Figure 1 ( A , B ) Representative images of the expression of NEDD4L in the skins from IMQ - treated mice ( A ) or psoriasis patients ( B ) as detected by IHC on the left . The NEDD4L expression level was calculated by multiplying the staining intensity score and the extent score and is shown on the right . ( A , n = 5 , B , normal , n = 15 , psoriasis , n = 36 ) . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance . ( C , D ) Representative images of the expression of EZH2 in the skin epidermis from the IMQ - treated mice ( C ) and the psoriasis patients ( D ) . The number of EZH2 - positive cells per HPF ( high power field ) in each mouse and patient is shown on the right . ( C , control , n = 3 , IMQ , n = 6 . D , normal , n = 13 , psoriasis , n = 27 ) . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance . ( E ) WB analysis of NEDD4L , EZH2 and EZH1 expression in the EZH2 - specific siRNA ( siEZH2 ) - or negative control siRNA ( siNC ) - transfected NHEKs with IMQ stimulation . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) .

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Figure 1(A, B) Representative images of the expression of NEDD4L in the skins from IMQ-treated mice (A) or psoriasis patients (B) as detected by IHC on the left. The NEDD4L expression level was calculated by multiplying the staining intensity score and the extent score and is shown on the right. (A, n=5, B, normal, n=15, psoriasis, n=36). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.(C, D) Representative images of the expression of EZH2 in the skin epidermis from the IMQ-treated mice (C) and the psoriasis patients (D). The number of EZH2-positive cells per HPF (high power field) in each mouse and patient is shown on the right. (C, control, n=3, IMQ, n=6. D, normal, n=13, psoriasis, n=27). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.(E) WB analysis of NEDD4L, EZH2 and EZH1 expression in the EZH2-specific siRNA (siEZH2)- or negative control siRNA (siNC)-transfected NHEKs with IMQ stimulation. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×).


Figure 2 ( D ) Representative images of HE staining of the back tissues are shown on the left . The epidermal thickness measured using the scale in the microscope is shown on the right . ( E ) Representative images of Ki67 staining in back tissues are shown on the left . The number of Ki67 - positive cells per HPF in each mouse is shown on the right . ( F ) Representative images of HE staining of the ear tissues are shown on the left . The epidermis thickness measured using the scale in the microscope is shown on the right . ( G ) Representative images of Ki67 staining in ear tissues ( same specimen as in F ) are shown on the left . The number of Ki67 positive cells per HPF in each mouse is shown on the right . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments . For back tissues , scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . For ear tissues , scale bar , 200 μm ( 100 × ) or 200 μm ( 200 × ) . Significant differences were tested using a two - tailed Student ' s t test Control , n = 3 , IMQ , n = 6 . * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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Figure 2(D) Representative images of HE staining of the back tissues are shown on the left. The epidermal thickness measured using the scale in the microscope is shown on the right. (E) Representative images of Ki67 staining in back tissues are shown on the left. The number of Ki67-positive cells per HPF in each mouse is shown on the right. (F) Representative images of HE staining of the ear tissues are shown on the left. The epidermis thickness measured using the scale in the microscope is shown on the right. (G) Representative images of Ki67 staining in ear tissues (same specimen as in F) are shown on the left. The number of Ki67 positive cells per HPF in each mouse is shown on the right. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Student's t test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance.


Figure 3NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs . WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L - WT or NEDD4L - C943S mutants . cell signaling pathway activation induced by IL - 6 were detected by WB analyses Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments ( G , H ) Primary keratinocytes were separated from the WT and Nedd4l KO mice . ( G ) WB analysis of the protein expression in IL - 6 - induced cell signaling pathway activation . ( H ) WB analysis of the proteins in the IL - 6 signaling pathway . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent

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Figure 3NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L-WT or NEDD4L-C943S mutants. cell signaling pathway activation induced by IL-6 were detected by WB analyses Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments(G, H) Primary keratinocytes were separated from the WT and Nedd4l KO mice. (G) WB analysis of the protein expression in IL-6-induced cell signaling pathway activation. (H) WB analysis of the proteins in the IL-6 signaling pathway. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments


Figure 4Six - to eight - weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment , the mice were injected intra - epidermally with 10 μg anti - GP130 antibody or with same dosage of isotype on every other day . Representative images of Ki67 staining in back tissues are on the left . The number of Ki67 - positive cells per HPF is shown on the right . Data information : Data are shown as mean ± s . e . m . , and are representative of two independent experiments . Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test C , control , n = 5 , IMQ , n = 6 . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance . Six - to eight - weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment , the mice were injected intra - epidermally with 10 μg anti - GP130 antibody or with same dosage of isotype on every other day . Keratinocyte proliferation associated genes in the skin tissues are detected using real - time PCR analysis . Data information : Data are shown as mean ± s . e . m . , and are representative of two independent experiments . Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test D , control , n = 3 , IMQ , n = 6 . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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Figure 4Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Representative images of Ki67 staining in back tissues are on the left. The number of Ki67-positive cells per HPF is shown on the right. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test C, control, n=5, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Keratinocyte proliferation associated genes in the skin tissues are detected using real-time PCR analysis. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test D, control, n=3, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.


Figure 5 ( A ) Representative pictures of the expression of NEDD4L , GP130 and p - STAT3 in the epidermis of a normal skin section ( same specimen ) and sections from psoriasis patients . Isotype are IgG antibodies from same species served as negative control . ( B ) Semiquantitative scoring of NEDD4L , GP130 and p - STAT3 in the epidermis detected by IHC . Data information : Each symbol represents one patient Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . All data are shown as the mean ± s . e . m . , and significant differences were analyzed using two - tailed Student ' s t test ( normal , n = 15 , psoriasis , n = 36 ) . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 .

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Figure 5(A) Representative pictures of the expression of NEDD4L, GP130 and p-STAT3 in the epidermis of a normal skin section (same specimen) and sections from psoriasis patients. Isotype are IgG antibodies from same species served as negative control. (B) Semiquantitative scoring of NEDD4L, GP130 and p-STAT3 in the epidermis detected by IHC. Data information: Each symbol represents one patient Scale bar, 200 μm (100×) or 50 μm (400×). All data are shown as the mean ± s.e.m., and significant differences were analyzed using two-tailed Student's t test (normal, n=15, psoriasis, n=36). * P＜0.05, ** P＜0.01, *** P＜0.001.


Figure 6 ( F ) Full length Flag - GP130 , GP130 - aa 616 - 918 , GP130 - aa 616 - 889 , GP130 - aa 616 - 764 , GP130 - aa 616 - 734 were transfected with Myc - NEDD4L into HEK293T cells , and whole cell lysates were co - IP with ANTI - FLAG M2 magnetic beads , followed by WB with anti - Flag or anti - Myc antibodies .

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Figure 6(F) Full length Flag-GP130, GP130-aa 616-918, GP130-aa 616-889, GP130-aa 616-764, GP130-aa 616-734 were transfected with Myc-NEDD4L into HEK293T cells, and whole cell lysates were co-IP with ANTI-FLAG M2 magnetic beads, followed by WB with anti-Flag or anti-Myc antibodies.


Figure 7 ( C ) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L - WT , NEDD4L WW domain truncated mutant ( NEDD4L - ΔWW ) or its enzymatic activity mutant ( NEDD4L - C943S ) , the cells were followed by denaturation - IP with anti - GP130 antibody and WB analysis with anti - Ub antibodies . ( E ) WB analysis of the polyubiquitination of GP130 in HEK293T cells co - transfected with full length Myc - NEDD4L , HA - Ub and Flag - GP130 or its lysine mutants ( K - R ) in the intracellular domain , followed by denaturation - IP with ANTI - FLAG M2 magnetic beads and WB analysis with anti - HA antibodies .

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Figure 7(C) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L-WT, NEDD4L WW domain truncated mutant (NEDD4L-ΔWW) or its enzymatic activity mutant (NEDD4L-C943S), the cells were followed by denaturation-IP with anti-GP130 antibody and WB analysis with anti-Ub antibodies.(E) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with full length Myc-NEDD4L, HA-Ub and Flag-GP130 or its lysine mutants (K-R) in the intracellular domain, followed by denaturation-IP with ANTI-FLAG M2 magnetic beads and WB analysis with anti-HA antibodies.


Figure 1 ( F ) Comparison of protein degradation efficiency using different degrons . Cells were incubated for indicated time periods with Asv or DOX / IAA or both ( DIA , see methods ) then collected for western blot analysis . Doxycycline was added 2 hours before addition of IAA or Asv / IAA . To determine protein degradation , either cyclin A2 or cyclin B1 were probed as indicated . Anti - myc antibody was used to check OsTIR1 - myc expression . Asterisks indicate non - specific protein crossreacting with the cyclin B1 antibody . ( G ) Timecourse of DIA induced cyclin A and B degradation , cross checking levels of each mitotic cyclin . Treatments were performed as for DIA treated Cell extracts were checked for both cyclin A2 and B1 levels following DIA

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Figure 1(F) Comparison of protein degradation efficiency using different degrons. Cells were incubated for indicated time periods with Asv or DOX/IAA or both (DIA, see methods) then collected for western blot analysis. Doxycycline was added 2 hours before addition of IAA or Asv/IAA. To determine protein degradation, either cyclin A2 or cyclin B1 were probed as indicated. Anti-myc antibody was used to check OsTIR1-myc expression. Asterisks indicate non-specific protein crossreacting with the cyclin B1 antibody.(G) Timecourse of DIA induced cyclin A and B degradation, cross checking levels of each mitotic cyclin. Treatments were performed as for DIA treated Cell extracts were checked for both cyclin A2 and B1 levels following DIA addition


Figure 2A ) Cyclin B2 knock - out and induced degradation by immunoblotting . Indicated cell lines were analysed 24 hours after mock or Dox / IAA / Asv ( DIA ) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation . ( ( D ) Cell proliferation of A2dd , CycB1dd and B1dd / B2ko following mock or DIA treatment . 1000 cells were plated in each well ( diameter 3 . 5cm ) and incubated for 10 days before methanol fixation and Crystal Violet staining . ( E ) Kinetics of mitotic entry as measured by time lapse microscopy in A2dd and B1dd / B2ko cells following mock or four - hour DIA treatment of asynchronous cells . The cells were imaged for 16h with 5min intervals using widefield DIC , mitotic entry was manually scored by detecting cell rounding . Curves display the cumulative mitotic index ( data from three repeats , n > 500 cells per condition , s . d . indicated by shaded area ) . ( I ) Cyclin A2 siRNA depletion causes endoreplication . Following 72 hours of siRNA transfection MCF7 and MCF10A cells were analysed by PI staining and FACS . The histograms show the changes in DNA content ( PI Int . ) towards > 4N following cyclin A2 depletion . ( J ) Cyclin A2 siRNA and degron depletion in RPE - 1 cells . RPE - 1 OsTIR1 and RPE - 1 A2dd cells were subjected to 72 hours of cyclin A2 siRNA depletion and / or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting . The longer exposure ( L . E . ) reveals incomplete depletion of cyclin 2 by siRNA .

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Figure 2A) Cyclin B2 knock-out and induced degradation by immunoblotting. Indicated cell lines were analysed 24 hours after mock or Dox/IAA/Asv (DIA) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation. ( (D) Cell proliferation of A2dd, CycB1dd and B1dd/B2ko following mock or DIA treatment. 1000 cells were plated in each well (diameter 3.5cm) and incubated for 10 days before methanol fixation and Crystal Violet staining.(E) Kinetics of mitotic entry as measured by time lapse microscopy in A2dd and B1dd/B2ko cells following mock or four-hour DIA treatment of asynchronous cells. The cells were imaged for 16h with 5min intervals using widefield DIC, mitotic entry was manually scored by detecting cell rounding. Curves display the cumulative mitotic index (data from three repeats, n>500 cells per condition, s. d. indicated by shaded area).(I) Cyclin A2 siRNA depletion causes endoreplication. Following 72 hours of siRNA transfection MCF7 and MCF10A cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards >4N following cyclin A2 depletion.(J) Cyclin A2 siRNA and degron depletion in RPE-1 cells. RPE-1 OsTIR1 and RPE-1 A2dd cells were subjected to 72 hours of cyclin A2 siRNA depletion and/or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting. The longer exposure (L.E.) reveals incomplete depletion of cyclin 2 by siRNA.


Figure 3 ( C ) Representative images from live - cell imaging of SiR - Tubulin labelled ( red ) mitotic B1dd / B2ko cells expressing FusionRed - H2B ( green ) , Mis12 - GFP ( white ) ; time in mins , scale bar = 5µm . Cells were imaged four hours after DIA treatment . ( F ) Cyclin A2 levels were assessed by immunoblotting after 18 hours in 100ng / ml Nocodazole and / or 5µM STLC . 6µM proTAME was added to the indicated samples for the final six hours . ( G ) Upper panel , confirmation of Cdc27 depletion by immunoblotting 72 hours after siRNA transfection in B1dd / B2ko cells . Lower panel , following 40 - hour siRNA transfection B1dd / B2ko cells were treated for 24 hours with Thymidine , released for 10 hours and treated with proTAME and Apcin for additional four hours . At this point , the cells were fixed and stained with tubulin and pericentrin for immunofluorescence analysis . ( H ) Representative images of mitotic spindles in control - and DIA - treated B1dd / B2ko cells following arrest in mitosis by proTAME / Apcin treatment Tubulin staining is shown in white , pericentrin in green and DAPI in red , scale bar = 5µm . ( L ) Immunofluorescence images from mitotic Ctr and DIA treated B1dd / B2ko cells after P / A synchronisation stained with anti - alpha - tubulin ( green ) , anti - gamma - tubulin ( red ) antibodies and DAPI ( blue ) , scale bar = 5µm . Cells were exposed to ice - cold medium and either fixed immediately after cold exposure , or incubated for 5 , 10 , or 20 minutes at 37 ° C before fixation .

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Figure 3(C) Representative images from live-cell imaging of SiR-Tubulin labelled (red) mitotic B1dd/B2ko cells expressing FusionRed-H2B (green), Mis12-GFP (white); time in mins, scale bar = 5µm. Cells were imaged four hours after DIA treatment.(F) Cyclin A2 levels were assessed by immunoblotting after 18 hours in 100ng/ml Nocodazole and/or 5µM STLC. 6µM proTAME was added to the indicated samples for the final six hours.(G) Upper panel, confirmation of Cdc27 depletion by immunoblotting 72 hours after siRNA transfection in B1dd/B2ko cells. Lower panel, following 40-hour siRNA transfection B1dd/B2ko cells were treated for 24 hours with Thymidine, released for 10 hours and treated with proTAME and Apcin for additional four hours. At this point, the cells were fixed and stained with tubulin and pericentrin for immunofluorescence analysis.(H) Representative images of mitotic spindles in control- and DIA-treated B1dd/B2ko cells following arrest in mitosis by proTAME/Apcin treatment Tubulin staining is shown in white, pericentrin in green and DAPI in red, scale bar = 5µm.(L) Immunofluorescence images from mitotic Ctr and DIA treated B1dd/B2ko cells after P/A synchronisation stained with anti-alpha-tubulin (green), anti-gamma-tubulin (red) antibodies and DAPI (blue), scale bar = 5µm. Cells were exposed to ice-cold medium and either fixed immediately after cold exposure, or incubated for 5,10, or 20 minutes at 37° C before fixation.


Figure 4 ( A ) Time - lapse images of PCNA - mRuby tagged A2dd cells . The imaging sequence was started at the time of Doxycycline addition , and degron activity was triggered three hours later by addition of IAA and Asv or PBS ( indicated by dashed line ) . Time is shown as hh : min , scale bar equals 10µm . For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase . ( D ) Images from time - lapse sequence of mitosis showing cell division in controls and after addition of 0 . 5µM PD - 166285 in DIA treated A2dd labelled with SiR - DNA ( time is indicated as hh : min , the scale bars represent 20µm ) .

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Figure 4(A) Time-lapse images of PCNA-mRuby tagged A2dd cells. The imaging sequence was started at the time of Doxycycline addition, and degron activity was triggered three hours later by addition of IAA and Asv or PBS (indicated by dashed line). Time is shown as hh:min, scale bar equals 10µm. For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase.(D) Images from time-lapse sequence of mitosis showing cell division in controls and after addition of 0.5µM PD-166285 in DIA treated A2dd labelled with SiR-DNA (time is indicated as hh:min, the scale bars represent 20µm).


Figure 5 ( D ) Cyclin A2dd cells with inducible CycB1 - YFP ( B1 - WT ) and CycB1 - YFP - NLS ( B1 - NLS ) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion . Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and cyclin A2 antibodies ( I ) Immunoprecipitation of YFP from extract from A2dd following induction of YFP ( Y ) , CycB1 - YFP ( BY ) and CycB1 - YFP - NLS ( BYN ) . The samples in the odd lanes ( IAA / Asv - ) were treated with 1µg / ml doxycycline alone , while samples in even lanes ( IAA / Asv + ) were treated with Auxin and Asv to induce cyclin A2 depletion . Total cell extract and immune - precipitates were probed by immunoblotting with the indicated antisera .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(D) Cyclin A2dd cells with inducible CycB1-YFP (B1-WT) and CycB1-YFP-NLS (B1-NLS) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion. Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and cyclin A2 antibodies(I) Immunoprecipitation of YFP from extract from A2dd following induction of YFP (Y), CycB1-YFP (BY) and CycB1-YFP-NLS (BYN). The samples in the odd lanes (IAA/Asv-) were treated with 1µg/ml doxycycline alone, while samples in even lanes (IAA/Asv+) were treated with Auxin and Asv to induce cyclin A2 depletion. Total cell extract and immune-precipitates were probed by immunoblotting with the indicated antisera.


Figure 6 ( E ) Quantification of changes in selected cyclin B - dependent mitotic phosphorylations including Ki - 67 ( left panel ) , INCENP / Borealin ( middle panel ) and Top2B , CDCA2 and Tpx2 . ( F ) Representative images of Ki - 67 immunofluorescent staining of chromosome spreads from control ( Ctr ) and DIA treated P / A synchronised B1dd / B2ko cells and quantification of Ki - 67 staining intensity using cross - sections of chromosomes ( G ) Representative images from live - cell imaging of DIA - treated SiR - Tubulin labelled ( red ) B1dd / B2ko cells expressing FusionRed - H2B ( green ) and AurB - GFP ( white ) ; time is indicated in minutes , scale bar = 5µm . Bottom panel shows frequencies of aberrant AurB localisation in P / A synchronised B1dd / B2ko cells . ( J ) Tpx2 shows reduced spread on mitotic chromosomes in DIA treated B1dd / B2ko cells . The top panel shows immunofluorescence images , for quantification we measured the mean intensity of Tpx2 staining on the centrosomes and spindle and plotted the ratio of these values per cell . We also analysed the centrosome / spindle distribution of AurA that does not change following DIA treatment .

O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(E) Quantification of changes in selected cyclin B-dependent mitotic phosphorylations including Ki-67 (left panel), INCENP/Borealin (middle panel) and Top2B, CDCA2 and Tpx2.(F) Representative images of Ki-67 immunofluorescent staining of chromosome spreads from control (Ctr) and DIA treated P/A synchronised B1dd/B2ko cells and quantification of Ki-67 staining intensity using cross-sections of chromosomes(G) Representative images from live-cell imaging of DIA-treated SiR-Tubulin labelled (red) B1dd/B2ko cells expressing FusionRed-H2B (green) and AurB-GFP (white); time is indicated in minutes, scale bar = 5µm. Bottom panel shows frequencies of aberrant AurB localisation in P/A synchronised B1dd/B2ko cells.(J) Tpx2 shows reduced spread on mitotic chromosomes in DIA treated B1dd/B2ko cells. The top panel shows immunofluorescence images, for quantification we measured the mean intensity of Tpx2 staining on the centrosomes and spindle and plotted the ratio of these values per cell. We also analysed the centrosome/spindle distribution of AurA that does not change following DIA treatment.


Figure 7 ( B ) Confirmation of ENSA / ARPP19 S67 phosphorylation in mitotic extracts from Ctr and DIA treated cells , before and after ENSA / ARPP19 siRNA depletion . ( E ) Dynamics of Greatwall , PP1 , and Cdc27 dephosphorylation following Cdk1 inhibition in proTAME / Apcin treated mitotic cell extracts from Ctr or DIA treated cells . Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol and analysed by

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Figure 7(B) Confirmation of ENSA/ARPP19 S67 phosphorylation in mitotic extracts from Ctr and DIA treated cells, before and after ENSA/ARPP19 siRNA depletion.(E) Dynamics of Greatwall, PP1, and Cdc27 dephosphorylation following Cdk1 inhibition in proTAME/Apcin treated mitotic cell extracts from Ctr or DIA treated cells. Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol and analysed by immunoblotting


Figure 8A ) Representative images from live - cell imaging of siRNA transfected B1dd / B2ko cells ( FusionRed - H2B in red , SiR - Tubulin in white , scale bar = 10µm ) . Bar - plot panels on the right show single - cell analysis of 10 cells manually scored for length mitosis pre - NEBD and post - NEBD . Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus . ( B ) Widefield Imaging . DIC ( Grey ) , SiR - DNA ( b / w ) , of ENSA / ARPP19 siRNA transfected DIA treated B1dd / B2ko cells . Time is indicated in hh : min , scale bar = 10µm . ( C ) Panels of representative immunofluorescence images of siRNA transfected and P / A treated cells treated as indicated , scale bar = 10µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) Representative images from live-cell imaging of siRNA transfected B1dd/B2ko cells (FusionRed-H2B in red, SiR-Tubulin in white, scale bar = 10µm). Bar-plot panels on the right show single-cell analysis of 10 cells manually scored for length mitosis pre-NEBD and post-NEBD. Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus.(B) Widefield Imaging. DIC (Grey), SiR-DNA (b/w), of ENSA/ARPP19 siRNA transfected DIA treated B1dd/B2ko cells. Time is indicated in hh:min, scale bar = 10µm.(C) Panels of representative immunofluorescence images of siRNA transfected and P/A treated cells treated as indicated, scale bar = 10µm.


Figure 1The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : Spinning disc confocal live cell imaging of Alexa 555 - EGF ( 555 - EGF ) shown as vesicle tracking with time represented as a colour spectrum . Tracking started 5 min after addition of 20 ng / mL 555 - EGF for the indicated durations . Scale bar : 10 μm . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . B : Immunofluorescence staining of EGFR following 5 or 15 min stimulation with 20 ng / mL EGF . Scale bar : 20 μm . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : Quantification of EGFR vesicles in a perinuclear region ( within 30 μm diameter of the centre of the nucleus ) Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . D : Cells were stimulated with 20 ng / mL 555 - EGF for 15 or 30 min before immunofluorescence staining against EGFR . Scale bar : 10 μm . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : Quantification of percentage of total EGFR vesicles that colocalise with 555 - EGF Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . F : Cells stably expressing mCherry - Rab5 were stimulated with 20 ng / mL EGF for indicated times before immunofluorescence staining against EGFR . Scale bar : 10 μm . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . G : Pearson ' s colocalisation coefficient between mCherry - Rab5 and EGFR Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Spinning disc confocal live cell imaging of Alexa 555-EGF (555-EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/mL 555-EGF for the indicated durations. Scale bar: 10 μm. The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). B: Immunofluorescence staining of EGFR following 5 or 15 min stimulation with 20 ng/mL EGF. Scale bar: 20 μm.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Quantification of EGFR vesicles in a perinuclear region (within 30 μm diameter of the centre of the nucleus) Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Cells were stimulated with 20 ng/mL 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Quantification of percentage of total EGFR vesicles that colocalise with 555-EGF Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). F: Cells stably expressing mCherry-Rab5 were stimulated with 20 ng/mL EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm.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.


Figure 2The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . A : Control , sgAtg7 , or sgAtg16l1 cells were treated with 2 ng / mL EGF for 15 min . Cells were then processed for staining using anti - EEA1 antibodies and PI ( 3 ) P probe ( Alexa 488 - labelled 2XFYVE domains ) . To ensure the specificity of the probe , control cells were pre - treated with 5 mM 3 ' MA for 30 min . Scale bar : 10 μm . B : Quantification of PI ( 3 ) P + label intensity per cell ( in A ) . C : Pearson ' s colocalisation coefficient between PI ( 3 ) P and EEA1 ( in A ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . D : Control and sgAtg7 cells were stimulated 20 ng / mL EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR . Scale bar : 10 μm . E : Pearson ' s colocalisation coefficient between EEA1 and EGFR ( in D ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . F : Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng / mL EGF for 15 min then lysed in CHAPS - containing detergent buffer and binding partners detected by western blotting . G : Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 ( in F ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. A: Control, sgAtg7, or sgAtg16l1 cells were treated with 2 ng/mL EGF for 15 min. Cells were then processed for staining using anti-EEA1 antibodies and PI(3)P probe (Alexa 488-labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm. B: Quantification of PI(3)P+ label intensity per cell (in A). C: Pearson's colocalisation coefficient between PI(3)P and EEA1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. D: Control and sgAtg7 cells were stimulated 20 ng/mL EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR. Scale bar: 10 μm. E: Pearson's colocalisation coefficient between EEA1 and EGFR (in D). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.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.


Figure 3The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : Cells transiently expressing YFP - Galectins ( YFP - Gal3 , - Gal8 , or - Gal9 ) were stimulated with 2 ng / mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 . Scale bar : 10 μm . B : Quantification of percentage of total EEA1 vesicles that colocalise with YFP - Galectins ( in A ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : Cells transiently expressing YFP - Gal8 were stimulated with 20 ng / mL Alexa 555 - EGF ( 555 - EGF ) for 15 min . Scale bar : 10 μm . D : Quantification of the percentage of YFP - Gal8 - labelled vesicles that colocalise with 555 - EGF in control or ATG7 - deficient cells ( in C ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : Cells transiently expressing YFP - Gal8 were treated 100 μM monensin for 1 hr and stimulated with 2 ng / mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 . Scale bar : 10 μm . F : Quantification of percentage of total EEA1 vesicles that colocalise with YFP - Gal8 upon monensin treatment ( in E ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells transiently expressing YFP-Galectins (YFP-Gal3, -Gal8, or -Gal9) were stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Galectins (in A).Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells transiently expressing YFP-Gal8 were stimulated with 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min. Scale bar: 10 μm. D: Quantification of the percentage of YFP-Gal8-labelled vesicles that colocalise with 555-EGF in control or ATG7-deficient cells (in C). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). 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.


Figure 4The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : Cells stably expressing GFP - LC3 were treated with 100 μM monensin in the presence or absence of bafilomycin A1 ( 20 nM ) for 1 hr and stimulated with 2 ng / mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 . White arrows indicate colocalisation . Scale bar : 10 μm . B : Quantification of percentage of total EEA1 vesicles that colocalise with GFP - LC3 ( in A ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : Cells were treated for 1 hr with 100 μM monensin then stimulated with 2 ng / mL EGF for 15 min . LAMP2 and EEA1 were then detected by immunofluorescence staining . White arrows indicate colocalisation . Scale bar : 10 μm . D : Quantification of the percentage of total EEA1 vesicles that colocalise with LAMP2 ( in C ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : Cells were treated with 2 ng / mL EGF for 15 min before immunofluorescence against endogenous ATG16L1 and EEA1 . Scale bar : 10 μm . F : Quantification of percentage of total EEA1 puncta that colocalise with ATG16L1 ( in E ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . G : Cells stably expressing Flag - S - ATG16L1 were treated for 1 hr with either 100 μM monensin or 30 μM Dynasore , then stimulated with 2 ng / mL EGF for 15 min . Cells were then stained by immunofluorescence against EEA1 and Flag tag . Scale bar : 10 μm . H : Quantification of percentage of total EEA1 vesicles that colocalise with Flag - S - ATG16L1 ( in G ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . I : Untreated control or sgAtg7 cells , or control cells pre - treated with 100 μM monensin 1 hr , stably expressing Flag - S - ATG16L1 were stimulated 20 ng / mL Alexa 555 - EGF ( 555 - EGF ) for 15 min before fixation and immunofluorescence staining against Flag tag and EEA1 . Cells were then imaged by structured illumination microscopy ( SIM ) and images were reconstructed in Nikon Elements software . Scale bar : 10 μm . J : Quantification of the percentage of EGF - EEA1 colocalised vesicles that stained triple - positive with ATG16L1 by SIM ( in I ) . Due to the low throughput nature of this assay , the following cell numbers were counted : control untreated ( 9 cells ) , sgAtg7 # 1 ( 10 cells ) , sgAtg7 # 2 ( 9 cells ) , and control + monensin : ( 11 cells ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . K : Cells were treated with 2 ng / mL EGF for 15 min before immunofluorescence against endogenous WIPI2 and EEA1 . Scale bar : 10 μm . L : Quantification of percentage of total EEA1 puncta that colocalise with WIPI2 ( in K ) . Data information : White arrows indicate colocalisation . Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells stably expressing GFP-LC3 were treated with 100 μM monensin in the presence or absence of bafilomycin A1 (20 nM) for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. White arrows indicate colocalisation. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with GFP-LC3 (in A). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells were treated for 1 hr with 100 μM monensin then stimulated with 2 ng/mL EGF for 15 min. LAMP2 and EEA1 were then detected by immunofluorescence staining. White arrows indicate colocalisation. Scale bar: 10 μm. D: Quantification of the percentage of total EEA1 vesicles that colocalise with LAMP2 (in C). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Cells were treated with 2 ng/mL EGF for 15 min before immunofluorescence against endogenous ATG16L1 and EEA1. Scale bar: 10 μm. F: Quantification of percentage of total EEA1 puncta that colocalise with ATG16L1 (in E). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). G: Cells stably expressing Flag-S-ATG16L1 were treated for 1 hr with either 100 μM monensin or 30 μM Dynasore, then stimulated with 2 ng/mL EGF for 15 min. Cells were then stained by immunofluorescence against EEA1 and Flag tag. Scale bar: 10 μm. H: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 (in G). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). I: Untreated control or sgAtg7 cells, or control cells pre-treated with 100 μM monensin 1 hr, stably expressing Flag-S-ATG16L1 were stimulated 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min before fixation and immunofluorescence staining against Flag tag and EEA1. Cells were then imaged by structured illumination microscopy (SIM) and images were reconstructed in Nikon Elements software. Scale bar: 10 μm. J: Quantification of the percentage of EGF-EEA1 colocalised vesicles that stained triple-positive with ATG16L1 by SIM (in I). Due to the low throughput nature of this assay, the following cell numbers were counted: control untreated (9 cells), sgAtg7#1 (10 cells), sgAtg7#2 (9 cells), and control + monensin: (11 cells). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). K: Cells were treated with 2 ng/mL EGF for 15 min before immunofluorescence against endogenous WIPI2 and EEA1. Scale bar: 10 μm. L: Quantification of percentage of total EEA1 puncta that colocalise with WIPI2 (in K). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.


Figure 5The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . A : Cells were treated for 15 min with 2 ng / mL EGF before fixation and immunofluorescence staining against EEA1 and p - TBK1 . White arrows indicate colocalisation . Scale bar : 10 μm . B : Quantification of percentage of total EEA1 vesicles that colocalise with p - TBK1 ( in A ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . C : Cells stably expressing Flag - S - ATG16L1 were pre - treated for 1 hr with TBK1 inhibitors ( 100 μM MRT68601 or 5 μM momelotinib ) . Control cells were also treated 100 μM monensin for 1 hr as indicated . All cells were stimulated for 15 min with 2 ng / mL EGF followed by fixation and immunofluorescence staining against Flag tag and EEA1 . Scale bar : 10 μm . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . D : Quantification of total EEA1 vesicles that colocalise with Flag - S - ATG16L1 Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . E : Western blotting of shNf - 1 / shTp53 glial cells expressing gRNA sequences targeting Gal8 . The following experiments were performed in glial shNf - 1 / shTp53 glial cells serum starved 4 hr before assaying . Cells were expressing either Cas9 alone ( control ) or Cas9 and sgRNA targeting Atg7 ( sgAtg7 # 1 or # 2 ) . F : Control and sgGal8 cells stably expressing Flag - S - ATG16L1 were treated 100 μM monensin for 1 hr and stimulated with 2 ng / mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 and Flag tag . White arrows indicate colocalisation . Scale bar : 10 μm . G : Quantification of percentage of total EEA1 vesicles that colocalise with Flag - S - ATG16L1 in sgGal8 cells ( in F ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells were treated for 15 min with 2 ng/mL EGF before fixation and immunofluorescence staining against EEA1 and p-TBK1. White arrows indicate colocalisation. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with p-TBK1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells stably expressing Flag-S-ATG16L1 were pre-treated for 1 hr with TBK1 inhibitors (100 μM MRT68601 or 5 μM momelotinib). Control cells were also treated 100 μM monensin for 1 hr as indicated. All cells were stimulated for 15 min with 2 ng/mL EGF followed by fixation and immunofluorescence staining against Flag tag and EEA1. Scale bar: 10 μm.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Quantification of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Western blotting of shNf-1/shTp53 glial cells expressing gRNA sequences targeting Gal8.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.


Figure 6A : Glial shNf - 1 / shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 20 ng / mL TexasRed - EGF ( TxRed - EGF ) for 30 min . Scale bar : 10 μm . B : Quantification of TxRed - EGF puncta per cell , relative to the number taken up by control cells ( in A ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 . C : Endocytosis rate of wild type ( WT ) EGFR was assayed in control , sgAtg7 , or sgAtg16l1 glial shNf - 1 / shTp53 or EGFRvIII - expressing cells by cell surface biotinylation and one application of 2 ng / mL EGF for 15 min . Monensin treatment was added as indicated . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 . D : Plasma membrane recycling rate of WT EGFR in control , sgAtg7 , or sgAtg16l1 glial shNf - 1 / shTp53 or EGFRvIII - expressing cells was assayed by cell surface biotinylation and successive applications of 2 ng / mL EGF treatments for 15 min Monensin treatment was added as indicated . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 . E : Glial shNf - 1 / shTp53 control and sgAtg7 cells stably overexpressing mCherry - Rab11 were serum starved 4 hr then stimulated with 20 ng / mL EGF for 15 min then fixed and stained by immunofluorescence against EGFR . White arrows indicate colocalisation . Scale bar : 10 μm . F : Quantification of Pearson ' s colocalisation coefficient between mCherry - Rab11 and EGFR ( in E ) . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 20 ng/mL TexasRed-EGF (TxRed-EGF) for 30 min. Scale bar: 10 μm. B: Quantification of TxRed-EGF puncta per cell, relative to the number taken up by control cells (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.C: Endocytosis rate of wild type (WT) EGFR was assayed in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells by cell surface biotinylation and one application of 2 ng/mL EGF for 15 min. Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.D: Plasma membrane recycling rate of WT EGFR in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells was assayed by cell surface biotinylation and successive applications of 2 ng/mL EGF treatments for 15 min Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.E: Glial shNf-1/shTp53 control and sgAtg7 cells stably overexpressing mCherry-Rab11 were serum starved 4 hr then stimulated with 20 ng/mL EGF for 15 min then fixed and stained by immunofluorescence against EGFR. White arrows indicate colocalisation. Scale bar: 10 μm. F: Quantification of Pearson's colocalisation coefficient between mCherry-Rab11 and EGFR (in E). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.


Figure 7Glial shNf - 1 / shTp53 sgAtg7 ( A ) cells were either left untreated ( + serum ) or serum starved ( - serum ) 4 hr then stimulated 2 ng / mL EGF for 15 min before analyses by western blotting . Glial shNf - 1 / shTp53 sgAtg16l1 ( B ) cells were either left untreated ( + serum ) or serum starved ( - serum ) 4 hr then stimulated 2 ng / mL EGF for 15 min before analyses by western blotting . Glial shNf - 1 / shTp53 sgGal8 ( C ) cells were either left untreated ( + serum ) or serum starved ( - serum ) 4 hr then stimulated 2 ng / mL EGF for 15 min before analyses by western blotting . D : Glial shNf - 1 / shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 2 ng / mL EGF for the indicated times before analysis of EGFR signalling pathway activity by western blotting . E : Phosphorylated : total protein ratios for EGFR and AKT calculated from densitometry analyses of western blots were made relative to their respective points of maximal stimulation ( 100 % ) ( for EGFR : t = 5 min in control cells and for AKT : t = 7 min in control cells ) Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 . F : Glial shNf - 1 / shTp53 control and sgAtg7 cells were serum starved 4 hr with or without 20 ng / mL EGF 30 min stimulation before subcellular fractionation and analysis by western blotting . G : Radar chart indicating the relative levels of pAKT ( pink line ) , pEGFR ( light blue line ) , or total EGFR levels ( dark blue line ) , in autophagy - deficient cell lines relative to their control counterparts . The following knockouts were introduced : glial cells ( sgAtg7 ) , HT29 ( crATG7 ) , MCF - 7 ( sgATG5 ) , A549 ( sgATG5 ) , U2OS ( sgATG5 ) , and MEFs ( Atg16l1 - / - ) . H : Control or sgAtg7 glial shNf - 1 / shTp53 cells were serum starved for 24 hr in the presence or absence of 20 ng / mL EGF . Quantification of the percentage of propidium iodide ( PI ) - positive cells is shown . Data information : Statistical analyses were performed on at least 3 independent experiments , where error bars represent SEM and p values represent a two - tailed Student ' s t test : NS p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Glial shNf-1/shTp53 sgAtg7 (A) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.Glial shNf-1/shTp53 sgAtg16l1 (B) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.Glial shNf-1/shTp53 sgGal8 (C) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.D: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 2 ng/mL EGF for the indicated times before analysis of EGFR signalling pathway activity by western blotting. E: Phosphorylated:total protein ratios for EGFR and AKT calculated from densitometry analyses of western blots were made relative to their respective points of maximal stimulation (100%) (for EGFR: t=5 min in control cells and for AKT: t=7 min in control cells) Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.F: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr with or without 20 ng/mL EGF 30 min stimulation before subcellular fractionation and analysis by western blotting.G: Radar chart indicating the relative levels of pAKT (pink line), pEGFR (light blue line), or total EGFR levels (dark blue line), in autophagy-deficient cell lines relative to their control counterparts. The following knockouts were introduced: glial cells (sgAtg7), HT29 (crATG7), MCF-7 (sgATG5), A549 (sgATG5), U2OS (sgATG5), and MEFs (Atg16l1-/-).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.


Figure 1 ( A ) Coimmunoprecipitation of SMO - HA and Myc - Smaug proteins . Extracts of Cl8 cells expressing combinations of SMOWT - HA or SMOPKA - SD - HA with Myc - SmaugWT in the absence or presence of HH were immunoprecipitated ( IP ) with anti - HA . The input ( lower panel ) and the IP complexes ( upper panel ) were analyzed by western blot with anti - Myc ( αMyc ) or anti - HA ( αHA ) antibodies . Here and in the other figures , the names of the proteins detected are indicated on the left and the molecular weights on the right , in kDa ; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs . * corresponds to higher molecular weight forms of SMO of unknown origin . Here and in panel F , the black arrow indicates unphosphorylated SMOWT - HA and the bracket indicates the phosphorylated forms of SMOWT - HA or SMOPKA - SD . ( D , E ) Mapping of the Smaug interaction domain in SMO . Extracts from Cl8 cells transfected with Myc - SmaugWT and various regions of SMO fused to either HA ( D ) or to GFP ( E ) were immunoprecipitated with αMyc ( D ) or αGFP ( E ) and analyzed by western blotting with αMyc ( lower panel in D and upper panel in E ) , αHA ( D , upper panel ) or αGFP ( E , lower panel ) . In : Input ; Sup : supernatant . See also Fig EV1D - F . ( F ) Hyperphosphorylated forms of SMO do not interact with Smaug . Left panel : Extracts of Cl8 cells expressing SMOWT - HA with or without Myc - SmaugWT , in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA ( upper panel ) or αMyc antibodies ( lower panel ) . Right panel : Cl8 cells expressing SNAP - SMOWT with Myc - SmaugWT in the presence of HH were extracellularly labelled with a membrane - nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation ( IP ) with anti - Myc . Labelled SNAP - SMO was directly visualized after electrophoresis . Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms . See also Fig EV1G - J . ( G ) SMO and Smaug colocalize . Representative fluorescent images of Cl8 cells expressing GFP - SmaugWT alone ( 1 - 2 ) or together with SMOWT - mCherry ( 3 - 3 ' ' and 4 - 4 ' ' ) either without ( 1 , 3 - 3 " ) or with HH ( 2 , 4 - 4 " ) . The merge images in 3 " and 4 " show GFP - SmaugWT in green and SMOWT - mCh in red . The same results were seen with different fluorescent tags as well ( Fig EV2B ) . The lack of effect of HH on GFP - SmaugWT in absence of SMOWT - mCh is likely due to limiting amounts of endogeneous SMO . A least 20 cells were assayed for each condition . In absence of HH , all co - transfected cells exhibited greater than 90 % colabelled SMOWT - mCherry . Scale bar ( shown in G1 , identical for all panels ) : 10μm . See also Fig EV2 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Coimmunoprecipitation of SMO-HA and Myc-Smaug proteins. Extracts of Cl8 cells expressing combinations of SMOWT-HA or SMOPKA-SD-HA with Myc-SmaugWT in the absence or presence of HH were immunoprecipitated (IP) with anti-HA. The input (lower panel) and the IP complexes (upper panel) were analyzed by western blot with anti-Myc (αMyc) or anti-HA (αHA) antibodies. Here and in the other figures, the names of the proteins detected are indicated on the left and the molecular weights on the right, in kDa; the samples loaded for the input and the supernatants are equivalent to a tenth of the volume loaded for the IPs. * corresponds to higher molecular weight forms of SMO of unknown origin. Here and in panel F, the black arrow indicates unphosphorylated SMOWT-HA and the bracket indicates the phosphorylated forms of SMOWT-HA or SMOPKA-SD.(D, E) Mapping of the Smaug interaction domain in SMO. Extracts from Cl8 cells transfected with Myc-SmaugWT and various regions of SMO fused to either HA (D) or to GFP (E) were immunoprecipitated with αMyc (D) or αGFP (E) and analyzed by western blotting with αMyc (lower panel in D and upper panel in E), αHA (D, upper panel) or αGFP (E, lower panel). In: Input; Sup: supernatant. See also Fig EV1D-F.(F) Hyperphosphorylated forms of SMO do not interact with Smaug. Left panel: Extracts of Cl8 cells expressing SMOWT-HA with or without Myc-SmaugWT, in the absence or presence of HH were IP with an αMyc antibody before analysis by western blot with αHA (upper panel) or αMyc antibodies (lower panel). Right panel: Cl8 cells expressing SNAP-SMOWT with Myc-SmaugWT in the presence of HH were extracellularly labelled with a membrane-nonpermeable fluorescent SNAP substrate before cell lysis and immunoprecipitation (IP) with anti-Myc. Labelled SNAP-SMO was directly visualized after electrophoresis. Note its presence in the IP fraction with a relative enrichment of the less phosphorylated forms compared to the hyperphosphorylated forms. See also Fig EV1G-J.(G) SMO and Smaug colocalize. Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT alone (1-2) or together with SMOWT-mCherry (3-3'' and 4-4'') either without (1, 3-3") or with HH (2, 4-4"). The merge images in 3" and 4" show GFP-SmaugWT in green and SMOWT-mCh in red. The same results were seen with different fluorescent tags as well (Fig EV2B). The lack of effect of HH on GFP-SmaugWT in absence of SMOWT-mCh is likely due to limiting amounts of endogeneous SMO. A least 20 cells were assayed for each condition. In absence of HH, all co-transfected cells exhibited greater than 90% colabelled SMOWT-mCherry. Scale bar (shown in G1, identical for all panels):10μm. See also Fig EV2.


Figure 2 ( B , C ) Smaug downregulates reporter expression . Mean values of the relative levels of SNAP - GUS reporter ( SNAP - GUS / GFP - SNAP ratio , in arbitrary units ) in extracts of transfected Cl8 cells in the absence ( black ) or presence of λN - SNAP - Smaug ( red ) , λN - SNAP ( pale grey ) or SNAP - Smaug ( dark grey ) . See Fig EV3A for the representative gel and Fig EV3B which shows that λN - SNAP - Smaug has no effect on a SNAP - GUS construct that lacks the 5BoxB . N = 3 ( biological replicates ) . See also Fig EV3A - C . Here and in D - F : SNAP - GUS / GFP - SNAP ratios were set to 1 in cells expressing λN ‐ SNAP - Smaug . The error bars correspond to the standard deviation of the mean ( SD ) . Statistical analysis was done by a Kruskal - Wallis test by ranks followed by a Dunn test and p values are : black bracket < 0 . 05 , red bracket < 0 . 01 , green bracket < 0 . 0001 . Here and in D - E : 15 ng of pAct . λN - SNAP - smaug plasmid were used per transfection . , E ) HH / SMO reduces Smaug levels and its overall negative effect on reporter expression . Mean values of the relative levels of reporter expression ( determined as above ) ( D ) or of λN - SNAP - Smaug ( ratio λN - SNAP - Smaug / GFP - SNAP ) ( E ) in the absence of SMO constructs ( red ) or in the presence of SMOWT - HA ( green ) , SMOPKA - SD - HA ( blue ) or SMO ∆ 958 - HA ( yellow ) , without HH ( plain boxes ) or in presence of HH ( striped boxes ) . N = 6 ( independent biological replicates ) , except for " no SMO , no HH " and " SMOWT with HH " conditions where n = 10 ( independent biological replicates ) . A representative gel is shown in Fig EV3D . Note that SMOWT - HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant . See also Fig EV3E - F . ( F ) HH / SMO reduces Smaug ' s intrinsic repressive ability . A similar experiment was conducted with different amounts of λN - SNAP - smaug expressing plasmid ( ranging from 0 to 50 ng ) as indicated ) . The mean values of the relative levels of reporter ( y - axis ) are plotted against the relative levels of λN - SNAP - Smaug ( x - axis ) . SNAP - GUS / GFP - SNAP ratios were set to 1 in cells expressing λN ‐ SNAP - Smaug in absence of SMOPKA - SD / HH . The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with ( blue ) and without ( red ) SMOPKA - SD / HH , respectively . N = 3 ( biological triplicates ) . See Fig EV3H and also a representative gel in Fig EV3G .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B, C) Smaug downregulates reporter expression. Mean values of the relative levels of SNAP-GUS reporter (SNAP-GUS/GFP-SNAP ratio, in arbitrary units) in extracts of transfected Cl8 cells in the absence (black) or presence of λN-SNAP-Smaug (red), λN-SNAP (pale grey) or SNAP-Smaug (dark grey). See Fig EV3A for the representative gel and Fig EV3B which shows that λN-SNAP-Smaug has no effect on a SNAP-GUS construct that lacks the 5BoxB. N=3 (biological replicates). See also Fig EV3A-C. Here and in D-F: SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug. The error bars correspond to the standard deviation of the mean (SD). Statistical analysis was done by a Kruskal-Wallis test by ranks followed by a Dunn test and p values are: black bracket <0.05, red bracket <0.01, green bracket <0.0001. Here and in D-E: 15 ng of pAct.λN-SNAP-smaug plasmid were used per transfection., E) HH/SMO reduces Smaug levels and its overall negative effect on reporter expression. Mean values of the relative levels of reporter expression (determined as above) (D) or of λN-SNAP-Smaug (ratio λN-SNAP-Smaug/GFP-SNAP) (E) in the absence of SMO constructs (red) or in the presence of SMOWT-HA (green), SMOPKA-SD-HA (blue) or SMO∆958-HA (yellow), without HH (plain boxes) or in presence of HH (striped boxes). N=6 (independent biological replicates), except for "no SMO, no HH" and "SMOWT with HH" conditions where n=10 (independent biological replicates). A representative gel is shown in Fig EV3D. Note that SMOWT-HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant. See also Fig EV3E-F.(F) HH/SMO reduces Smaug's intrinsic repressive ability. A similar experiment was conducted with different amounts of λN-SNAP-smaug expressing plasmid (ranging from 0 to 50 ng) as indicated). The mean values of the relative levels of reporter (y-axis) are plotted against the relative levels of λN-SNAP-Smaug (x-axis). SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug in absence of SMOPKA-SD/HH. The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with (blue) and without (red) SMOPKA-SD/HH, respectively. N=3 (biological triplicates). See Fig EV3H and also a representative gel in Fig EV3G.