IP: anti-gfp VPS29-GFP. IP: anti-vps26. IP: anti-gfp - + +
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1 FAM21 Strump. WASH1 IP: anti FAM21 Strump. FKBP IP: anti-gfp VPS29- GFP GFP-FAM21 tail H H/P P H H/P P c FAM21 FKBP Strump. VPS29-GFP IP: anti-gfp FKBP VPS VPS VPS VPS29 1 = VPS29-GFP 2 = VPS29 V90D-GFP 3 = HeLa control 4 = HeLa 5 = GFP-VPS 6 = GFP-VPS endogenous VPS KD CAPZα VPS29 H = HEPES lysis f H/P = HEPES/PBS mix P = PBS lysis f Lysates. (1% total) FAM21 FKBP Strump. VPS d FAM21 Strump. WASH1 FKBP GFP-VPS IP: anti-gfp Lysates (1% total) - mcherry- mcherry- FKBP FKBP FKBP Strump. PBS lysis uffer 1 = control 2 = FAM21 KD 3 = FKBP KD Supplementary Figure 1. Interactions of the retromer CSC. (a) Cells staly expressing GFP-tagged wild-type or point mutant versions of VPS29 or VPS along with untransfected HeLa cells were lysed and immunoprecipitated with anti- antisera. () Cells staly expressing VPS29-GFP or GFP-FAM21 tail were lysed using either the low stringency HEPES uffer (H), the higher stringency PBS uffer (P) or the mixed HEPES/PBS uffer (H/P) as in Figure 2. Lysates were treated with anti-gfp to recover the respective GFP-tagged protein. (c) FKBP or FAM21 expression was aolished with sirna treatment in the VPS29-GFP expressing cells. Cells were lysed and the VPS29-GFP recovered y native IP. (d) Cells staly expressing either wild-type GFP-VPS or the mutant were transiently transfected with mcherry-fkbp or a control plasmid 48 hours prior to lysis in the higher stringency PBS uffer. GFP-VPS and associated proteins were recovered y native IP.
2 HeLa control HeLa VPS KD GFP-VPS VPS KD SNX1 SNX1 SNX1 SNX1 GFP-VPS VPS KD GFP GFP GFP GFP GFP CIMPR c GFP-VPS GFP-VPS LAMP1 GFP-VPS GFP-VPS LAMP1 Supplementary Figure 2. The VPS mutant localizes normally to endosomes. (a) Untransfected HeLa cells and HeLa cells staly expressing GFP-VPS wild-type () and were treated with sirna to silence endogenous VPS expression. 72 hours post-transfection, cells were fixed and stained with antiodies against GFP, SNX1 and. The loss of VPS results in loss of. Expression of either or VPS rescues the staining and oth and VPS appear to localize to SNX1-positive structures. () Cells expressing either wild-type GFP-VPS or were treated with sirna to silence expression of endogenous VPS. 72 hours post-transfection, cells were fixed and laeled with antiodies against and CIMPR. (c) HeLa cells staly expressing GFP-VPS wild-type () and were fixed and laeled with antiody against LAMP1. LAMP1 staining did not appear markedly different from cells expressing VPS. Scale ars in (a)-(c) = 20 µm.
3 2 hrs 4 hrs 6 hrs HeLa GFP-VPS GFP-VPS Average Cell Perimeter, as proportion of perimeter at 2 hrs hours after plating HeLa GFP-VPS GFP-VPS HeLa control HeLa VPS KD GFP-VPS VPS KD GFP-VPS VPS KD GLUT-1 SNX1 Supplementary Figure 3. The VPS mutant impacts on WASH-associated phenotypes. (a) Untransfected HeLa cells and HeLa cells staly expressing GFP-VPS wild-type () and were plated on glass coverslips and fixed at 2, 4, and 6 hours after plating. Representative images are shown for each condition. Scale ar = µm. The graph represents the average cell perimeter quantified y Cellomics automated fluorescence microscopy. A minimum of 5000 cells per coverslip were analysed. () Untransfected HeLa cells and HeLa cells staly expressing GFP-VPS wild-type () and were treated with sirna to silence endogenous VPS expression. 72 hours post-transfection, cells were fixed and stained with antiodies against SNX1 and GLUT-1. Cells were selected for imaging y viewing in the SNX1 channel without viewing the GLUT-1 staining so as to avoid iasing the oserved GLUT-1 localisation. The knockdown of VPS shifts the localization of GLUT1 to intracellular structures. The expression of GFP-tagged VPS can rescue GLUT-1 localisation ut the mutant displays GLUT-1 localisation more similar to a VPS KD in untransfected HeLa cells.
4 a GFP-VPS clone 9 c DMSO GFP-VPS GFP-VPS clone 8 control KD GFP-VPS GFP-VPS GFP-VPS clone 9 DMSO endog VPS KD Bafilomycin GFP-VPS GFP-VPS clone 8 GFP-VPS tuulin LC3-II GFP-VPS LC3-II/tuulin normalised to wt GFP-VPS GFP-VPS clone9 Bafilomycin endog control KD VPS KD GFP-VPS * ** GFP-VPS clone8 DMSO Baf tuulin LC3-II (low exp) LC3-II (high exp) d LC3-II/tuulin, normalised to control DMSO Baf e 0 sictrl control KD sictrl sivps sivps DMSO endog VPS KD Bafilomycin endog control KD VPS KD GFP-VPS GFP-VPS GFP-VPS GFP-VPS GFP-VPS GFP-VPS GFP-VPS GFP-VPS GFP-VPS endog VPS tuulin Supplementary Figure 4. VPS impairs autophagy in independent clones and is unaffected y knockdown of the endogenous protein. (a) HeLa cells staly expressing GFP-VPS wild-type () and were derived from clones different than those in Fig. 1. The cells were treated with Bafilomycin A1, lysed, and assessed for LC3-II and tuulin levels as descried previously. A representative lot is shown. () The experiment in triplicate shown in (a) is quantified with LC3-II levels normalised to tuulin and expressed as a ratio of levels in wild-type. Error ars indicate SEM. * indicates p=0.03 and ** indicates p=0.004 y 2-tailed Student s t-test. (c) The original clones of HeLa cells staly expressing GFP-VPS and were treated once with 40 nm sirna to silence endogenous human VPS without affecting the GFP-tagged murine construct. Cells were treated with Bafilomycin A1 as previously descried and assessed for LC3-II and tuulin levels. (low exp: low exposure: high exp: high exposure) (d) Quantification of the experiment in triplicate shown in (c). Error ars indicate SEM. The p-values (all two-tailed) etween wild-type and cells with a control knockdown are and for DMSO and Baf, respectively. The p-values etween wild-type and cells depleted of endogenous VPS are and for DMSO and Baf, respectively. The p-value for the comparison of cells with and without depletion of endogenous VPS in DMSO is All other comparisons etween cells with and without depletion of endogenous VPS are nonsignificant.
5 anti-vps/lank anti-vps/anti- anti-vps/anti- PLA signal GFP-VPS DAPI PLA signal GFP-VPS DAPI anti-vps/lank anti-vps/anti- anti-vps/anti- Supplementary Figure 5 (continued on next page)
6 nti-vps/lank anti-vps/anti- anti-vps/anti- PLA signal DAPI ATG9-GFP mrfp-rab5 WASH1 Supplementary Figure 5. resides in the same compartment as VPS and the WASH complex. (a) Untransfected HeLa cells, or cells staly expressing GFP-VPS or GFP-VPS were fixed and analysed using the proximity ligation assay (PLA), with primary antiodies as indicated. The cells were imaged y epifluorescence microscopy. Scale ar = 20 µm. () HeLa cells were transfected with -GFP and mrfp-rab5, immunostained for endogenous WASH1, and sujected confocal microscopy. Scale ar = µm.
7 TGN46 GFP-VPS GFP-VPS 2 hr stv 1 hr stv 30 min stv asal TGN46 TGN46 TGN46 TGN46 TGN46 TGN46 TGN46 TGN46 TGN46 Supplementary Figure 6. VPS affects trafficking and localization of. (a) HeLa cells staly expressing GFP-VPS wild-type () and were depleted of endogenous VPS using 40 nm of sirna, and susequently immunostained for TGN46 and endogenous and sujected to confocal microscopy, as in Fig. 6d. Compressed z-stack images are shown. () HeLa cells staly expressing GFP-VPS wild-type () and were starved in HBSS for the time indicated or kept in full medium ( asal ). Following fixation, cells were immunostained for TGN46 and endogenous and sujected to confocal microscopy. Confocal slices are shown. Scale ars in (a) and () = µm.
8 GFP-VPS Golgin-97 GFP-VPS Golgin-97 GFP-VPS GM GFP-VPS GM Supplementary Figure 7. VPS alters Golgi and TGN morphology. (a) HeLa cells staly expressing GFP-VPS wild-type () and were depleted of endogenous VPS using 40 nm of sirna, and susequently immunostained for Golgin-97 and sujected to confocal microscopy. () HeLa cells staly expressing GFP-VPS wild-type () and were fixed and immunostained for GM and sujected to confocal microscopy. Scale ars in (a) and () = µm.
9 VPS TGN46 WASH1 KD starvation WASH1 KD asal control KD starvation control KD asal VPS TGN46 VPS TGN46 VPS TGN46 Golgin-97 c control GM Golgin-97 WASH1 KD WASH1 KD control KD GM Supplementary Figure 8. WASH1 depletion affects ATG9 localization, as well as TGN and Golgi morphology. (a) HeLa cells depleted of WASH1 with two successive sirna treatments were either starved in HBSS for one hour or kept in full medium. Following fixation, cells were immunostained for TGN46,, and VPS and sujected to confocal microscopy, as in Fig. 7a. Compressed z-stack images are shown. () HeLa cells were depleted of WASH1 and fixed as in (a), and immunostained for and Golgin-97. Confocal slices are shown. (c) HeLa cells were depleted of WASH1 and fixed as in (a), and immunostained for GM. Confocal slices are shown. Scale ars in (a)-(c) = µm.
10 GFP-VPS RFP-LC3 GFP-VPS RFP-LC3 GFP-LC3 control GFP-LC3 WASH1 KD Supplementary Figure 9. VPS and WASH1 depletion impair trafficking to autophagosomes. (a) HeLa cells staly expressing GFP-VPS wild-type and were transfected with mrfp-lc3 for 24 hours, immunostained for endogenous, and imaged y confocal microscopy. Compressed z-stack images are shown. () HeLa cells depleted of WASH1 with two successive sirna treatments were then transfected with GFP-LC3 for 24 hours, immunostained for endogenous, and imaged y confocal microscopy. Compressed z-stack images are shown. Scale ars in (a) and () = µm.
11 GFP-VPS mstr-atg16l1, asal GFP-VPS mstr-atg16l1, asal /mstr-atg16l1 d /mstr-atg16l1 Proportion of ATG16L1 signal that contains *** Proportion of ATG16L1 signal that contains control WASH1 KD c control KD mstr-atg16l1 WASH1 KD ns mstr-atg16l1 Supplementary Figure. VPS and WASH1 depletion do not alter ATG16 distriution. (a) HeLa cells staly expressing GFP-VPS wild-type and were transfected with mstrawerry-atg16l1 for 24 hours, immunostained for endogenous, and imaged y confocal microscopy. () Colocalization is expressed in terms of Mander s coefficient M1 to indicate the proportion of ATG16L1 intensities that also contain intensities. n=34 cells () and 27 cells (). Error ars represent SEM and *** indicates p= y 2-tailed Student s t-test. (c) HeLa cells depleted of WASH1 with two successive sirna treatments were then transfected with mstrawerry-atg16l1 for 24 hours, immunostained for endogenous, and imaged y confocal microscopy. (d) Colocalization expressed as Mander s coefficent, as in (). n=18 cells (control) and 19 cells (WASH1 knockdown). Error ars indicate SEM, p=0.14 y 2-tailed Student s t-test. Scale ars in (a) and (c) = µm.
12 GFP-VPS mch-rab11 GFP-VPS mch-rab11 WASH1 KD control mch-rab11 mch-rab11 Supplementary Figure 11. VPS and WASH1 depletion alter recycling endosome morphology. (a) HeLa cells staly expressing GFP-VPS wild-type and were transfected with mcherry-rab11 for 24 hours, immunostained for endogenous, and imaged y confocal microscopy. () HeLa cells depleted of WASH1 with two successive sirna treatments were then transfected with mcherry-rab11 for 24 hours, immunostained for endogenous, and imaged y confocal microscopy. Scale ars in (a) and () = µm.
13 Figure FKBP Strump WASH1 VPS29 VPS WB: FKBP, Strumpellin, WASH1, Figure 1c WB: FKBP, Strumpellin, WASH1,, VPS29 (phosphoimager image) WB: VPS FKBP VPS FAM21 Strump WASH1 Strump VPS29 WB: FKBP, VPS,, FAM21, Strumpellin, WASH1 (higher exp.) WB: FKBP, VPS,, FAM21, Strumpellin, WASH1 (lower exp.) WB: Strumpellin,, VPS29 Figure 1d Figure 2a FKBP Strump WASH1 WB: FAM21, Strumpellin, FKBP, VPS,, VPS29 (phosphoimager image) 31 WB: FKBP, Strumpellin, WASH1,, VPS29 VPS29 WB: FAM21, Strumpellin, FKBP, VPS,, VPS29 Figure 2a, continued WB: FAM21, Strumpellin, WASH1, WB: FKBP, VPS,, VPS29 Supplementary Figure 12. Full scans of uncropped lots
14 Figure 2 Figure 2c FAM21 Strump WASH1 FKBP Strump WASH1 VPS29 WB: FAM21, Strumpellin, WASH1, (right image is from phosphoimager) WB: FKBP, Strumpellin, WASH1,, VPS29 (lower exp) FKBP GFP-VPS FKBP Strump WASH1 VPS29 VPS29 WB: FKBP, Strumpellin, WASH1,, VPS29 (higher exp) FAM21 GFP-VPS WB: FKBP, VPS, VPS29 (right image is from phosphoimager) WB: FAM21, VPS (phosphoimager image) Figure 3d Supplementary Figure 12. continued
15 Figure 4a WB: tuulin (DMSO) WB: tuulin (Baf) WB: LC3 (DMSO) WB: LC3 (Baf) Figure 4f Figure 4h non-specific GFP-α-synuclein A53T GFP WB: GFP (lower exp) Figure 4h, cont WB: GFP (higher exp) WB: tuulin (DMSO) WB: tuulin (Baf) WB: LC3 (DMSO) WB: LC3 (Baf) Figure 4j WB: VPS,, VPS29 (DMSO, lower exp.) WB: VPS,, VPS29 (DMSO, higher exp.) WB: tuulin (DMSO - top, Baf - ottom, shown upside down) WB: VPS,, VPS29 (Baf, lower exp.) WB: VPS,, VPS29 (Baf, higher exp.) Supplementary Figure 12. continued
16 Figure 5a WB: tuulin (DMSO) WB: tuulin (Baf) WB: LC3 (DMSO) WB: LC3 (Baf) Figure 5c Figure 5e WB: tuulin WB: LC3 (lower exp) WB: LC3 (higher exp) WB: tuulin (DMSO) WB: tuulin (Baf) WB: LC3 (DMSO) WB: LC3 (Baf) WB: WASH1 & GAPDH (DMSO, lower exp) WB: WASH1 & GAPDH (DMSO, higher exp) WB: WASH1 & GAPDH (Baf, lower exp) WB: WASH1 & GAPDH (Baf, higher exp) Supplementary Figure 12. continued
17 Figure 5j WB: tuulin (DMSO) WB: tuulin (Baf) WB: LC3 (DMSO, lower exp) WB: LC3 (DMSO, higher exp) WB: LC3 (Baf, lower exp) WB: LC3 (Baf, higher exp) WB: tuulin (DMSO) WB: tuulin (Baf) WB: FKBP (DMSO) WB: FKBP (Baf) Figure 9a 70 WB: WASH1, GAPDH (high exp) WB: WASH1, GAPDH (medium exp) WB: WASH1, GAPDH (low exp) 25 WB: tuulin WB: LC3 Supplementary Figure 12. continued
18 Supplementary Figure 1a WB: FAM21, Strumpellin, WASH1, FKBP, VPS,, VPS29 (lower exp) WB: FAM21, Strumpellin, WASH1, FKBP, VPS,, VPS29 (higher exp) Supplementary Figure 1 Supplementary Figure 1c WB: FAM21, Strumpellin, VPS,, CAPZa, VPS29 (right image is from phosphoimager) Supplementary Figure 1d WB: FKBP, Strumpellin, VPS, VPS29,, FAM21, TBC1D5 (lower exp) (higher exp) WB: FAM21, Strumpellin WB: FKBP, VPS WB: WB: WASH1, Supplementary Figure 12. continued
19 Supplementary Figure 4a WB: tuulin (DMSO) WB: tuulin (Baf) WB: LC3 (DMSO) WB: LC3 (Baf) Supplementary Figure 4c WB: tuulin (DMSO) WB: tuulin (Baf) WB: LC3 (DMSO, lower exp) WB: LC3 (Baf, lower exp) WB: LC3 (DMSO, higher exp) WB: LC3 (Baf, higher exp) Supplementary Figure 4e WB: tuulin (DMSO) WB: tuulin (Baf) WB: VPS (DMSO) WB: VPS (Baf) Supplementary Figure 12. continued
Ctrl CCT7 CCT2 GAPDH * * ** ** * Ctrl Size of LC3 dots (a.u.) mrfp GFP mrfp-gfp-lc3 CCT5 KD CCT7 KD
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