from fixed GFP - Rab34 transfected showing contacts between GFP - Rab34 positive peri - membranes lysosomes . ( d ) A cryo - immuno - EM image of a GFP - Rab34 transfected HeLa . GFP - Rab34 ( black dots ) is identified by immunostaining for GFP detection with gold conjugated A . lysosome is intimately associated with Rab34 positive membranes . Sites of contact are highlighted by arrows .

O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O

Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(d) A cryo-immuno-EM image of a GFP-Rab34 transfected HeLa cell. GFP-Rab34 (dark black dots) is identified by immunostaining for GFP and detection with gold conjugated protein A. The lysosome is intimately associated with Rab34 positive membranes. Sites of contact are highlighted by arrows.


( a ) Confocal immunofluorescence microscopy of HeLa transfected with Rab34 - dsRED - Mito , FLCN - GFP HA - FNIP2 . arrows highlight FLCN - GFP / HA - FNIP2 co - localisation distinct from Rab34 - mitochondria / FLCN - GFP localisation . Scale bar = 10μm .

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(a) Confocal immunofluorescence microscopy images of HeLa cells transfected with Rab34-dsRED-Mito, FLCN-GFP and HA-FNIP2. White arrows highlight FLCN-GFP/HA-FNIP2 co-localisation distinct from Rab34-mitochondria/FLCN-GFP localisation. Scale bar = 10μm.


( b ) Confocal immunofluorescence microscopy of Rab34 - dsRED - Mito ( WT ) FLCN - GFP DENN only or ΔDENN transfected HeLa showing DENN domain dependent recruitment to mitochondria . Scale bar = 10μm .

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(b) Confocal immunofluorescence microscopy images of Rab34-dsRED-Mito (WT) and FLCN-GFP DENN only or ΔDENN transfected HeLa cells showing DENN domain dependent recruitment to mitochondria. Scale bar = 10μm.


( c ) Western blot of GST - RILP pull down showing association of endogenous FLCN , Rab34 Rab7 with RILP . Input lane shows 1 % input extract .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O

(c) Western blot analysis of GST-RILP pull down showing association of endogenous FLCN, Rab34 and Rab7 with RILP. Input lane shows 1% input cell extract.


( d ) Western blot of GST - RILP pull down from UOK257 showing DENN domain dependent association of FLCN with RILP N - terminal , Rab binding domain ( RBD ) C - terminal fragments .

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(d) Western blot analysis of GST-RILP pull down from UOK257 cells showing DENN domain dependent association of FLCN with RILP N-terminal, Rab binding domain (RBD) and C-terminal fragments.


( e ) Western blot showing binding of His - tagged FLCN - DENN domain in E . coli extract to GST - RILP .

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(e) Western blot showing binding of His-tagged FLCN-DENN domain in E.coli extract to GST-RILP.


( f ) Western blot of GST - RILP pull down showing direct interaction between RILP GTP hydrolysis deficient ( Q111L ) Rab34 ( at 200nM , in presence of 1mM GTPγS ) FLCN - DENN domain ( 1μM ) , but not low nucleotide affinity ( T66N ) Rab34 . FLCN - DENN enhances association of RILP with Q111L Rab34 . Graph shows of relative RILP / Rab34 Q111L binding . Error bars show . * * = p < 0 . 01 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

(f) Western blot analysis of GST-RILP pull down showing direct interaction between RILP and GTP hydrolysis deficient (Q111L) Rab34 (at 200nM, in the presence of 1mM GTPγS) and FLCN-DENN domain (1μM), but not low nucleotide affinity (T66N) Rab34. FLCN-DENN enhances association of RILP with Q111L Rab34. Graph shows quantification of relative RILP / Rab34 Q111L binding. Error bars show SEM. ** = p<0.01.


( g ) Western blot of GST - RILP / Rab34 Q111L pull down ( Rab34 at 200nM ) showing that enhanced association of with RILP is achieved with low concentrations of FLCN .

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(g) Western blot analysis of GST-RILP / Rab34 Q111L pull down (Rab34 at 200nM) showing that enhanced association of with RILP is achieved with low concentrations of FLCN.


( h ) Western blot of GST - RILPpull down experiments with GTPγS loaded ( in presence of 1mM GTPγS ) GDP loaded ( in presence of 1mM GDP ) wildtype Rab34 , showing FLCN - DENN ( 500nM ) induced interaction with RILP .

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(h) Western blot analysis of GST-RILPpull down experiments with GTPγS loaded (in the presence of 1mM GTPγS) and GDP loaded (in the presence of 1mM GDP) wildtype Rab34, showing FLCN-DENN (500nM) induced interaction with RILP.


( i ) Western blot of a GFP - Trap immunoprecipitation experiment from HeLa showing that expression of HA - RILP promotes association of GFP - Rab34 HA - Rab7 .

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(i) Western blot analysis of a GFP-Trap immunoprecipitation experiment from HeLa cells showing that expression of HA-RILP promotes the association of GFP-Rab34 and HA-Rab7.


( a ) Western blot showing endogenous Rab7 Rab34 expression in UOK257 UOK257 - 2 extracts relative of active forms as measured by GST - RILP pull down . HSC70 is used as a loading control . Graphs show 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 showing intracellular distribution of LAMP1 in UOK257 UOK257 - 2 . Dashed highlights periphery in UOK257 - 2 . Scale bar = 10μm . ( bottom left ) Graph showing cumulative distribution of LAMP1 in UOK257 vs UOK257 - 2 . Error bars show S . E . M . from 30 in 3 replicates . ( bottom right ) Graph showing Lysotracker - Red PCC decay with time from confocal movies of UOK257 UOK257 - 2 . Mean values from 5 . Error bars show S . E . M .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O

(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 showing early endosomes ( EEA1 ) Golgi ( golgin - 97 ) in UOK257 UOK257 - 2 . 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 FLCNΔDENN expression in stable UOK257 derived lines .

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


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

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_LINE O B-CELL_LINE O B-GENEPROD B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-CELL_LINE O B-GENEPROD B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O

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

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O B-CELL_LINE O B-CELL_LINE O B-GENEPROD B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(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 indicated lines . Graph shows 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 Dmnt3b in spinal cords of AR - 97Q , AR - 24Q , wild - type mice . ( B ) Quantitative densitometry indicated that Dnmt1 spinal cord level was up - regulated in AR - 97Q mice ( n = 3 ) . Dnmt3a Dnmt3b spinal cord level was not significantly different among wild - type , AR - 24Q , AR - 97Q mice ( n = 3 ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O B-GENEPROD B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O B-GENEPROD O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O

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

O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

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

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O

(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 for Dnmt1 1C2 in 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 skeletal muscle of wild - type AR - 97Q mice . ( H ) of signal of Dnmt1 bands ( n = 3 ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE O O O O B-GENEPROD O O B-ORGANISM O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O

(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 expressing AR - 24Q or AR - 97Q ( NSC24Q NSC97Q , respectively ) that were treated with DHT for Dnmt1 , Dnmt3a Dnmt3b , of signal intensities of bands corresponding to Dnmt1 , Dnmt3a 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 of Dnmt1 in NSC24Q NSC97Q measured with RT - qPCR ( n = 3 ) .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

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


( C ) of Dnmt1 , Dnmt3a Dnmt3b in DHT - treated - untreated NSC97Q by Western blotting .

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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 of Dnmt1 in DHT - treated - untreated NSC97Q ( 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 viability changes due to siRNA - mediated knock down of Dnmt1 , Dnmt3a or Dnmt3b in NSC97Q with DHT ( E ) or without DHT ( F ) using WST - 8 ( 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 viability of NSC97Q treated with DHT RG108 using WST - 8 ( 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 treated with DHT RG108 for Dnmt1 , Dnmt3a Dnmt3b .

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


( I , J ) viability with siRNA - mediated knockdown of Dnmts in DHT - untreated NSC97Q ( I ) 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 ) , ( B ) , rotarod performance ( C ) ( Two - way ANOVA with Tukey ' s ) survival rate ( D ) ( Kaplan - Meier log - rank ) of AR - 97Q mice treated with or without RG108 ( DMSO , n = 20 ; 0 . 5 mg / dL , n = 20 ; 2 . 0 mg / dL , n = 20 ) . 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 ( ) ; * P < 0 . 05 ( rotarod ) ; * P < 0 . 05 ( survival ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O 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-GENEPROD O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-EXP_ASSAY O O

(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 ) an - matched AR - 97Q mouse treated with 2 . 0 mg / dL RG108 ( right ) . AR - 97Q mouse medicated with RG108 maintained muscle volume compared with DMSO - treated AR - 97Q mouse of same . compact masses ( arrow ) of mice head are cement covering 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 ) relative intensities ( G ) of 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 spinal cords of AR - 97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody . ( B ) of 1C2 - positive motor neurons in spinal cords ( n = 5 per group ) .

O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O

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

O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O

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

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-GENEPROD O B-GENEPROD O O B-ORGANISM B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O

(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 spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) .

B-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O B-ORGANISM O O O O O O O O

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


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

O O B-CELL_TYPE B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O B-ORGANISM O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O

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

O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O

(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 ) data ( D ) ( n = 3 ) .

O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

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


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

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

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


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

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(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 indicated decrease of Hes5 mRNA level in DHT - treated SH97Q compared with DHT - untreated SH97Q ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O

(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 SH97Q that were treated with DHT .

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(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 , AR - 97Q mice ( n = 3 ) .

O O O B-GENEPROD O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O

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


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

O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O

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


methylation of HES5 promoter region of SH97Q treated with DHT RG108 .

B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

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


HES5 mRNA of SH97Q treated with DHT RG108 .

B-GENEPROD B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

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


methylation of Hes5 promoter region in DHT - treated NSC97Q 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 of Hes5 ( n = 3 ) in DHT - treated NSC97Q 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 in spinal cords of AR - 97Q mice treated with or without RG108 ( n = 3 ) .

O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O

(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 ) .

O O O O O B-GENEPROD O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O

(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 treated with Hes5 control siRNA measured by WST - 8 ( n = 3 ) .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(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 treated with Hes5 siRNA ( n = 3 ) .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O

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


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

O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(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.


viability measured with WST - 8 of DHT - treated NSC97Q transfected with a mock plasmid or 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).


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

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O

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


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

O O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O

(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 transfected with a mock plasmid or Hes5 vector for AR . ( H ) AR aggregation as quantified by densitometry ( n = 3 ) .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O

(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 treated with Hes5 siRNA . ( B ) of of pSmad2 , Smad2 , phospho - IkBα heat shock factor - 1 ( Hsf1 ) ( n = 3 ) .

O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O

(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 level ( n = 3 ) .

O O O O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O

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


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

O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O

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


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

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O O B-GENEPROD I-GENEPROD O O O O O O

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


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

O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O O

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


( H ) Relative mRNA of Hes5 in motor neurons expressing AR24Q 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 , Hes5 in cortical neurons expressing AR97Q treated with mock plasmid or Hes5 - containing lentiviral vector .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O B-GENEPROD O O B-ORGANISM O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD 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-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY B-SUBCELLULAR O B-EXP_ASSAY O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O

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


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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE B-TISSUE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 ) were either left untreated ( + serum ) or serum starved ( - serum ) 4 hr then stimulated 2 ng / mL EGF for 15 min before analyses by western blotting .

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


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

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


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

O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 relative of pAKT ( pink ) , pEGFR ( blue ) , or total EGFR ( blue ) , in autophagy - deficient lines relative to their control counterparts . following knockouts were introduced : glial ( sgAtg7 ) , HT29 ( crATG7 ) , MCF - 7 ( sgATG5 ) , A549 ( sgATG5 ) , U2OS ( sgATG5 ) , MEFs ( Atg16l1 - / - ) .

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

O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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

O O O B-EXP_ASSAY O B-GENEPROD O O O B-DISEASE I-DISEASE I-DISEASE O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O B-DISEASE I-DISEASE I-DISEASE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

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


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

O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O

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


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

B-SMALL_MOLECULE O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O B-CELL_LINE O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O B-SUBCELLULAR O B-GENEPROD O O

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


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

B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O B-CELL_LINE O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O B-SUBCELLULAR O B-GENEPROD O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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