Ctrl CCT7 CCT2 GAPDH * * ** ** * Ctrl Size of LC3 dots (a.u.) mrfp GFP mrfp-gfp-lc3 CCT5 KD CCT7 KD

Transcription

1 CCT7 Size of dots (a.u.) CCT7 CCT7 /GAPH deitometry (a.u.) /GAPH deitometry (a.u.) a CCT7 Primary cortical neuro 55 CCT5 55 CCT7 55 CCT c DMSO 2 8 Bafilomycin A d mrfpgfp Synthesis Fusion Phagophore Autophagosome f mrfp GFP mrfp Autolysosome e CCT5 CCT7 GFP dots mrfp dots mrfpgfp mrfp GFP mrfpgfp Supplementary Fig.. CCT knockdown efficiency. (a) CCT depletion efficiency in HeLa cells. HeLa cells treated with sirnas targeting individual CCT suunits were lotted for various suunits of the complex. Knockdown of one suunit can also impact on the levels of the others. was used as loading control. () CCT depletion efficiency in mouse primary cortical neuro. Primary cortical neuro were traduced with two different shrnas targeting control ( sh# and sh#2) or the mouse suunit ( sh# and sh#2). was used as loading control. (c) II/ deitometry for HeLa cells, related to Fig. a. The graphs show the mean ± SEM of independent iological replicates in the asence (n=6) or presence (n=4) of Bafilomycin A. (P<., P<.5; twotailed ttest).

2 (d) Schematic model for HeLa cells staly expressing mrfpgfp. In this cell line, due to the different pka of the two tags (< 4.5 for mrfp and around 6 for GFP), the autophagosomes emit oth red (mrfp) and green (GFP) fluorescence, while the autolysosomes lose the GFP signal due to its rapid quenching in the acidic lysosomal compartment and are visualized as redonly vesicles y microscopy (Kimura et al., 27; Sarkar et al., 2). (e) Size of GFP dots (autophagosomes/nonacidified lysosomes) and mrfp only dots (autolysosomes). HeLa cells staly expressing mrfpgfp were treated as in Fig. e, then seeded in a 96 well plate and sujected to Cellomics visualization analysis. The graphs show the results for one representative experiment: 2 wells of 2 cells each were analysed per sample. Similar data was seen in other two independent experiments. Bars represent the mea ± SEM (n=2; P<.; twotailed ttest). (f) Representative Cellomics fields for the experiment in (e). Scale ar throughout the panel is µm. 2

3 sh#2 sh# sh#2 LAMP colocalization (Pearson s coefficient) sh# CCT7 LAMP colocalization (Pearson s coefficient) a Basal LAMP merged Nocodazole Basal Nocodazole CCT7 c Lamp merged d sh# sh#2 sh# sh#2 Supplementary Fig. 2. Loss of CCT function does not alter the autophagosomelysosome fusion (a) (antimouse Alexa 488) and LAMP (antirait Alexa 568) colocalization in HeLa cells. Cells depleted for or CCT7 were coimunostained for LAMP and. The cells were then exposed to nocodazole treatment 2 µm, 3 hours. Scale ar throughout the panel is µm. () Quantification of colocalization for the experiment in (a). The Pearson s coefficient was quantified for more than 3 cells per each condition for oth asal and nocodazole treated cells. Bars represent the mean ± SEM (P<., P<.5, not significant; twotailed ttest). Similar results were achieved in at least two independent experiments. (c) (antimouse Alexa 488) and LAMP (antirait Alexa 647) colocalization in mouse primary cortical neuro. The cortical neuro were traduced with two independent shrnas targeting control or the mouse. Scale ar throughout the panel is µm. (d) Pearson s coefficients for the experiment in (c). 235 neuro were coidered per condition. Bars represent the mean ± SEM (P<., not significant; twotailed ttest). The experiment was repeated with similar results. 3

4 p62cd63 colocalization coefficients, BafA, BafA a CD63 p62 merged CD63 p62 merged , BafA, BafA.3.2. PC M Supplementary Fig. 3. CCT depletion increases the p62 delivery into lysosomes. (a) P62 (antirait Alexa 488) and CD63 (antimouse Alexa 568) colocalization in HeLa cells. Cells depleted for CCT5, in the asence or presence of Bafilomycin A (BafA), were coimunostained for CD63 and p62. Scale ar throughout the panel is µm. () Quantification of colocalization for the experiment in (a). The Pearson s (PC) and Mander s (M represents the p62 localization in the lysosomal compartment) coefficients were quantified for more than 4 cells per each condition. Bars represent the mean ± SEM (P<., P<.5; twotailed ttest). The experiment was repeated with similar results. 4

5 a merged CD63 p62 GFP, BafA, BafA Pearson s colocalization coefficients Mander s colocalization coefficients , BafA, BafA p62/cd63 GFP/ 2 p62/ 3 CD63 GFP p62 in CD63 GFP 2 in p62 3 in CD63 GFP Supplementary Fig. 4. CCT depletion increases the p62 delivery into autophagosomes/autolysosomes. (a) P62 (antirait Alexa 594) CD63 (antimouse Alexa 647) GFP colocalization in HeLa cells staly expressing GFP. Cells depleted for CCT5, in the asence or presence of Bafilomycin A (BafA), were coimunostained for CD63 and p62. Scale ar throughout the panel is µm. () Quantification of colocalization for the experiment in (a). The Pearson s and Mander s coefficients were quantified for more than 2 cells per each condition. Bars represent the mean ± SEM (P<., P<.5; twotailed ttest). The experiment was repeated with similar results. 5

6 Ctsl Ctsl activity (a.u.) CTSL CTSL activity (a.u) a pro. im. m. Bafilomycin A CCT CCT7 c Bafilomycin A d 2 pro. im. m. M.Ctsl/ ratio: M.Ctsl/pro.Ctsl ratio: sh# sh# sh#2 BafA Inhiitor Supplementary Fig. 5. Loss of CCT function reduces cathepsin L maturation. (a) Cathepsin L (CTSL) maturation immunolot in and 7 knockdown HeLa cells. was used as loading control. () In vitro cathepsin L activity. HeLa cells depleted for and 7 were sujected to the cathepsin L activity kit and the fluorescence inteity was expressed as percentage of control. Bars represent the mean ± SD for one representative experiment (n=3; P<.; twotailed ttest). (c) Cathepsin L (Ctsl) maturation immunolot in depleted primary mouse cortical neuro. was used as loading control. (d) In vitro cathepsin L activity in primary cortical neuro exposed to shrna lentiviral treatment as indicated. Bars represent the mean ± SD (n=3; P<., P<., P<.5; twotailed ttest). 6

7 c a LAMP CTSD Calnexin merged CTSD merged CCT7 g sh# sh# sh#2 merged sh#2 Lysoseor TPMT YB CCT7 CTSD Lamp Va sh# f Calnexin.5 e.35 d.6 sh#2 CTSD/LAMP colocalization (Pearson s coeffcient) CTSD/Calnexin colocalization (Pearson s coefficient) CCT7 CCT7 merged BafA sh# sh#2 sh# sh#2 Colocalization coefficients Fluorescence ratio Yellow/Blue (a.u.) BafA sh# sh#2 sh# PC sh#2 M Supplementary Fig. 6. Cathepsin L maturation and lysosomal ph defects in CCT depleted cells. (a) Cathepsin D (CTSD, antimouse Alexa 488) LAMP (antirait Alexa 568) doule immunostaining in or CCT7 knockdown HeLa cells. Scale ar throughout the panel is µm. 7

8 () CTSDLAMP colocalization for the experiment in (a). At least 4 cells were quantified per experiment (each experiment was performed at least twice with similar results). Bars represent the mean ± SEM (P<.; twotailed ttest). (c) Cathepsin D (antimouse Alexa 488) Calnexin (antirait Alexa 568) doule immunostaining in or CCT7 knockdown HeLa cells. Scale ar throughout the panel is µm. (d) CTSDCalnexin colocalization for the experiment in (c). At least 4 cells were quantified per experiment (each experiment was performed at least twice with similar results). Bars represent the mean ± SEM (P<., P<.; twotailed ttest). (e) Enlarged image of cathepsin D (antimouse Alexa 488) calnexin (antirait Alexa 568) doule immunostaining from Fig. 2d. Scale ar throughout the panel is µm. (f) Representative images for the Lysosomal ph assay performed in depleted primary cortical neuro using the Lysoseor YellowBlue. TPMT states for the Bright Field/DIC image. Scale ar throughout the panel is µm. See Fig. 2f. (g) Voa (antirait Alexa555) and Lamp (antirat Alexa647) doule immunostaining in mouse primary cortical neuro. Neuro were plated and treated as in Fig. 2g. At least 3 neuro were counted per condition. PC states for the Pearson s coefficient, while M is the Mander s coefficient, which indicates the Voa localization in the Lamp compartment. Bars represent the mean ± SEM (P<.; twotailed ttest). Scale ar throughout the panel is µm. 8

9 Cytochalasin D DMSO CTSD and Calnexin colocalization (PC) Cytochalasin D CTSD and LAMP colocalization (PC) DMSO Ctsl M.Ctsl / Pro. Ctsl deitometry (a.u.) Ctsl/ deitometry (a.u.) II/ deitometry (a.u.) II/ deitometry (a.u.) Actin Actin Tuulin Size of dots (a.u.) a CCT7.6 GFP dots mrfp dots c e g Bafilomycin A Latrunculin A I II Latrunculin A Higher exp. pro. im. m. LAMP CSTD merged d f DMSO DMSO DMSO Latrunculin A Latrunculin A DMSO Latrunculin A Cytochalasin D Bafilomycin A DMSO DMSO Latrunculin A Latrunculin A m. Ctsl/ pro.ctsl/.6 Pearson s coefficient DMSO Cytochalasin D h Calnexin CSTD merged.3 Pearson s coefficient DMSO Cytochalasin D Supplementary Fig. 7. Actin depolymerising drugs locks autophagy. 9

10 Supplementary Fig. 7. Actin depolymerising drugs locks autophagy. (a) Cytoskeleton immunostaining in CCT knockdown cells. Scale ar throughout the panel is µm. () Size of GFP dots (autophagosomes/nonacidified lysosomes) and mrfp only dots (autolysosomes) in mrfpgfp stale HeLa cells independently treated with the actin depolymerising drugs latrunculin A and cytochalasin D for 3 hours at µm concentration each, and sujected to Cellomics visualization analysis. The graphs show the results for one representative experiment: 8 wells of 2 cells each were analysed per sample. The experiment was repeated with similar results. Bars represent the mea ± SEM (n=8; P<., P<.5; twotailed ttest). (c) II levels in mouse primary cortical neuro treated with latrunculin A. Primary cortical neuro (DIV 7) were exposed to Latrunculin A ( µm) for 6 hours in the presence or asence of Bafilomycin A (4 nm, 6 hours). (d) II/ deitometry for the experiment in (c). Bars represent the mean ± SD (n=3; P<.5; twotailed one sample ttest). (e) Cathepsin L (Ctsl) maturation immunolot in primary neuro treated with Latrunculin A ( µm) for 6 hours (pro = procathepsin L, im = intermediate Cathepsin L, m = mature Cathepsin L). (f) Quantification of m.ctsl/pro.ctsl, m.ctsl/ and pro.ctsl/ ratios in primary cortical neuro treated as in (e). For the first two, the statistical analysis was ased on twotailed one sample ttest and for the pro.ctsl/ ratio, onetailed one sample ttest (n=3; P<.5). (g) Cathepsin D (CTSD, antimouse Alexa 488) and LAMP (antirait Alexa 568) doule immunostaining in µm cytochalasin Dtreated cells for 3 hours. The quantification of colocalization is displayed on the right panel. At least 3 cells were counted per experiment (each experiment was performed at least twice). Bars represent the mean ± SEM (P<.; twotailed ttest). Scale ar throughout the panel is µm. (h) Cathepsin D (antimouse Alexa 488) and calnexin (antirait Alexa 568) doule immunostaining in µm cytochalasin D treated cells for 3 hours. The quantification of colocalization is displayed on the right panel. At least 3 cells were counted per experiment (each experiment was performed at least twice). Bars represent the mean ± SEM (P<.; twotailed ttest). Scale ar throughout the panel is µm.

11 % GFPpositive HeLa cells with HTT(Q3)GFP aggregates % GFPpositive HeLa cells with HTT(Q97)GFP aggregates Chymotrypsinlike proteasome activity ATG6L ATG6L % EGFP positive Atg6L / MEFs with EGFPHTT(Q74) aggregates % EGFP positive HeLa cells with EGFPHTT(Q74) aggregates % EGFPpositive cells with EGFPHTT(Q74) aggregates normalized to control % EGFPpositive ATG6L cells with EGFPHTT(Q74) aggregates normalized to control % EGFPpositive ATG6L cells with EGFPHTT(Q74) aggregates (.5 µg DNA) a d g ATG6L ATG6L (lower exp.) ATG6L I II HeLa (ATG6L) EGFPQ74 CCT5 CCT e I II ATG6L WT MEFs CCT5 CCT7 c Atg6L / MEFs DMSO MG32 DMSO MG f h ATG6L 2 DMSO MG32 5 CCT5 CCT7 45 DMSO 4 MG ATG6L ATG6L 5 i ATG6L ATG6L ATG6L ATG6L 2 Supplementary Fig. 8. CCT knockdown monitors the EGFPHTT(Q74) aggregation through autophagy. (a) EGFPHTT(Q74) aggregation in HeLa (ATG6L) cells exposed to sirna targeting different CCT suunits. The ars represent the mean of data from 3 independent experiments (iological replicates) ± SD (n=3; P<., P<.5; one sample ttest), while each experiment was performed in triplicates. () EGFPHTT(Q74) aggregation in (ATG6L) CRISPR/Cas9 HeLa cells exposed to sirna targeting different CCT suunits. The ars represent the mean of data from 3 independent experiments ± SD (n=3; not significant; one sample ttest), while each experiment was performed in triplicates. j (min) CtrL MG32 MG32 µg 2 µg

12 (c) EGFPHTT(Q74) aggregation in (ATG6L) CRISPR/Cas9 HeLa cells exposed to sirna targeting different CCT suunits. The experiment was performed in parallel with the one shown in Fig. 5. The cells were seeded on coverslips in triplicates, trafected with sirnas targeting control or individual CCT suunits and followed y.5 µg of EGFPHTT(Q74). The ars represent the mean of the percentages of cells with aggregates ± SD (n=3; not significant; twotailed ttest). (d) Biochemical assessment of ATG6L HeLa. CRISPR/Cas9 mediated degradation of ATG6L in HeLa cells shows no residual autophagy or ATG6L. was used as loading control. (e) Validation of depletion in WT and Atg6L / MEFs. The MEFs were traduced with lentiviral particles targeting control or the mouse suunit (two independent shrnas). II and levels were assessed y western lot. was used as loading control. (f) ATG6L and ATG6L HeLa cells were seeded on coverslips in triplicates and trafected with µg of EGFPHTT(Q74) and treated with vehicle (DMSO) or MG32 ( µm) for the last 2 hours. The ars represent the mean of the percentages of cells with aggregates ± SD (n=3; P<., P<.5; twotailed t test). Treatment with MG32 further increased the percentage of ATG6L HeLa cells with EGFP HTT(Q74) aggregates. (g) Representative images for DMSO and MG32 treated cells as in (f). Scale ar throughout the panel is µm. The cells with aggregates are indicated with white lines or arrows. (h) Atg6L/ MEFs were seeded on coverslips in triplicates and trafected with µg or 2 µg of EGFP HTT(Q74) and treated with vehicle (DMSO) or MG32 ( µm) for the last 2 hours. Left: The ars represent the mean of the percentages of cells with aggregates ± SD (n=3; P<., P<.5; twotailed t test). Treatment with MG32 increased the percentage of Atg6L/ MEFs with Q74GFP aggregates. (i) N73QGFP (left) and N797QPGFP (right) aggregation in CCTdepleted ATG6L and ATG6L HeLa cells from one representative experiment performed in triplicates. The ars represent the mean of the percentages of cells with aggregates ± SD (n=3; P<.5, not significant; one sample t test). Similar results were achieved in other three independent experiments. (j) Chymotrypsinlike proteasome activity in CCT5 knockdown HeLa cells (n=3; not significant; twotailed ttest). 2

13 Total Lysates Total Lysates Total Lysates ATG6L HeLa (ATG6L) Atg6L / MEFs WT MEFs a sh# sh# sh#2 EGFPQ74 DAPI CCT7 EGFPQ74 EGFPQ74 c Htt(Q38)GFP GFPvector CCT5 sirna IB: Htt(Q38)GFP ATG6L ATG6L 3 d ATG6L MG32 (2 hours) IB: GFPHtt(Q38) IP: FlagHtt(Q38) 3 3 ATG6L MG32 (2 hours) IB: FlagHtt(Q38) IP: GFPHtt(Q38) 3 3 IP: FlagHtt(Q38) 3 GFPHtt(Q38) 3 FlagHtt(Q38) 3 Htt(Q38)GFP 3 FlagHtt(Q38) 3 GFPHtt(Q38) 3 FlagHtt(Q38) CCT %GFPHtt(Q38) immunoprecipitated relative to total lysates. 3.4 %FlagHtt(Q38) immunoprecipitated relative to total lysates Supplementary Fig. 9. EGFPHTT(Q74) aggregates in CCT knockdown cells (a) Representative images of WT and Atg6L / MEFs with EGFPHTT(Q74) aggregates for the experiment presented in Fig. 5c. Cells were seeded on coverslips in triplicates, traduced with lentiviral particles targeting control or the mouse suunit (two independent shrnas) and trafected with EGFPHTT(Q74). Similar data was achieved in other two independent experiments. () Upper: Representative images of HeLa (ATG6L) cells with Q74GFP aggregates treated with sirna targeting various CCT suunits, as indicated in Fig. 5a. 3

14 Bottom: Representative images of (ATG6L) CRISPR/Cas9 HeLa cells with EGFPHTT(Q74) aggregates treated with sirna targeting various CCT suunits, as indicated in Fig. B. The dotted lines indicate the cells with EGFPHTT(Q74) aggregates. Scale ar throughout the panel is µm. (c) Full westernlot for Fig. 6a. (d) Oligomerization of mutant htt in ATG6L HeLa cells exposed to either vehicle (DMSO) or MG32 ( µm) for the last 2 hours (see Methods and Fig. 6ac). Treatment with MG32 enhanced the amount of coimunoprecipitated of mutant HTT(548) tagged to GFP with Flagtagged mutant HTT(588) see left panel, and viceversa see right panel, in the autophagy null cells. 4

15 WT MEFs WT MEFs Proportion of EGFP positive cells with EGFPQ74 aggregates (a.u.) P62/ deitometry (a.u.) % GFPpositive HeLa cells with GFPATXN3(Q84) aggregates a 35 3 c Primary cortical neuro BafA p62 64 p62 64 d p62 5 ATG6L ATG6L 2 WT MEFs Atg6L / MEFs WT MEFs 5 5 P62/: Atg6L / MEFs e WT Atg6L / p62 MG32 64 f sh# EGFPA9 DAPI sh# sh#2 sh#2 g sh# sh# HeLa (ATG6L) sh# ATG6L sh# sh# sh#2 HeLa (ATG6L) ATG6L Supplementary Fig.. CCT depletion increases the accumulation of autophagy sustrates (a) GFPATXN3(Q84) aggregation in ATG6L and ATG6L HeLa cells. Cells seeded on coverslips in triplicates, were exposed to either control or CCT5 sirna and trafected with GFPATXN3(Q84). The ars represent the mean of percentages of cells with aggregates ± SD (n=3; P<., not significant; twotailed ttest). Similar results were achieved in other three independent experiments. () P62 levels in HeLa cells traiently trafected with sirna targeting individual CCT suunits. (c) P62 levels in mouse primary cortical neuro. Primary cortical neuro were traduced with lentiviral particles targeting. Two independent shrnas were used per each condition. Bafilomycin A (4nM for 2 hours) was used as a positive control for the accumulation of the autophagy sustrates, p62. P62/ deitometry is shown ellow. (d) P62 levels in WT and Atg6L/ MEFs traduced with shrna lentiviral particles targeting control or the mouse suunit. The ars represent the mean of three independent experiments ± SD (n=3; P<.5, not significant; twotailed one sample ttest). (e) P62 levels in WT and Atg6L / MEFs treated with vehicle (DMSO) or MG32 ( µm) for 4 hours. MG32 increases the p62 levels in oth WT and Atg6L / MEFs. 5

16 (f) Representative images of EGFPA9 aggregates in WT MEFs traduced with shrna lentiviral particles targeting control or the mouse suunit. Similar data was achieved in other two independent experiments. (g) EGFPHTT(Q74) aggregation in ATG6L and ATG6L HeLa cells exposed to either control or CCT5 sirna and treated with the autophagy inducers: caramazepine (5 µm) and TatBeclin peptide (2 µm). HeLa cells were exposed to two round of sirna, trafected with EGFPHTT(Q74) and treated with autophagy inducers for the last hours. The percentage of trafected cells with aggregates was scored and the control data were normalised to to enale compariso. Note that the effects in CCTdepleted cells were oserved in two independent triplicate experiments. Data are shown for representative triplicate experiments with ttests. 6

17 Deitometry (a.u.) Deitometry (a.u.) a IGFR integrin 3.5 EGFR IGFR β integrin EGFR Time (h) EGF CHX EGFR Time post EGF wash (h) 2 Supplementary Fig.. Accumulation of endocytic sustrates upon CCT5 depletion (a) Protein levels of known endocytic route cargos in CCT5 sirna knockdown HeLa cells. significantly increased the levels of IGFR, βintegrin and EGFR. The ars represent the mean of data ± SD (n=3; P<.5; twotailed one sample ttest). () EGFR degradation assay in HeLa cells exposed to either or CCT5 sirna. HeLa cells were serum starved (DMEM with.2% FBS) for 4 hours efore loading with the receptor ligand EGF ( ng/ml for 3 min) in the presence of cyclohexamide (CHX, 4 µg/ml). After, the cells were washed twice with PBS, and further incuated in serumfree media in the presence of cyclohexamide for another or 2 hours. At time the cells were lysed immediately after the PBS wash. The graph represents the quantification of EGFR degradation in HeLa cells exposed to either or CCT5 sirna. For each individual time point we normalized the data to time (EGF/CHX) and used twotailed ttest to assess the protein level differences etween and CCT5 depleted cells. ANOVA two ways was performed to assess the overall significance of EGFR degradation. The ars represent the mean of data ± SD (n=3; P<., P<.; two tailed one sample ttest). reduces the EGFR degradation. See Materials and Methods. 7

18 a CgGAL4 UASGFPRef(2)P Atg8a KG7569 CgGAL4 UASGFPRef(2)P c w 8 GFPRef(2)P TOPRO3 GFPRef(2)P endog. Ref(2)P 3 GFPRef(2)P endogenous Ref(2)P (light exp.) Ref(2)p 3 3 (light exp.) Ref(2)P 3 Act Atg8aI x Atg8aII Cct Ponceau Red Cct7 KK Act d CgGAL4 UASGFPRef(2)P w 8 Atg8a KG7569 Cct7 KK Atg8a KG7569, Cct7 KK KK Atg8a KG7569, KK NIG Supplementary Fig. 2. CCT depletion locks autophagy in Drosophila. (a) Biochemical assessment of autophagy in the fat ody of CgGAL4 UASGFPRef(2)P third itar larvae upon Cct7 knockdown, either alone (Cct7 KK ) or in an Atg8a mutant ackground (Atg8a KG7569 ). The VDRC w 8 line (stock numer 6) was used as ackground control. Ref(2)P (oth GFPtagged and endogenous forms) accumulated upon Cct7 knockdown (Cct7 KK ) and in Atg8a mutants. Atg8II levels increase in Cct7depleted fat odies. There was no ovious effect of Cct7 knockdown on Ref(2)P levels in Atg8a mutant fat odies. Actin was used as loading control. () Biochemical assessment of endogenous and GFPtagged Ref(2)P upon knockdown in the fat ody of third itar larvae expressing GFPRef(2)P (CgGAL4::UASGFPRef(2)P) and RNAi ( KK or NIG ). The VDRC w 8 (stock numer 6) was used as a ackground control. 8

19 The western lot analysis was performed using a % polyacrylamide gel, to etter separate the GFPtagged from the endogenous Ref2(P). PonceauRed staining validates actin as a loading control in Drosophila fat odies. (c) Representative confocal images showing GFPRef(2)P in the fat ody larval progeny of CgGAL4 UAS GFPRef2(P) crossed to either w 8 or Cct7 KK. GFPRef2(P) positive aggregates accumulates in Cct7 depleted cells. Scale ar throughout the panel is µm. (d) Macroscopic images showing GFPRef2(P) aggregates in CgGAL4::UASGFPRef(2)P and Atg8a KG75569 ;CgGAL4::UAS GFPRef(2)P larvae depleted for Cct7 (Cct7 KK ) or (either KK or NIG ). Scale ar throughout the panel is 2 µm. 9

20 (Total lysates) (Total lysates) GFP (Total lysates) (IP) GFP (Total lysates) GFP (IP) (IP) GFP (IP) Cytochalasin D Supplementary Fig. 3 Fig. a Fig. a Fig. 2 Fig. 3a Latrunculin A Cathepsin L Fig. 3a Fig. c Primary cortical neuro Fig. a CCT7 Fig. 4e Fig. 4g Fig. 5f Fig. 4a CCT5 PFD6 PFD6 Fig. 5g GFP (IP) Fig. 6a Fig. 6a GFP (Total lysates) Flag (IP) Flag (Total lysates) 2

21 Supplementary Fig. 3 continued Fig. 6 Fig. 6d IP Total lysates Fig. 7d (right) Flag (IP) (IP) (IP) GFP (IP) GFP (Total lysates) Flag (Total lysates) Fig. 7d (left) IP Total lysates GFP (Total lysates) (Total lysates) (Total lysates) Fig. 7e CCT5 GFP Fig. 7d (right) GFP (IP) GFP (Total lysates) P62 () P62 (CCT7 ) () (CCT7 ) CCT7 Drosophila Fig. 8a (Drosophila) Atg8a Ref(2)P (Lighter exp.) Ref(2)P Atg8a 2

22 Cathepsin L Supplementary Fig. a CCT7 CCT7 CCT7 Supplementary Fig. 5c Supplementary Fig. 3 continued Supplementary Fig. a Primary cortical neuro Supplementary Fig. 5a Cathepsin L Cathepsin L CCT7 Supplementary Fig. 8d Supplementary Fig. 7c Supplementary Fig. 7e ATG6L Cathepsin L Cathepsin L ATG6L Supplementary Fig. 8e Supplementary Fig. 9d GFP (IP) ATG6L Flag (IP) ATG6L Flag (Total lysates) ATG6L Flag (IP) ATG6L GFP (IP) ATG6L GFP (Total lysates) ATG6L Flag (Total lysates) ATG6L GFP (Total lysates) ATG6L Supplementary Fig. Supplementary Fig. d Supplementary Fig. c MEFs p62 p62 p62 p62 22

23 (Lighter exp.) integrin EGFR Ref(2)P (Ligtherr exp.) Supplementary Fig. 3 continued Supplementary Fig. e Supplementary Fig. a Supplementary Fig. EGFR p62 IGFR Supplementary Fig. 2a (Lighter exp.) EGFR Ref(2)P Cct7 Supplementary Fig. 2 Atg8a Ref(2)P Ref(2)P Atg8a Supplementary Fig. 3. Full sca of uncropped lots. 23