( a ) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 and UOK257 - 2 cell extracts and relative levels of the active forms as measured by GST - RILP pull down . HSC70 is used as a loading control . Graphs show quantification of bound Rab7 / Rab34 from 3 independent experiments , error bars show S . E . M . , * = p < 0 . 05 .

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(a) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 and UOK257-2 cell extracts and relative levels of the active forms as measured by GST-RILP pull down. HSC70 is used as a loading control. Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments, error bars show S.E.M., * = p<0.05.


( b ) ( top ) Representative confocal immunofluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257 and UOK257 - 2 cells . Dashed line highlights cell periphery in UOK257 - 2 cells . Scale bar = 10μm . ( bottom left ) Graph showing cumulative distribution of LAMP1 intensity in UOK257 vs UOK257 - 2 cells . Error bars show S . E . M . from 30 cells in 3 replicates . ( bottom right ) Graph showing Lysotracker - Red PCC decay with time from confocal movies of UOK257 and UOK257 - 2 cells . Mean values from 5 cells . Error bars show S . E . M .

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(b) (top) Representative confocal immunofluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257 and UOK257-2 cells. Dashed line highlights cell periphery in UOK257-2 cells. Scale bar = 10μm. (bottom left) Graph showing cumulative distribution of LAMP1 intensity in UOK257 vs UOK257-2 cells. Error bars show S.E.M. from 30 cells in 3 replicates. (bottom right) Graph showing Lysotracker-Red PCC decay with time from confocal movies of UOK257 and UOK257-2 cells. Mean values from 5 cells. Error bars show S.E.M.


( c ) Widefield immunofluorescence images showing early endosomes ( EEA1 ) and Golgi ( golgin - 97 ) in UOK257 and UOK257 - 2 cells . Scale bar = 10μm .

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(c) Widefield immunofluorescence images showing early endosomes (EEA1) and Golgi (golgin-97) in UOK257 and UOK257-2 cells. Scale bar = 10μm.


( d ) Western blot showing FLCN and FLCNΔDENN expression in stable UOK257 derived cell lines .

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(d) Western blot showing FLCN and FLCNΔDENN expression in stable UOK257 derived cell lines.


( e ) Representative widefield fluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257 , UOK257 - FLCN and UOK257 - FLCNΔDENN cells . Scale bar = 10μm . Graphs showing cumulative distribution of LAMP1 intensity in UOK257 , UOK257 - FLCN and UOK257 - FLCNΔDENN cells . Error bars show S . E . M from 30 cells in 3 replicates .

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(e) Representative widefield fluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Scale bar = 10μm. Graphs showing cumulative distribution of LAMP1 intensity in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Error bars show S.E.M from 30 cells in 3 replicates.


( f ) ( left ) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 , UOK257 - FLCN and UOK257 - FLCNΔDENN cells and relative levels of activity as measured by GST - RILP pull down . HSC70 is used as a loading control . ( right ) Graphs show quantification of bound Rab7 / Rab34 from 3 independent experiments ( p < 0 . 05 , * * * = p < 0 . 001 ) .

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(f) (left) Western blot showing endogenous Rab7 and Rab34 expression in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells and relative levels of activity as measured by GST-RILP pull down. HSC70 is used as a loading control. (right) Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments (p<0.05, *** = p<0.001).


( g ) Western blot showing endogenous RILP expression in the indicated cell lines . Graph shows quantification of RILP expression from 3 independent experiments ( * = p < 0 . 05 , * * * = p < 0 . 001 ) .

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(g) Western blot showing endogenous RILP expression in the indicated cell lines. Graph shows quantification of RILP expression from 3 independent experiments (* = p<0.05, *** = p<0.001).


( A ) Immunoblotting for Dnmt1 , Dmnt3a and Dmnt3b in the spinal cords of AR - 97Q , AR - 24Q , and wild - type mice . ( B ) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up - regulated in AR - 97Q mice ( n = 3 ) . Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) .

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(A) Immunoblotting for Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of AR-97Q, AR-24Q, and wild-type mice. (B) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up-regulated in AR-97Q mice (n = 3). Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild-type, AR-24Q, and AR-97Q mice (n = 3).


( C ) Quantification of the Dnmts mRNA levels of the spinal cord in wild - type , AR - 24Q , and AR - 97Q mice using RT - qPCR ( n = 3 ) .

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(C) Quantification of the Dnmts mRNA levels of the spinal cord in wild-type, AR-24Q, and AR-97Q mice using RT-qPCR (n = 3).


( D and E ) Representative immunohistochemical images depicting the expression of Dnmt1 , Dmnt3a and Dmnt3b in the spinal cords of male AR - 97Q , AR - 24Q , and wild - type mice and quantification of the immunoreactivity for Dnmts ( n = 25 neurons from three mice of each group ) .

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(D and E) Representative immunohistochemical images depicting the expression of Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of male AR-97Q, AR-24Q, and wild-type mice and quantification of the immunoreactivity for Dnmts (n = 25 neurons from three mice of each group).


( F ) Double - immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR - 97Q mouse .

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(F) Double-immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR-97Q mouse.


( G ) Immunoblotting for Dnmt1 of the skeletal muscle of wild - type and AR - 97Q mice . ( H ) Quantification of the signal intensity of Dnmt1 bands ( n = 3 ) .

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(G) Immunoblotting for Dnmt1 of the skeletal muscle of wild-type and AR-97Q mice. (H) Quantification of the signal intensity of Dnmt1 bands (n = 3).


( A ) Immunoblotting of NSC34 cells expressing AR - 24Q or AR - 97Q ( NSC24Q and NSC97Q , respectively ) that were treated with DHT for Dnmt1 , Dnmt3a and Dnmt3b , and quantification of the signal intensities of bands corresponding to Dnmt1 , Dnmt3a and Dnmt3b ( n = 3 ) .

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(A) Immunoblotting of NSC34 cells expressing AR-24Q or AR-97Q (NSC24Q and NSC97Q, respectively) that were treated with DHT for Dnmt1, Dnmt3a and Dnmt3b, and quantification of the signal intensities of bands corresponding to Dnmt1, Dnmt3a and Dnmt3b (n = 3).


( B ) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT - qPCR ( n = 3 ) .

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(B) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT-qPCR (n = 3).


( C ) Protein levels of Dnmt1 , Dnmt3a and Dnmt3b in DHT - treated and - untreated NSC97Q cells by Western blotting analysis .

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(C) Protein levels of Dnmt1, Dnmt3a and Dnmt3b in DHT-treated and -untreated NSC97Q cells by Western blotting analysis.


( D ) RT - qPCR analysis of Dnmt1 in DHT - treated and - untreated NSC97Q cells ( n = 3 ) .

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(D) RT-qPCR analysis of Dnmt1 in DHT-treated and -untreated NSC97Q cells (n = 3).


( E , F ) Quantitative assessment of cell viability changes due to the siRNA - mediated knock down of Dnmt1 , Dnmt3a or Dnmt3b in NSC97Q cells with DHT ( E ) or without DHT ( F ) using the WST - 8 assay ( n = 3 ) .

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(E, F) Quantitative assessment of cell viability changes due to the siRNA-mediated knock down of Dnmt1, Dnmt3a or Dnmt3b in NSC97Q cells with DHT (E) or without DHT (F) using the WST-8 assay (n = 3).


( G ) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST - 8 assay ( n = 3 per group ) .

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(G) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST-8 assay (n = 3 per group).


( H ) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1 , Dnmt3a and Dnmt3b .

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(H) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1, Dnmt3a and Dnmt3b.


( I , J ) Cell viability analysis with siRNA - mediated knockdown of Dnmts in DHT - untreated NSC97Q ( I ) and DHT - treated NSC24Q ( J ) .

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(I, J) Cell viability analysis with siRNA-mediated knockdown of Dnmts in DHT-untreated NSC97Q (I) and DHT-treated NSC24Q (J).


( A - D ) Grip power ( A ) , body weight ( B ) , rotarod performance ( C ) ( Two - way ANOVA with Tukey ' s test ) and survival rate ( D ) ( Kaplan - Meier analysis and log - rank test ) of AR - 97Q mice treated with or without RG108 ( DMSO , n = 20 ; 0 . 5 mg / dL , n = 20 ; 2 . 0 mg / dL , n = 20 ) . All parameters were significantly improved in AR - 97Q mice treated with 2 . 0 mg / dL RG108 compared with those treated with DMSO : * P < 0 . 05 ( grip ) ; * P < 0 . 05 ( body weight ) ; * P < 0 . 05 ( rotarod ) ; and * P < 0 . 05 ( survival ) .

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(A-D) Grip power (A), body weight (B), rotarod performance (C) (Two-way ANOVA with Tukey's test) and survival rate (D) (Kaplan-Meier analysis and log-rank test) of AR-97Q mice treated with or without RG108 (DMSO, n =20; 0.5 mg/dL, n = 20; 2.0 mg/dL, n = 20). All parameters were significantly improved in AR-97Q mice treated with 2.0 mg/dL RG108 compared with those treated with DMSO: *P < 0.05 (grip); *P < 0.05 (body weight); *P < 0.05 (rotarod); and *P < 0.05 (survival).


( E ) Representative photograph of a 13 - week - old AR - 97Q mouse treated with DMSO ( left ) and an age - matched AR - 97Q mouse treated with 2 . 0 mg / dL RG108 ( right ) . AR - 97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO - treated AR - 97Q mouse of same age . The white compact masses ( arrow ) of mice head are the cement covering the surgical site .

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(E) Representative photograph of a 13-week-old AR-97Q mouse treated with DMSO (left) and an age-matched AR-97Q mouse treated with 2.0 mg/dL RG108 (right). AR-97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO-treated AR-97Q mouse of same age. The white compact masses (arrow) of mice head are the cement covering the surgical site.


( F , G ) Immunoblots ( F ) and relative intensities ( G ) of the Dnmt1 - immunoreactive bands of spinal cords from AR - 97Q mice treated with or without RG108 ( n = 3 per group ) .

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(F,G) Immunoblots (F) and relative intensities (G) of the Dnmt1-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3 per group).


( H , I ) Dnmt1 immunohistochemistry in spinal cord sections from AR - 97Q mice treated with or without RG108 ( n = 3 per group ) .

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(H,I) Dnmt1 immunohistochemistry in spinal cord sections from AR-97Q mice treated with or without RG108 (n = 3 per group).


( A ) Immunohistochemistry of the spinal cords of AR - 97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody . ( B ) Quantification of 1C2 - positive motor neurons in the spinal cords ( n = 5 per group ) .

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(A) Immunohistochemistry of the spinal cords of AR-97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody. (B) Quantification of 1C2-positive motor neurons in the spinal cords (n = 5 per group).


( C ) Immunoblotting of the spinal cords of AR - 97Q mice treated with or without RG108 for AR . ( D ) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR - 97Q mice treated with DMSO or RG108 ( n = 3 ) .

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(C) Immunoblotting of the spinal cords of AR-97Q mice treated with or without RG108 for AR. (D) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR-97Q mice treated with DMSO or RG108 (n = 3).


( E ) Relative mRNA expression levels of human AR in AR - 97Q mice spinal cord with or without RG108 treatment ( n = 3 ) .

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(E) Relative mRNA expression levels of human AR in AR-97Q mice spinal cord with or without RG108 treatment (n = 3).


Immunohistochemistry for ChAT in the spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) .

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Immunohistochemistry for ChAT in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).


quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) .

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quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).


( H , I ) Western blot analysis ( H ) and relative signal intensities ( I ) of the ChAT - immunoreactive bands of spinal cords from AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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(H,I) Western blot analysis (H) and relative signal intensities (I) of the ChAT-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3).


( A ) Immunoblotting of SH - SY5Y cells stably expressing AR - 24Q ( SH24Q ) or AR - 97Q ( SH97Q ) for Dnmt1 , Dnmt3a and Dnmt3b . ( B ) Densitometry quantification showing that Dnmt1 level was up - regulated in SH97Q cells compared with SH24Q cells ( n = 3 ) .

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(A) Immunoblotting of SH-SY5Y cells stably expressing AR-24Q (SH24Q) or AR-97Q (SH97Q) for Dnmt1, Dnmt3a and Dnmt3b. (B) Densitometry quantification showing that Dnmt1 level was up-regulated in SH97Q cells compared with SH24Q cells (n = 3).


( C , D ) Immunoblots for Dnmts of SH97Q with or without DHT treatment ( C ) and quantification data ( D ) ( n = 3 ) .

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(C, D) Immunoblots for Dnmts of SH97Q with or without DHT treatment (C) and quantification data (D) (n = 3).


( E ) Seven genes with DNA hyper - methylation in their CpG islands were identified by DNA methylation array analysis .

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(E) Seven genes with DNA hyper-methylation in their CpG islands were identified by DNA methylation array analysis.


( F ) The relative mRNA levels of CDC25B , GFRA3 , NPY , HES5 , SCTR , LEF1 - AS1 and CABS1 , normalized to beta - 2 microglobulin , in DHT - treated SH24Q and SH97Q cells were measured using RT - qPCR ( n = 3 ) .

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(F) The relative mRNA levels of CDC25B, GFRA3, NPY, HES5, SCTR, LEF1-AS1 and CABS1, normalized to beta-2 microglobulin, in DHT-treated SH24Q and SH97Q cells were measured using RT-qPCR (n = 3).


( G ) RT - qPCR analysis indicated the decrease of Hes5 mRNA level in DHT - treated SH97Q cells compared with DHT - untreated SH97Q cells ( n = 3 ) .

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(G) RT-qPCR analysis indicated the decrease of Hes5 mRNA level in DHT-treated SH97Q cells compared with DHT-untreated SH97Q cells (n = 3).


( H ) Methylation - specific PCR of SH24Q and SH97Q cells that were treated with DHT .

(H) Methylation-specific PCR of SH24Q and SH97Q cells that were treated with DHT.


( I ) Hes5 immunoreactivity of spinal motor neurons in wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) .

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(I) Hes5 immunoreactivity of spinal motor neurons in wild-type, AR-24Q, and AR-97Q mice (n = 3).


( J ) Hes5 mRNA levels in the spinal cords of wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) .

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(J) Hes5 mRNA levels in the spinal cords of wild-type, AR-24Q, and AR-97Q mice (n = 3).


DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108 .

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DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108.


HES5 mRNA levels of SH97Q cells treated with DHT and RG108 .

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HES5 mRNA levels of SH97Q cells treated with DHT and RG108.


DNA methylation levels of the Hes5 promoter region in DHT - treated NSC97Q cells treated with or without RG108 .

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DNA methylation levels of the Hes5 promoter region in DHT-treated NSC97Q cells treated with or without RG108.


mRNA levels of Hes5 ( n = 3 ) in DHT - treated NSC97Q cells treated with or without RG108 .

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mRNA levels of Hes5 (n = 3) in DHT-treated NSC97Q cells treated with or without RG108.


( E ) Hes5 mRNA levels in the spinal cords of AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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(E) Hes5 mRNA levels in the spinal cords of AR-97Q mice treated with or without RG108 (n = 3).


( F , G ) Hes5 immunoreactivity of spinal motor neurons in AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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(F, G) Hes5 immunoreactivity of spinal motor neurons in AR-97Q mice treated with or without RG108 (n = 3).


( A ) Viability of DHT - treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST - 8 assay ( n = 3 ) .

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(A) Viability of DHT-treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST-8 assay (n = 3).


( B ) Immunoblots of DHT - administrated NSC97Q cells treated with Hes5 siRNA ( n = 3 ) .

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(B) Immunoblots of DHT-administrated NSC97Q cells treated with Hes5 siRNA (n = 3).


( C ) Quantitative cell viability analysis revealed that siRNA - mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT ( n = 3 ) , as determined by the WST - 8 assay .

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(C) Quantitative cell viability analysis revealed that siRNA-mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT (n = 3), as determined by the WST-8 assay.


Cell viability measured with the WST - 8 assay of DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 3 ) .

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Cell viability measured with the WST-8 assay of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).


Cell viability measured with immunoblotting of DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 3 ) .

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Cell viability measured with immunoblotting of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).


( F ) Quantification of LDH release from DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 4 ) .

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(F) Quantification of LDH release from DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 4).


( G ) Immunoblots of DHT - administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR . ( H ) AR aggregation as quantified by densitometry ( n = 3 ) .

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(G) Immunoblots of DHT-administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR. (H) AR aggregation as quantified by densitometry (n = 3).


( A ) Immunoblots of NSC34 cells treated with Hes5 siRNA . ( B ) Quantification of protein levels of pSmad2 , Smad2 , phospho - IkBα and heat shock factor - 1 ( Hsf1 ) ( n = 3 ) .

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(A) Immunoblots of NSC34 cells treated with Hes5 siRNA. (B) Quantification of protein levels of pSmad2, Smad2, phospho-IkBα and heat shock factor-1 (Hsf1) (n = 3).


( C ) siRNA - mediated knockdown of Hes5 down - regulated Hes5 protein level ( n = 3 ) .

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(C) siRNA-mediated knockdown of Hes5 down-regulated Hes5 protein level (n = 3).


( D ) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment ( n = 3 ) .

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(D) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment (n = 3).


( E ) Protein levels of Smad2 , pSmad2 , and Hes5 in NSC97Q cells transfected with Hes5 vector ( n = 3 ) .

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(E) Protein levels of Smad2, pSmad2, and Hes5 in NSC97Q cells transfected with Hes5 vector (n = 3).


( F ) Immunoblots of Smad2 , pSmad2 , and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q ( n = 3 ) .

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(F) Immunoblots of Smad2, pSmad2, and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q (n = 3).


( G ) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q ( n = 3 ) .

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(G) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q (n = 3).


( H ) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q ( n = 6 ) .

(H) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q (n = 6).


( I ) Western blots of Smad2 , pSmad2 , and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5 - containing lentiviral vector .

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(I) Western blots of Smad2, pSmad2, and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5-containing lentiviral vector.


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

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


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

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


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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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


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

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

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

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O

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


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

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

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


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

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

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 .

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


Glial shNf - 1 / shTp53 sgAtg16l1 ( B ) 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 .

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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 .

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


G : Radar chart indicating 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 - / - ) .

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


H : Control or sgAtg7 glial shNf - 1 / shTp53 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 .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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