeted to the MAM (FACL4 HyPer) or the cytosol (Cyto HyPer). Imaging was performed after serum starvation for 12h. Representative photomicrographs of the fluorescence ratio are shown to the right and mean data for changes in fluorescence ratio presented to the left. The signal obtained using corresponding ROS-insensitive SypHer probes was used to correct for any pH-induced changes in fluorescence; the change in fluorescence ratio between HyPer and corresponding SypHer probe (ΔR) for each condition is reported. n=5 independent cell preparations/group, with at least 20 cells imaged/preparation. Scale bars: 10 µm.


B Basal mitochondrial calcium in serum - starved WT Nox4KO MEFs in presence or absence of Akti ( 1 µmol / L ) normalized versus WT MEF control . n = 6 / group with > 20 imaged per experiment .

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B Basal mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs in the presence or absence of Akti (1 µmol/L) normalized versus WT MEF control. n=6/group with >20 cells imaged per experiment.


C Effect of Akti ( 1 µmol / L ) or XeC ( 1 µmol / L ) on histamine - induced changes in mitochondrial calcium in MEFs . time course of changes in calcium is shown on left mean data for peak mitochondrial calcium on right . n = 3 / group with > 40 imaged per experiment .

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C Effect of Akti (1 µmol/L) or XeC (1 µmol/L) on histamine-induced changes in mitochondrial calcium levels in MEFs. The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right. n=3/group with >40 cells imaged per experiment.


D of in serum - starved WT Nox4KO MEFs in absence or presence of Akti or XeC . n = 6 / group .

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D Quantification of cell death in serum-starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC. n=6/group.


E Cardiac troponin I ( cTnI ) as a marker of cardiomyocyte necrosis in perfusate of isolated WT Nox4KO hearts subjected to ischemia - reperfusion ( I / R ) . XeC was added at a final of 2 µmol / L prior to ischemia . n = 6 - 7 / group .

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E Cardiac troponin I (cTnI) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia-reperfusion (I/R). XeC was added at a final concentration of 2 µmol/L prior to ischemia. n=6-7/group.


F Immunoblotting for phosphorylation of Akt InsP3R in crude mitochondrial fractions from WT Nox4KO hearts after I / R . InsP3R was first immunopreciptated then precipitate was immunoblotted for total InsP3R for phosphorylated Akt - substrate motif RXRXX ( pS / T ) ( p - InsP3R ) . Mean data shown to right . n = 3 / group .

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F Immunoblotting for the phosphorylation levels of Akt and InsP3R in crude mitochondrial fractions from WT and Nox4KO hearts after I/R. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Mean data shown to the right. n=3/group.


G Cardiac left ventricular contractile function in isolated Langendorff - perfused WT Nox4KO hearts at baseline ( Basal ) then after I / R . Hearts were treated with XeC ( 1 μmol / L ) or vehicle control for 20 min prior to ischemia . RPP , heart rate x left ventricular pressure product ; DEVP , left ventricular developed pressure . n = 6 - 7 / group .

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G Cardiac left ventricular contractile function in isolated Langendorff-perfused WT and Nox4KO hearts at baseline (Basal) and then after I/R. Hearts were treated with XeC (1 μmol/L) or vehicle control for 20 min prior to ischemia. RPP, heart rate x left ventricular pressure product; DEVP, left ventricular developed pressure. n=6-7/group.


A of MNase - digested fragments from wild - type ( WT , sizes indicated in black ) nap1Δ ( N , sizes indicated in red ) by agarose gel electrophoresis . Diagram illustrating reduction of array in nap1Δ .

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A Analysis of MNase-digested chromatin DNA fragments from wild-type (WT, sizes indicated in black) and nap1Δ (N, sizes indicated in red) cells by agarose gel electrophoresis. Diagram illustrating reduction of array length in nap1Δ cells.


B Frequency distribution of nucleosomal ( WT in grey fill with nap1Δ in blue traces ) from H3 ChIP - seq .

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B Frequency distribution of nucleosomal DNA size (WT in grey fill with nap1Δ in blue traces) from H3 ChIP-seq.


C Calculation of nucleosome fuzziness as standard deviation of nucleosome ( or tag ) positions . See also Appendix Fig S1 .

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C Calculation of nucleosome fuzziness as the standard deviation of nucleosome (or tag) positions. See also Appendix Fig S1.


B Ribbon view of asymmetric crystallographic unit containing a ( yNap1c2 - H2AΔ14 - H2BΔ28 ) 6 assembly . For clarity only layer 1 of is shown . Layer 1 contains three protomers A - C that are related by two perpendicular non - crystallographic dyads ( ) . C Side - view of . layer 2 omitted from panel B is shown in grey . yNap1 dimer is shown in green yellow , H2A in red H2B in blue . See also Fig EV1 . D View of yNap1 - H2A - H2B protomer with labeling of secondary structure elements .

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B Ribbon view of the asymmetric crystallographic unit containing a (yNap1c2-H2AΔ14-H2BΔ28)6 assembly. For clarity only layer 1 of the complex is shown. Layer 1 contains three protomers A-C that are related by two perpendicular non-crystallographic dyads ().C Side-view of the complex. The layer 2 omitted from panel B is shown in grey. The yNap1 dimer is shown in green and yellow, H2A in red and H2B in blue. See also Fig EV1.D View of the yNap1-H2A-H2B protomer complex with labeling of secondary structure elements.


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A Overview of the yNap1c2-H2AΔ14-H2BΔ28 complex.B Residues R17, R20, R29, R32, R35 and K36 of H2A make contacts with the acidic HBR1 in yNap1 comprising residues D201, D205 and E310.C Residues K75, R77 and R81 of H2A make polar contacts with the HBR2 comprising residues E332, D333 and E339 in α8 of yNap1.


D In vitro GST - pulldown showing role of residues in HBR interfaces in H2A - H2B binding . mutant labeled HBR1 ( lane 6 ) contains triple mutation D201R , D205R , E310R HBR2 ( lane 12 ) triple mutations E332 , D333 , E336 . HBR1 + 2 ( lane 13 ) contains six mutations . mutant labeled HBR1 + 2A ( lane 14 ) has Alanine replacements in six HBR residues . Top panel : Bound . Bottom panel : Input .

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D In vitro GST-pulldown assay showing the role of residues in the HBR interfaces in H2A-H2B binding. The mutant labeled HBR1 (lane 6) contains the triple mutation D201R, D205R, E310R and HBR2 (lane 12) the triple mutations E332, D333, E336. HBR1+2 (lane 13) contains all six mutations. The mutant labeled HBR1+2A (lane 14) has Alanine replacements in the six HBR residues. Top panel: Bound. Bottom panel: Input.


E Disruption of Nap1 - histone interaction by H2A mutation . Bottom panel : Inputs of coexpressed ( soluble extract ) top panel material bound to glutathione affinity column were analyzed by SDS - PAGE Coomassie .

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E Disruption of the Nap1-histone interaction by H2A mutation. Bottom panel: Inputs of the coexpressed proteins (soluble extract) and top panel the material bound to the glutathione affinity column were analyzed by SDS-PAGE and Coomassie staining.


F Controls showing input ( bottom ) bound ( top ) fractions . See also Fig EV2 .

F Controls showing input (bottom) and bound (top) fractions. See also Fig EV2.


A EM of yNap1c2 - H2AΔ14 - H2BΔ28 . Representative rawimage of negatively - stained particles . B Representative particles ( 1st row ) , class averages coming from projection matching procedure ( 2nd row ) comparison of class averages with projections from a final model generated after four projection matching cycles ( 3rd row ) .

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A EM analysis of the yNap1c2-H2AΔ14-H2BΔ28 complex. Representative rawimage of negatively-stained particles.B Representative particles (1st row), class averages coming from the projection matching procedure (2nd row) and comparison of the class averages with projections from a final model generated after four projection matching cycles (3rd row).


C From left to right : Crystal structure of ( yNap1c2 - H2A - H2B ) 6 in two different orientations ( brown ) ; 60 Å - filtered structure of ( magenta ) ; 3D reconstruction of from negatively stained particles ( green ) fit of crystal structure into EM map .

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C From left to right: Crystal structure of the (yNap1c2-H2A-H2B)6 complex in two different orientations (brown); 60 Å-filtered structure of the complex (magenta); 3D reconstruction of the complex from negatively stained particles (green) and fit of the crystal structure into the EM map.


D - I Mass spectra of yNap1 variants in absence presence of H2A - H2B . yNap1 in in absence ( D ) or presence of H2A - H2B ( E ) . yNap1c in absence ( F ) or bound to H2A - H2B ( G ) . oligomerization mutant yNap1 _ IF1 alone ( H ) or in with H2A _ IF2Δ14 - H2BΔ28 ( I ) . yNap1 is shown in green circles H2A - H2B as red blue asterisks .

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D-I Mass spectra of yNap1 variants in the absence and presence of H2A-H2B. yNap1 in complex in the absence (D) or presence of H2A-H2B (E). yNap1c in the absence (F) or bound to H2A-H2B (G). The oligomerization mutant yNap1_IF1 alone (H) or in complex with H2A_IF2Δ14-H2BΔ28 (I). yNap1 is shown in green circles and H2A-H2B as red and blue asterisks.


A Native PAGE gel showing result of an affinity shift scanned to detect AF488 - ( left ) or AF647 - labeled ( right ) . 31 nM - 4 µM of yNap1 - H2A - H2B ( lanes 6 - 13 ) were incubated with a mixture of 0 . 8 µM AF488 - labeled H3 - H4 - tetrasome complexes 0 . 8 µM AF647 - labeled ( lane 5 ) . Lanes 1 - 3 show migration of tetrasomes ( tetra ) , nucleosomes ( nucl ) or free H2A - H2B obtained by salt deposition onto AF647 - labeled .

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A Native PAGE gel showing the result of an affinity shift assay scanned to detect AF488- (left) or AF647-labeled DNA (right). 31 nM - 4 µM of yNap1-H2A-H2B (lanes 6-13) were incubated with a mixture of 0.8 µM AF488-labeled H3-H4-DNA tetrasome complexes and 0.8 µM AF647-labeled DNA (lane 5). Lanes 1-3 show migration of tetrasomes (tetra), nucleosomes (nucl) or free H2A-H2B obtained by salt deposition onto AF647-labeled DNA.


B of nucleosome band ( nucl ) is plotted . value at 4 µM of yNap1 - H2A - H2B ( lane 13 ) was set to 100 % . For non - specific binding , value in absence of H2A - H2B ( lane 5 ) was set to 100 % disappearance of free AF647 band was plotted . Each curve is representative of three independent experiments performed on different days . Standard deviations are shown .

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B Intensity of the nucleosome band (nucl) is plotted. The value at 4 µM of yNap1-H2A-H2B (lane 13) was set to 100%. For non-specific DNA binding, the value in the absence of H2A-H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown.


C of MNase - digested fragments from wild - type ( BY4741 ) , nap1Δ , nap1Δ complemented with different yNap1 expression plasmids . Total MNase units per 50 ml of culture are indicated on bottom of lanes . Kb + : Marker .

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C Analysis of MNase-digested chromatin DNA fragments from wild-type (BY4741), nap1Δ, and nap1Δ cells complemented with different yNap1 expression plasmids. Total MNase units per 50 ml of culture are indicated on the bottom of the lanes. Kb+: DNA Marker.


a ) Kaplan - Meier to first fever ( ≥ 38 ° C ) episode within 24 hours of receiving vaccines administered at 4 months of ; control n = 88 4CMenB n = 89 .

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a) Kaplan-Meier to first fever (≥38°C) episode within 24 hours of receiving vaccines administered at 4 months of age; control n=88 and 4CMenB n=89.


b ) Principal component ( PC ) of - sequencing data ( 14837 genes , n = 253 ) from study time points . lines connect participant ' s pre - post - vaccination samples . ellipses are two - dimensional ( PC1 PC2 ) 95 % confidence intervals for each study time point . A contribution plot ( top right ) displays genes contributing most to PC1 / PC2 , i . e . 0 . 15 implies 0 . 15 % contribution of that variable to principal components displayed .

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b) Principal component (PC) analysis of RNA-sequencing data (14837 genes, n= 253) from all study time points. The lines connect participant's pre- and post-vaccination samples. The ellipses are the two-dimensional (PC1 and PC2) 95% confidence intervals for each study time point. A contribution plot (top right) displays the genes contributing most to PC1/PC2, i.e. 0.15 implies 0.15% contribution of that variable to the principal components displayed.


c ) Plotted are CIBERSORTx neutrophil fractions from whole blood - sequencing data , with median interquartile range . P - values were determined from a two sample Wilcoxon rank sum . number of individuals in each group is display in x - axis .

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c) Plotted are the CIBERSORTx neutrophil fractions from whole blood RNA-sequencing data, with median and interquartile range. P-values were determined from a two sample Wilcoxon rank sum test. The number of individuals in each group is display in the x-axis.


d ) Spearman ' s rank correlation between neutrophil measure by full blood ( n = 142 ) those estimated by CIBERSORTx , using LM22 signature matrix .

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d) Spearman's rank correlation between neutrophil counts measure by full blood counts (n= 142) and those estimated by CIBERSORTx, using the LM22 signature matrix.


a ) Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated blue downregulated ) at each study time point versus pre - vaccination ( 4 months of ) 4 hours post - vaccination ( 719 DEGs , n = 28 ) . P - values were obtained from moderated t - statistic , after adjustment for multiple testing ( Benjamini Hochberg ' s method ) . top 10 genes , ranked by FDR , are labelled .

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a) Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination (4 months of age) 4 hours post-vaccination (719 DEGs, n=28). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). The top 10 genes, ranked by FDR, are labelled.


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated blue downregulated ) at each study time point versus pre - vaccination b ) Same as ( a ) but 24 hours post - vaccination ( 5553 DEGs , n = 31 )

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Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination b) Same as (a) but 24 hours post-vaccination (5553 DEGs, n=31)


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated blue downregulated ) at each study time point versus pre - vaccination c ) Same as ( a ) but 3 days post - vaccination ( 159 DEGs , n = 30 )

Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination c) Same as (a) but 3 days post-vaccination (159 DEGs, n=30)


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated blue downregulated ) at each study time point versus pre - vaccination d ) Same as ( a ) but 7 days post - vaccination ( 6 DEGs , n = 36 ) .

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Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination d) Same as (a) but 7 days post-vaccination (6 DEGs, n=36).


a ) Top genes differentially regulated between vaccine groups 4 hours post - vaccination . Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup . plotted lines are LOESS ( locally estimated scatterplot smoothing ) regression curves with 95 % confidence intervals in grey . FDR was derived by comparing fold - changes in gene expression in control group with group , from baseline to time point designated , is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from moderated t - statistic , after adjustment for multiple testing ( Benjamini Hochberg ' s method ) . Pre - vaccination samples n = 125 , 4 hour samples n = 28 , 24 hour samples n = 31 , 3 day sample n = 30 , 7 day samples n = 36 . expression E value , are gene expression value derived from voom - limma workflow ( Law et al . , 2014 ) .

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a) Top genes differentially regulated between vaccine groups 4 hours post-vaccination. Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). Pre-vaccination samples n= 125, 4 hour samples n= 28, 24 hour samples n = 31, 3 day sample n= 30, 7 day samples n= 36. The expression E value, are the gene expression value derived from the voom-limma workflow (Law et al., 2014).


Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup . plotted lines are LOESS ( locally estimated scatterplot smoothing ) regression curves with 95 % confidence intervals in grey . FDR was derived by comparing fold - changes in gene expression in control group with group , from baseline to time point designated , is reported if statistically significant ( FDR < 0 . 05 ) . b ) Same as ( a ) but top genes differentially regulated between vaccine groups 24 hours post - vaccination .

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Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). b) Same as (a) but top genes differentially regulated between vaccine groups 24 hours post-vaccination.