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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Cells were stimulated with 20 ng/mL 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Cells stably expressing mCherry-Rab5 were stimulated with 20 ng/mL EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

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.


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 .

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

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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O 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.


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 .

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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

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

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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Western blotting of shNf-1/shTp53 glial cells expressing gRNA sequences targeting Gal8.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

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 ) 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) 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 ) 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

F: 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 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 - / - ) .

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

G: 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 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 .

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

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.


( A ) Coimmunoprecipitation of SMO - HA and Myc - Smaug proteins . Extracts of Cl8 cells expressing combinations of SMOWT - HA or SMOPKA - SD - HA with Myc - SmaugWT in the absence or presence of HH were immunoprecipitated ( IP ) with anti - HA . The input ( lower panel ) and the IP complexes ( upper panel ) were analyzed by western blot with anti - Myc ( αMyc ) or anti - HA ( αHA ) antibodies . Here and in the other figures , the names of the proteins detected are indicated on the left and the molecular weights on the right , in kDa ; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs . * corresponds to higher molecular weight forms of SMO of unknown origin . Here and in panel F , the black arrow indicates unphosphorylated SMOWT - HA and the bracket indicates the phosphorylated forms of SMOWT - HA or SMOPKA - SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coimmunoprecipitation of SMO-HA and Myc-Smaug proteins. Extracts of Cl8 cells expressing combinations of SMOWT-HA or SMOPKA-SD-HA with Myc-SmaugWT in the absence or presence of HH were immunoprecipitated (IP) with anti-HA. The input (lower panel) and the IP complexes (upper panel) were analyzed by western blot with anti-Myc (αMyc) or anti-HA (αHA) antibodies. Here and in the other figures, the names of the proteins detected are indicated on the left and the molecular weights on the right, in kDa; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs. * corresponds to higher molecular weight forms of SMO of unknown origin. Here and in panel F, the black arrow indicates unphosphorylated SMOWT-HA and the bracket indicates the phosphorylated forms of SMOWT-HA or SMOPKA-SD.


( D , E ) Mapping of the Smaug interaction domain in SMO . Extracts from Cl8 cells transfected with Myc - SmaugWT and various regions of SMO fused to either HA ( D ) or to GFP ( E ) were immunoprecipitated with αMyc ( D ) or αGFP ( E ) and analyzed by western blotting with αMyc ( lower panel in D and upper panel in E ) , αHA ( D , upper panel ) or αGFP ( E , lower panel ) . In : Input ; Sup : supernatant . See also Fig EV1D - F .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Mapping of the Smaug interaction domain in SMO. Extracts from Cl8 cells transfected with Myc-SmaugWT and various regions of SMO fused to either HA (D) or to GFP (E) were immunoprecipitated with αMyc (D) or αGFP (E) and analyzed by western blotting with αMyc (lower panel in D and upper panel in E), αHA (D, upper panel) or αGFP (E, lower panel). In: Input; Sup: supernatant. See also Fig EV1D-F.


( F ) Hyperphosphorylated forms of SMO do not interact with Smaug . Left panel : Extracts of Cl8 cells expressing SMOWT - HA with or without Myc - SmaugWT , in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA ( upper panel ) or αMyc antibodies ( lower panel ) . Right panel : Cl8 cells expressing SNAP - SMOWT with Myc - SmaugWT in the presence of HH were extracellularly labelled with a membrane - nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation ( IP ) with anti - Myc . Labelled SNAP - SMO was directly visualized after electrophoresis . Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms . See also Fig EV1G - J .

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

(F) Hyperphosphorylated forms of SMO do not interact with Smaug. Left panel: Extracts of Cl8 cells expressing SMOWT-HA with or without Myc-SmaugWT, in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA (upper panel) or αMyc antibodies (lower panel). Right panel: Cl8 cells expressing SNAP-SMOWT with Myc-SmaugWT in the presence of HH were extracellularly labelled with a membrane-nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation (IP) with anti-Myc. Labelled SNAP-SMO was directly visualized after electrophoresis. Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms. See also Fig EV1G-J.


( G ) SMO and Smaug colocalize . Representative fluorescent images of Cl8 cells expressing GFP - SmaugWT alone ( 1 - 2 ) or together with SMOWT - mCherry ( 3 - 3 ' ' and 4 - 4 ' ' ) either without ( 1 , 3 - 3 " ) or with HH ( 2 , 4 - 4 " ) . The merge images in 3 " and 4 " show GFP - SmaugWT in green and SMOWT - mCh in red . The same results were seen with different fluorescent tags as well ( Fig EV2B ) . The lack of effect of HH on GFP - SmaugWT in absence of SMOWT - mCh is likely due to limiting amounts of endogeneous SMO . A least 20 cells were assayed for each condition . In absence of HH , all co - transfected cells exhibited greater than 90 % colabelled SMOWT - mCherry . Scale bar ( shown in G1 , identical for all panels ) : 10μm . See also Fig EV2 .

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

(G) SMO and Smaug colocalize. Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT alone (1-2) or together with SMOWT-mCherry (3-3'' and 4-4'') either without (1, 3-3") or with HH (2, 4-4"). The merge images in 3" and 4" show GFP-SmaugWT in green and SMOWT-mCh in red. The same results were seen with different fluorescent tags as well (Fig EV2B). The lack of effect of HH on GFP-SmaugWT in absence of SMOWT-mCh is likely due to limiting amounts of endogeneous SMO. A least 20 cells were assayed for each condition. In absence of HH, all co-transfected cells exhibited greater than 90% colabelled SMOWT-mCherry. Scale bar (shown in G1, identical for all panels):10μm. See also Fig EV2.


( B , C ) Smaug downregulates reporter expression . Mean values of the relative levels of SNAP - GUS reporter ( SNAP - GUS / GFP - SNAP ratio , in arbitrary units ) in extracts of transfected Cl8 cells in the absence ( black ) or presence of λN - SNAP - Smaug ( red ) , λN - SNAP ( pale grey ) or SNAP - Smaug ( dark grey ) . See Fig EV3A for the representative gel and Fig EV3B which shows that λN - SNAP - Smaug has no effect on a SNAP - GUS construct that lacks the 5BoxB . N = 3 ( biological replicates ) . See also Fig EV3A - C . Here and in D - F : SNAP - GUS / GFP - SNAP ratios were set to 1 in cells expressing λN ‐ SNAP - Smaug . The error bars correspond to the standard deviation of the mean ( SD ) . Statistical analysis was done by a Kruskal - Wallis test by ranks followed by a Dunn test and p values are : black bracket < 0 . 05 , red bracket < 0 . 01 , green bracket < 0 . 0001 . Here and in D - E : 15 ng of pAct . λN - SNAP - smaug plasmid were used per transfection .

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

(B, C) Smaug downregulates reporter expression. Mean values of the relative levels of SNAP-GUS reporter (SNAP-GUS/GFP-SNAP ratio, in arbitrary units) in extracts of transfected Cl8 cells in the absence (black) or presence of λN-SNAP-Smaug (red), λN-SNAP (pale grey) or SNAP-Smaug (dark grey). See Fig EV3A for the representative gel and Fig EV3B which shows that λN-SNAP-Smaug has no effect on a SNAP-GUS construct that lacks the 5BoxB. N=3 (biological replicates). See also Fig EV3A-C. Here and in D-F: SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug. The error bars correspond to the standard deviation of the mean (SD). Statistical analysis was done by a Kruskal-Wallis test by ranks followed by a Dunn test and p values are: black bracket <0.05, red bracket <0.01, green bracket <0.0001. Here and in D-E: 15 ng of pAct.λN-SNAP-smaug plasmid were used per transfection.


, E ) HH / SMO reduces Smaug levels and its overall negative effect on reporter expression . Mean values of the relative levels of reporter expression ( determined as above ) ( D ) or of λN - SNAP - Smaug ( ratio λN - SNAP - Smaug / GFP - SNAP ) ( E ) in the absence of SMO constructs ( red ) or in the presence of SMOWT - HA ( green ) , SMOPKA - SD - HA ( blue ) or SMO ∆ 958 - HA ( yellow ) , without HH ( plain boxes ) or in presence of HH ( striped boxes ) . N = 6 ( independent biological replicates ) , except for " no SMO , no HH " and " SMOWT with HH " conditions where n = 10 ( independent biological replicates ) . A representative gel is shown in Fig EV3D . Note that SMOWT - HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant . See also Fig EV3E - F .

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, E) HH/SMO reduces Smaug levels and its overall negative effect on reporter expression. Mean values of the relative levels of reporter expression (determined as above) (D) or of λN-SNAP-Smaug (ratio λN-SNAP-Smaug/GFP-SNAP) (E) in the absence of SMO constructs (red) or in the presence of SMOWT-HA (green), SMOPKA-SD-HA (blue) or SMO∆958-HA (yellow), without HH (plain boxes) or in presence of HH (striped boxes). N=6 (independent biological replicates), except for "no SMO, no HH" and "SMOWT with HH" conditions where n=10 (independent biological replicates). A representative gel is shown in Fig EV3D. Note that SMOWT-HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant. See also Fig EV3E-F.


( F ) HH / SMO reduces Smaug ' s intrinsic repressive ability . A similar experiment was conducted with different amounts of λN - SNAP - smaug expressing plasmid ( ranging from 0 to 50 ng ) as indicated ) . The mean values of the relative levels of reporter ( y - axis ) are plotted against the relative levels of λN - SNAP - Smaug ( x - axis ) . SNAP - GUS / GFP - SNAP ratios were set to 1 in cells expressing λN ‐ SNAP - Smaug in absence of SMOPKA - SD / HH . The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with ( blue ) and without ( red ) SMOPKA - SD / HH , respectively . N = 3 ( biological triplicates ) . See Fig EV3H and also a representative gel in Fig EV3G .

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(F) HH/SMO reduces Smaug's intrinsic repressive ability. A similar experiment was conducted with different amounts of λN-SNAP-smaug expressing plasmid (ranging from 0 to 50 ng) as indicated). The mean values of the relative levels of reporter (y-axis) are plotted against the relative levels of λN-SNAP-Smaug (x-axis). SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug in absence of SMOPKA-SD/HH. The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with (blue) and without (red) SMOPKA-SD/HH, respectively. N=3 (biological triplicates). See Fig EV3H and also a representative gel in Fig EV3G.


( A ) HH / SMO promotes slow - migrating forms of Smaug . Western blot analysis of Cl8 cells that transiently express HA - SmaugWT alone , or together with either SMOWT - GFP or SMOPKA - SD - GFP , in the presence or in absence of HH . GMAP serves as a loading control . Here and in the other panels , the antibodies used are indicated on the left . Here and in ( B , C ) , the black arrows indicate the unphosphorylated form of HA - SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA - SmaugWT . NT : not transfected . See a phosphatase assay in Fig EV4A .

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(A) HH/SMO promotes slow-migrating forms of Smaug. Western blot analysis of Cl8 cells that transiently express HA-SmaugWT alone, or together with either SMOWT-GFP or SMOPKA-SD-GFP, in the presence or in absence of HH. GMAP serves as a loading control. Here and in the other panels, the antibodies used are indicated on the left. Here and in (B,C), the black arrows indicate the unphosphorylated form of HA-SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA-SmaugWT. NT: not transfected. See a phosphatase assay in Fig EV4A.


( B ) Smaug phosphorylation requires its interaction with SMO . Western blot analysis of Cl8 cells expressing HA - SmaugWT , in presence of HH , in combination with SMO ∆ 958 - GFP , SMOPKA - SD - GFP or SMOPKA - SD , ∆ 978 - GFP , as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Smaug phosphorylation requires its interaction with SMO. Western blot analysis of Cl8 cells expressing HA-SmaugWT, in presence of HH, in combination with SMO∆958-GFP, SMOPKA-SD-GFP or SMOPKA-SD, ∆978-GFP, as indicated.


( C ) FU controls Smaug phosphorylation . Extracts of Cl8 cells expressing SNAP - Smaug with SMOPKA - SD FU - SD - GFP ( with HH ) in combination with GFP - FUWT , GAP - GFP - FUWT , Myc - FUEE or GAP - null - FUDANA ( as indicated ) were labeled for the SNAP tag before electrophoresis . After quantification of the gels , the percentage ( % ) of phosphorylated Smaug ( % of phosphorylated forms of SNAP - Smaug total amounts of SNAP - Smaug ) was estimated . In C to E , the mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom , and the statistical analysis was done by one tailed bivariate Wilcoxon rank test . See also fig EV4C , D .

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(C) FU controls Smaug phosphorylation. Extracts of Cl8 cells expressing SNAP-Smaug with SMOPKA-SD FU-SD-GFP (with HH) in combination with GFP-FUWT, GAP-GFP-FUWT, Myc-FUEE or GAP-null-FUDANA (as indicated) were labeled for the SNAP tag before electrophoresis. After quantification of the gels, the percentage (%) of phosphorylated Smaug (% of phosphorylated forms of SNAP-Smaug total amounts of SNAP-Smaug) was estimated. In C to E, the mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom, and the statistical analysis was done by one tailed bivariate Wilcoxon rank test. See also fig EV4C, D.


( D ) Phosphorylation of Smaug by activated FU requires the co - expression of SMO . Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN - SNAP - Smaug with GAP - GFP - FUWT and SMOWT alone or together ( as indicated ) N = 3 ( billogical triplicates ) . Note that the effect of GAP - GFP - FUWT and SMOWT together is lower than the one observed with GAP - GFP - FUWT and SMOPKA - SD FU - SD - GFP together ( in panel C ) . This could have multiple nonexclusive causes , including the fact that SMOWT is present at much lower levels than SMOPKA - SD FU - SD [ 38 ] and / or the involvement of another kinase . See also Fig EV4B . Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2 .

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(D) Phosphorylation of Smaug by activated FU requires the co-expression of SMO. Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN-SNAP-Smaug with GAP-GFP-FUWT and SMOWT alone or together (as indicated) N=3 (billogical triplicates). Note that the effect of GAP-GFP-FUWT and SMOWT together is lower than the one observed with GAP-GFP-FUWT and SMOPKA-SD FU-SD-GFP together (in panel C). This could have multiple nonexclusive causes, including the fact that SMOWT is present at much lower levels than SMOPKA-SD FU-SD [38] and/or the involvement of another kinase. See also Fig EV4B. Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2.


( E ) Forcing the interaction of Smaug with activated FU leads to a SMO - independent phosphorylation of Smaug . Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN - SNAP - Smaug with GFP - FU , FU - SBR , FUEE - SBR or FUDANA - SBR , with or without HH , as indicated . N = 3 ( biological triplicates ) . See also Fig EV4C and D . Note that the levels of Smaug phosphorylation observed in presence of FUDANA - SBR are not lower than those seen with GFP - FU . This suggests an incomplete inhibition of endogenous FU , which may be due to the trapping of FUDANA - SBR by Smaug . See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D .

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(E) Forcing the interaction of Smaug with activated FU leads to a SMO-independent phosphorylation of Smaug. Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN-SNAP-Smaug with GFP-FU, FU-SBR, FUEE-SBR or FUDANA-SBR, with or without HH, as indicated. N=3 (biological triplicates). See also Fig EV4C and D. Note that the levels of Smaug phosphorylation observed in presence of FUDANA-SBR are not lower than those seen with GFP-FU. This suggests an incomplete inhibition of endogenous FU, which may be due to the trapping of FUDANA-SBR by Smaug. See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D.


( A and C ) Effect of FUEE - SBR on Smaug phosphorylation and levels . Extracts of Cl8 cells transfected with λN - SNAP - smaug ( 25 ng ) and SNAP - GUS - 5BoxB ( 300 ng ) plasmids in absence or with different amounts of FUEE expressing plasmid ( ranging from 15 to 60 μg , as indicated ) were analyzed by electrophoresis before quantification as above . See a reprenstive gel in Fig EV5A . The % of phosphorylated SNAP - Smaug protein ( determined as in Fig 3 ) ( A ) are represented as a function of the levels of FUEE - SBR . The mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom . The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value = 0 . 05 ( * ) . Here CLIP - GUS was used as an internal control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The % of phosphorylated SNAP-Smaug protein (determined as in Fig 3) (A) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.


( B ) Effect of FUEE - SBR on Smaug activity . The relative levels of reporter expression obtained in the same experiment as in ( A - C ) are represented as a function of the % of phosphorylated Smaug . Color of data points correspond to ng of transfected FUEE - SBR in A - C .

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(B) Effect of FUEE-SBR on Smaug activity. The relative levels of reporter expression obtained in the same experiment as in (A-C) are represented as a function of the % of phosphorylated Smaug. Color of data points correspond to ng of transfected FUEE-SBR in A-C.


( A and C ) Effect of FUEE - SBR on Smaug phosphorylation and levels . Extracts of Cl8 cells transfected with λN - SNAP - smaug ( 25 ng ) and SNAP - GUS - 5BoxB ( 300 ng ) plasmids in absence or with different amounts of FUEE expressing plasmid ( ranging from 15 to 60 μg , as indicated ) were analyzed by electrophoresis before quantification as above . See a reprenstive gel in Fig EV5A . The relative levels of of λN - SNAP - Smaug protein ( C ) ( determined as in Fig 2 ) are represented as a function of the levels of FUEE - SBR . The mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom . The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value = 0 . 05 ( * ) . Here CLIP - GUS was used as an internal control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The relative levels of of λN-SNAP-Smaug protein (C) (determined as in Fig 2) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.


( D ) Effect of FUEE - SBR on Smaug subcellular localization . Representative fluorescent images of Cl8 cells expressing GFP - SmaugWT ( 1 ) , FUEE - SBR - mCherry ( 2 ) , FUDANA - SBR - mCh ( 3 ) or expressing GFP - SmaugWT together with FUEE - SBR - mCherry ( 4 - 4 ' ' ) or GFP - SmaugWT together with FUDANA - SBR - mCh ( 5 - 5 ' ' ) . The merge images in 4 " and 5 " show GFP - SmaugWT in green and FUEE - SBR - mCh or FUDANA - SBR - mCh in red . ( 6 ) In presence of FUEE - SBR , 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci . In presence of FUDANA - SBR , 100 % of the cotransfected cells ( n = 241 ) show Smaug and FU distributed in bodies and colocalized . 250 ng of plasmid were used for each construct . Scale bar ( shown in D1 , identical for all panels ) :

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(D) Effect of FUEE-SBR on Smaug subcellular localization.Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT (1), FUEE-SBR-mCherry (2), FUDANA-SBR-mCh (3) or expressing GFP-SmaugWT together with FUEE-SBR-mCherry (4-4'') or GFP-SmaugWT together with FUDANA-SBR-mCh (5-5''). The merge images in 4" and 5" show GFP-SmaugWT in green and FUEE-SBR-mCh or FUDANA-SBR-mCh in red. (6) In presence of FUEE-SBR, 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci. In presence of FUDANA-SBR, 100% of the cotransfected cells (n=241) show Smaug and FU distributed in bodies and colocalized. 250 ng of plasmid were used for each construct. Scale bar (shown in D1, identical for all panels): 10μ


( A ) Effect of FUEE - SBR and HH / SMO on endogeneous Smaug target mRNAs in Cl8 cells . Relative mRNA levels estimated using semi - quantitative RT - PCR for various Smaug ' s targets in Cl8 cells expressing FUEE - SBR ( black ) , FUEE ( dark grey ) , or FUDANA - SBR ( light grey ) were reported with respect to levels of a GFP control . mRNA levels were set to 1 for the GFP control . The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3 . See also Fig EV5E . Here and in Fig EV5D : RNA encoding mitochondrial proteins are written in blue . N = 3 ( biological triplicates ) and the quantifications were repeated 3 times on each sample . The results are presented as the means ± standard error . The differences between groups were assessed using Student ' s t - tests with Prism V8 . 2 . 1 software . A p value < 0 . 05 was considered to indicate a statistically significant

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(A) Effect of FUEE-SBR and HH/SMO on endogeneous Smaug target mRNAs in Cl8 cells. Relative mRNA levels estimated using semi-quantitative RT-PCR for various Smaug's targets in Cl8 cells expressing FUEE-SBR (black) , FUEE (dark grey) , or FUDANA-SBR (light grey) were reported with respect to levels of a GFP control. mRNA levels were set to 1 for the GFP control. The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3. See also Fig EV5E. Here and in Fig EV5D: RNA encoding mitochondrial proteins are written in blue. N=3 (biological triplicates) and the quantifications were repeated 3 times on each sample.The results are presented as the means ± standard error. The differences between groups were assessed using Student's t-tests with Prism V8.2.1 software. A p value <0.05 was considered to indicate a statistically significant difference


( B ) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc . Relative levels of Smaug ' s targets mRNAs in MS1096 ; UAS - hh or hhts and their respective controls ( as in Fig EV5X ) . N = 3 , with xx disc for each phenotype .

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(B) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc. Relative levels of Smaug's targets mRNAs in MS1096; UAS-hh or hhts and their respective controls (as in Fig EV5X). N=3, with xx disc for each phenotype.


( C ) Effect of smaug mutation on [ fu1 ] wing class distribution . Percent distribution of phenotypic classes ( defined in Appendix Figure S4 ) in fu1 males in presence of zero ( fu1 ) , one ( fu1 ; ; smaug47 / + ) or two copies ( fu1 ; ; smaug47 / smaug47 ) of the smaug47 allele . The wings were double - blindly classified in " weak " and " strong " according to the LV3 - V4 defects ( see Fig EV6B ) . * Chi2 statistical analysis gave a p of 0 , 015 for fu1 and fu1 ; ; smaug47 / + distributions and of 0 , 0024 when comparing fu1 ; ; smaug47 / + to fu1 ; ; smaug47 / smaug47 .

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(C) Effect of smaug mutation on [fu1] wing class distribution. Percent distribution of phenotypic classes (defined in Appendix Figure S4) in fu1 males in presence of zero (fu1), one (fu1;;smaug47/+) or two copies (fu1;; smaug47/smaug47) of the smaug47 allele. The wings were double-blindly classified in "weak" and "strong" according to the LV3-V4 defects (see Fig EV6B). *Chi2 statistical analysis gave a p of 0,015 for fu1 and fu1;;smaug47/+ distributions and of 0,0024 when comparing fu1;;smaug47/+ to fu1;; smaug47/smaug47.


A . Volcano plot showing the fold change ( y - axis ) versus adjusted ( adj . ) p - value ( x - axis ) of the beta cell transcriptomes between chow and HFD - fed mice ( 16 weeks ) . Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj . P value < 0 . 01 ( two - tailed paired student ' s t - test ) . Genes with > 10 fold upregulated expression are labeled with their name . Pbk gene expression is indicated by the arrow . RNAseq data was available from https : / / doi . org / 10 . 1371 / journal . pone . 0213299 . t004 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.Volcano plot showing the fold change (y-axis) versus adjusted (adj.) p-value (x-axis) of the beta cell transcriptomes between chow and HFD-fed mice (16 weeks). Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj. P value < 0.01 (two-tailed paired student's t-test). Genes with >10 fold upregulated expression are labeled with their name. Pbk gene expression is indicated by the arrow. RNAseq data was available from https://doi.org/10.1371/journal.pone.0213299.t004.


L . Pbk KD in INS - 1 cells suppresses cell growth . Western blot data showed the decreased Pbk expression level in INS - 1 cells with Pbk - targeted shRNA transduction . Cell growth curve was from three independent experimets ( n = 3 ) . * * * P < 0 . 0001 ( Vector : shRNA - # 1 ) , * P = 0 . 0181 ( Vector : shRNA - # 5 ) , ( Two way ANOVA ) . M . The role of ectopic expression of Pbk - WT and Pbk - Mut on INS - 1 cell growth . Western blot data showed the overexpression of V5 - flaged wild type and mutant Pbk in INS - 1 cells . Cell growth curve was from three independent experimets ( n = 3 ) . * * P = 0 . 0069 ( Two way ANOVA ) . ns , not statistically significant difference .

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

L. Pbk KD in INS-1 cells suppresses cell growth. Western blot data showed the decreased Pbk expression level in INS-1 cells with Pbk-targeted shRNA transduction. Cell growth curve was from three independent experimets (n=3). ***P < 0.0001 (Vector:shRNA-#1), *P = 0.0181 (Vector:shRNA-#5) , (Two way ANOVA). M. The role of ectopic expression of Pbk-WT and Pbk-Mut on INS-1 cell growth. Western blot data showed the overexpression of V5-flaged wild type and mutant Pbk in INS-1 cells. Cell growth curve was from three independent experimets (n=3). **P = 0.0069 (Two way ANOVA). ns, not statistically significant difference.


F . Islet number in each pancreas section on 11 - week - old male PbkKI / KI and PbkWT / WT mice ( n = 4 for each group ) . * P = 0 . 0443 ( two - tailed unpaired student ' s t - test ) . G . Beta cell mass comparison between 11 - week - old male PbkKI / KI and PbkWT / WT mice ( n = 4 for each group ) . * * P = 0 . 0015 ( two - tailed unpaired student ' s t - test ) .

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F. Islet number in each pancreas section on 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). *P = 0.0443 (two-tailed unpaired student's t-test). G. Beta cell mass comparison between 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). **P = 0.0015 (two-tailed unpaired student's t-test).


K , L . Representative images of double immuno - staining for insulin ( green ) and BrdU ( red ) of pancreas from PbkWT / WT ( K ) or PbkKI / KI ( L ) mice with 5 weeks HFD feeding . Islet area was circled with white dashed line . Nuclei were labeled by DAPI ( blue ) . Scale bar : 50 μm .

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K, L. Representative images of double immuno-staining for insulin (green) and BrdU (red) of pancreas from PbkWT/WT (K) or PbkKI/KI (L) mice with 5 weeks HFD feeding. Islet area was circled with white dashed line. Nuclei were labeled by DAPI (blue). Scale bar: 50 μm.


E , F . JunD KD in INS - 1 cells using shRNA upregulated Pbk protein levels ( E ) and mRNA levels ( F ) . qPCR data was from three independent experimets ( n = 3 ) . * * * P = 0 . 0001 ( shRNA - # 1 ) , * * * P = 0 . 0007 ( shRNA - # 2 ) ( two - tailed unpaired student ' s t - test ) . G , H . JunD KD in PIME cells using shRNA increased Pbk protein levels ( G ) and mRNA levels ( H ) . qPCR data was from three independent experimets ( n = 3 ) . * P = 0 . 0236 ( shRNA - # 1 ) , * P = 0 . 0175 ( shRNA - # 2 ) ( two - tailed unpaired student ' s t - test ) .

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E, F. JunD KD in INS-1 cells using shRNA upregulated Pbk protein levels (E) and mRNA levels (F). qPCR data was from three independent experimets (n=3).***P = 0.0001 (shRNA-#1), ***P = 0.0007 (shRNA-#2) (two-tailed unpaired student's t-test). G, H. JunD KD in PIME cells using shRNA increased Pbk protein levels (G) and mRNA levels (H). qPCR data was from three independent experimets (n=3).*P = 0.0236 (shRNA-#1), *P = 0.0175 (shRNA-#2) (two-tailed unpaired student's t-test).


N . JunD overexpression in PIME cells suppressed Pbk protein levels ( M , Lane 2 vs Lane 1 ) , but did not affect Pbk expression in menin - null PIME cells ( M , Lane 4 vs Lane 3 ) .

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N. JunD overexpression in PIME cells suppressed Pbk protein levels (M, Lane 2 vs Lane 1), but did not affect Pbk expression in menin-null PIME cells (M, Lane 4 vs Lane 3).


J , K . The effect of T5224 on the binding of JunD , menin , HDAC3 , and acetylated histone3 at the Pbk locus in PIME cells ( J ) or INS - 1 cells ( K ) . Three independent experimets ( n = 3 ) . * P = 0 . 0490 ( J , JunD ) , * P = 0 . 0479 ( J , Menin ) , * P = 0 . 0154 ( J , HDAC3 ) , * * P = 0 . 0029 ( J , Ac - H3 ) , * P = 0 . 0104 ( K , JunD ) , * * P = 0 . 0024 ( K , Menin ) , * * * P = 0 . 0002 ( K , HDAC3 ) , * * * P = 0 . 0001 ( K , Ac - H3 ) , ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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

J, K. The effect of T5224 on the binding of JunD, menin, HDAC3, and acetylated histone3 at the Pbk locus in PIME cells (J) or INS-1 cells (K). Three independent experimets (n=3). *P = 0.0490 (J, JunD), *P = 0.0479 (J, Menin), *P = 0.0154 (J, HDAC3), **P = 0.0029 (J, Ac-H3), *P = 0.0104 (K, JunD), **P = 0.0024 (K, Menin), ***P = 0.0002 (K, HDAC3), ***P = 0.0001 (K, Ac-H3), (two-tailed unpaired student's t-test). ns, not statistically significant difference.


F . Mouse primary islets treated with MI - 503 for 3 days were subjected ChIP assay to detect the binding of menin , HDAC3 , JunD , as well as histone acetylation level at the Pbk locus . Three independent experimets ( n = 3 ) . * P = 0 . 0218 ( Menin ) , * P = 0 . 0145 ( HDAC3 ) , * P = 0 . 0184 ( Ac - H3 ) ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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

F. Mouse primary islets treated with MI-503 for 3 days were subjected ChIP assay to detect the binding of menin, HDAC3, JunD, as well as histone acetylation level at the Pbk locus. Three independent experimets (n=3). *P = 0.0218 (Menin), *P = 0.0145 (HDAC3), *P = 0.0184 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.


G . MI - 503 increases proliferation of menin - expressing PIME cells but not menin - null PIME cells . Three independent experimets ( n = 3 ) . * * * P < 0 . 0001 ( Two - way ANOVA ) .

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

G. MI-503 increases proliferation of menin-expressing PIME cells but not menin-null PIME cells. Three independent experimets (n=3). ***P < 0.0001 (Two-way ANOVA).


K - M . 5 days of MI - 503 treatment impacts the expression of Pbk at protein level ( K ) and mRNA level ( L ) , as well as ki67 mRNA levels ( M ) in human primary islets ( donor number is four ) . qPCR data was from three independent experimets ( n = 3 ) . * * P = 0 . 0036 ( L , 10 nM ) , * * P = 0 . 0024 ( L , 50 nM ) , * * P = 0 . 0073 ( L , 100 nM ) , * P = 0 . 0237 ( M , 50 nM ) ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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

K-M. 5 days of MI-503 treatment impacts the expression of Pbk at protein level (K) and mRNA level (L), as well as ki67 mRNA levels (M) in human primary islets (donor number is four). qPCR data was from three independent experimets (n=3). **P = 0.0036 (L, 10 nM), **P = 0.0024 (L, 50 nM), **P = 0.0073 (L, 100 nM), *P = 0.0237 (M, 50 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.


E - L . Representative images of insulin ( green ) and Ki67 ( red ) double staining on mouse islets with 8 wks MI treatment ( E - H ) , and insulin ( green ) or Pbk ( red ) double staining on mouse islets ( I - L ) . Nuclei were labeled by DAPI ( blue ) . Islet area was circled by white dashed line . Ki67 and Pbk staining are denoted by white arrows . Scale bars : 50 μm .

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E-L. Representative images of insulin (green) and Ki67 (red) double staining on mouse islets with 8 wks MI treatment (E-H), and insulin (green) or Pbk (red) double staining on mouse islets (I-L). Nuclei were labeled by DAPI (blue). Islet area was circled by white dashed line. Ki67 and Pbk staining are denoted by white arrows. Scale bars: 50 μm.


D , E . Double staining for insulin ( green ) and BrdU ( red ) of mice islets after 12 weeks of vehicle ( D ) or MI ( E ) administration . Islet area was circled by white dashed line . Nuclei were labeled by DAPI ( blue ) . BrdU staining is indicated by arrows . Scale bar : 50 μm . F , G . Quantification of BrdU positive beta cells ( F ) and beta cell mass ( G ) of MI - 463 treated HFD - fed mice or control mice . Five mice for each group , and at least 10 islet images per mouse were analyzed . * * P = 0 . 0059 , * P = 0 . 0103 ( two - tailed unpaired student ' s t - test ) .

O O O O 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 O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E. Double staining for insulin (green) and BrdU (red) of mice islets after 12 weeks of vehicle (D) or MI (E) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). BrdU staining is indicated by arrows. Scale bar:50 μm. F, G. Quantification of BrdU positive beta cells (F) and beta cell mass (G) of MI-463 treated HFD-fed mice or control mice. Five mice for each group, and at least 10 islet images per mouse were analyzed. **P = 0.0059, *P = 0.0103 (two-tailed unpaired student's t-test).


H , I . Double staining for insulin ( green ) and Pbk ( red ) of mice islets after 12 weeks of vehicle ( H ) or MI ( I ) administration . Islet area was circled by white dashed line . Nuclei were labeled by DAPI ( blue ) . Pbk staining is indicated by arrows . Scale bar : 50 μm . J . Quantification of Pbk staining - positive β - cells . Five mice for each group , and at least 10 islet images per mouse were analyzed . * P = 0 . 0120 ( two - tailed unpaired student ' s t - test ) .

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

H, I. Double staining for insulin (green) and Pbk (red) of mice islets after 12 weeks of vehicle (H) or MI (I) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). Pbk staining is indicated by arrows. Scale bar: 50 μm. J. Quantification of Pbk staining-positive β-cells. Five mice for each group, and at least 10 islet images per mouse were analyzed. *P = 0.0120 (two-tailed unpaired student's t-test).


K . Quantification of the percentage with pbk and BrdU co - staining cells among total Pbk positive cells in HFD - fed mouse islets . The mean values of five mice from 5 sections for each mouse were presented .

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

K. Quantification of the percentage with pbk and BrdU co-staining cells among total Pbk positive cells in HFD-fed mouse islets. The mean values of five mice from 5 sections for each mouse were presented.


O , P . GTT data for MI - 463 or vehicle treated PbkKI / KI or PbkWT / WT mice ( n = 3 for each group ) on 8 weeks HFD ( O ) ( * P = 0 . 0133 ( Two way ANOVA ) , ns , not statistically significant difference . ) and AUC of GTT data ( P ) ( * P = 0 . 0442 ( two - tailed unpaired student ' s t - test ) , ns , not statistically significant difference . ) .

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

O, P. GTT data for MI-463 or vehicle treated PbkKI/KI or PbkWT/WT mice (n=3 for each group) on 8 weeks HFD (O) (*P = 0.0133 (Two way ANOVA), ns, not statistically significant difference.) and AUC of GTT data (P) (*P = 0.0442 (two-tailed unpaired student's t-test), ns, not statistically significant difference.).


( A ) SARS - CoV - 2 genomic view showing the distribution of normalized 22nt small RNA reads from Caco - 2 and A549 - ACE2 cells at 24 and 48 hpi . The most abundant small RNAs are marked by the red and blue boxes . For all the experiments shown n = 2 .

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(A) SARS-CoV-2 genomic view showing the distribution of normalized 22nt small RNA reads from Caco-2 and A549-ACE2 cells at 24 and 48 hpi. The most abundant small RNAs are marked by the red and blue boxes. For all the experiments shown n=2.


( C ) Percentage of conservation along the nucleotide positions in the ORF7a among 4 , 055 , 609 sequenced SARS - CoV - 2 genomes . The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a . Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71 - 366 nt of ORF7a among 4 , 055 , 609 sequenced SARS - CoV - 2 genomes . Box plots display median ( line ) , first and third quartiles ( box ) , and 5th / 95th percentile value ( whiskers ) . Each dot represents the outliers . Two - tailed P values were calculated using the student ' s t - test .

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(C) Percentage of conservation along the nucleotide positions in the ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a. Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71-366 nt of ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. Box plots display median (line), first and third quartiles (box), and 5th /95th percentile value (whiskers). Each dot represents the outliers. Two-tailed P values were calculated using the student's t-test.


( A ) Log2 fold changes of the levels of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and COV2 - miR - O7a . 2 in DICER1 knockdown SARS - CoV - 2 - infected A549 - ACE2 cells compared to control cells analyzed by stem - loop RT - qPCR . Results from three independent replicates are shown .

(A) Log2 fold changes of the levels of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 in DICER1 knockdown SARS-CoV-2-infected A549-ACE2 cells compared to control cells analyzed by stem-loop RT-qPCR. Results from three independent replicates are shown.


( B ) In vitro kinetics of DICER1 mediated cleavage of pre - CoV2 - miR - O7a stem - loop precursor , pre - CoV2 - miR - O7a with mutations that prevent the formation of stem - loop structure and pre - hsa - miR21 . Folded conformations for the three precursor molecules are shown . Fold change in the production of mature CoV2 - miR - O7a . 2 is shown in presence of DICER1 compared to control reaction ( no DICER1 ) as measure by stem - loop RT - qPCR . Bars and error bars represent the average and standard deviation from three independent experiments .

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(B) In vitro kinetics of DICER1 mediated cleavage of pre-CoV2-miR-O7a stem-loop precursor, pre-CoV2-miR-O7a with mutations that prevent the formation of stem-loop structure and pre-hsa-miR21. Folded conformations for the three precursor molecules are shown. Fold change in the production of mature CoV2-miR-O7a.2 is shown in presence of DICER1 compared to control reaction (no DICER1) as measure by stem-loop RT-qPCR. Bars and error bars represent the average and standard deviation from three independent experiments.


( C ) Expression levels of the virus - derived miRNAs as a percentage of the viral genome . Absolute quantification of virus - derived miRNAs and viral genome from infected A549 - ACE2 cells was performed using two spike - in ( see methods ) . Line represents the average and individual dots represent data from two independent experiments .

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(C) Expression levels of the virus-derived miRNAs as a percentage of the viral genome. Absolute quantification of virus-derived miRNAs and viral genome from infected A549-ACE2 cells was performed using two spike-in (see methods). Line represents the average and individual dots represent data from two independent experiments.


( D ) copy number per cell of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and CoV2 - miR - O7a . 2 in infected A549 - ACE2 cells quantified using a synthetic small RNA spike - in standard curve by stem - loop RT - qPCR . Levels of hsa - miR - let - 7a were normalized for the percentage of infected cells . Bars and error bars represent the average and standard deviation from at least three independent experiments .

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(D) copy number per cell of hsa-miR-let-7a, CoV2-miR-O7a.1 and CoV2-miR-O7a.2 in infected A549-ACE2 cells quantified using a synthetic small RNA spike-in standard curve by stem-loop RT-qPCR. Levels of hsa-miR-let-7a were normalized for the percentage of infected cells. Bars and error bars represent the average and standard deviation from at least three independent experiments.


( E ) Loading of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and COV2 - miR - O7a . 2 into AGOs as measured by stem - loop RT - qPCR and analyzed as a percentage of input from the immunoprecipitates ( IPs ) of either pan - AGO IP or control IgG IP from infected A549 - ACE2 cells . Levels of miRNA were normalized for the percentage of infected cells .

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(E) Loading of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 into AGOs as measured by stem-loop RT-qPCR and analyzed as a percentage of input from the immunoprecipitates (IPs) of either pan-AGO IP or control IgG IP from infected A549-ACE2 cells. Levels of miRNA were normalized for the percentage of infected cells.