Figure S1.
ERdj8 localization and ERdj8 overexpression or knockdown on autophagy flux. (A) COS-7 cells were cotransfected with ERdj8-Flag and DsRed-KDEL, immunostained with anti-FLAG, and observed on the LSM700. Insets, enlargements of framed regions. White arrows show ERdj8-Flag puncta. Scale bar, 10 µm. (B) HeLa cells stably expressing GFP-ULK1 were starved for 1 h, immunolabeled for ERdj8, and imaged on an SP-8. The number is the percentage and SD of GFP-ULK1–positive structures among ERdj8 structures per cell (n = 14). Scale bar, 10 µm. (C) HeLa cells transfected with RFP-Sec61β and ERdj8-Flag were stained with anti-FIP200 and imaged on an SP-8. Insets, enlargements of framed regions. Scale bar, 1 µm. (D) HeLa cells stably expressing eGFP-LC3 were transfected with mCherry-STX17 and ERdj8-BFP or BFP only, starved for 2 h, and imaged on an SP-8. White arrows show co-localization of eGFP-LC3 and mCherry-STX17. Scale bar, 5 µm. (E) HeLa cells transfected with RFP-Sec61β, GFP-ATG14, and ERdj8-Flag were imaged on an SP-8. Insets, enlargements of framed regions. Scale bar, 1 µm. (F) HeLa cells stably expressing mRFP-eGFP-LC3 were transfected with ERdj8-BFP or BFP alone and then cultured under starvation conditions for 4 h. Images were acquired on a DeltaVision system and subjected to deconvolution. For deconvolution, five 0.2-µm–slice images were processed using SoftWoRx software (Applied Precision). Numbers of larger (red arrowheads, white bars) or smaller (gray bars) GFP-positive LC3 puncta are shown. Insets, enlargements of framed regions. Scale bar, 10 µm. Length is the distance to the most distal point in each of the eGFP-positive puncta. Graph shows numbers of eGFP-positive puncta longer or shorter than 0.4 µm in all cells. **, P < 0.005 by t test. Results are reported as the means of 33 cells ± SEM. (G) LC3 turnover monitored by Western blot in HeLa cells overexpressing ERdj8-Flag, either fed or starved for amino acids (STV), with (+) or without (−) BafilomycinA1 (BafA1). (H) LC3 turnover was monitored by Western blot in HeLa cells following knockdown of ERdj8 using siRNA (siERdj8no1 and siERdj8no2). Cells were either fed or starved for amino acids (STV), with (+) or without (−) BafilomycinA1 (BafA1).

ERdj8 localization and ERdj8 overexpression or knockdown on autophagy flux. ( A ) COS-7 cells were cotransfected with ERdj8-Flag and DsRed-KDEL, immunostained with anti-FLAG, and observed on the LSM700. Insets, enlargements of framed regions. White arrows show ERdj8-Flag puncta. Scale bar, 10 µm. (B) HeLa cells stably expressing GFP-ULK1 were starved for 1 h, immunolabeled for ERdj8, and imaged on an SP-8. The number is the percentage and SD of GFP-ULK1–positive structures among ERdj8 structures per cell (n = 14). Scale bar, 10 µm. (C) HeLa cells transfected with RFP-Sec61β and ERdj8-Flag were stained with anti-FIP200 and imaged on an SP-8. Insets, enlargements of framed regions. Scale bar, 1 µm. (D) HeLa cells stably expressing eGFP-LC3 were transfected with mCherry-STX17 and ERdj8-BFP or BFP only, starved for 2 h, and imaged on an SP-8. White arrows show co-localization of eGFP-LC3 and mCherry-STX17. Scale bar, 5 µm. (E) HeLa cells transfected with RFP-Sec61β, GFP-ATG14, and ERdj8-Flag were imaged on an SP-8. Insets, enlargements of framed regions. Scale bar, 1 µm. (F) HeLa cells stably expressing mRFP-eGFP-LC3 were transfected with ERdj8-BFP or BFP alone and then cultured under starvation conditions for 4 h. Images were acquired on a DeltaVision system and subjected to deconvolution. For deconvolution, five 0.2-µm–slice images were processed using SoftWoRx software (Applied Precision). Numbers of larger (red arrowheads, white bars) or smaller (gray bars) GFP-positive LC3 puncta are shown. Insets, enlargements of framed regions. Scale bar, 10 µm. Length is the distance to the most distal point in each of the eGFP-positive puncta. Graph shows numbers of eGFP-positive puncta longer or shorter than 0.4 µm in all cells. **, P < 0.005 by t test. Results are reported as the means of 33 cells ± SEM. (G) LC3 turnover monitored by Western blot in HeLa cells overexpressing ERdj8-Flag, either fed or starved for amino acids (STV), with (+) or without (−) BafilomycinA1 (BafA1). (H) LC3 turnover was monitored by Western blot in HeLa cells following knockdown of ERdj8 using siRNA (siERdj8no1 and siERdj8no2). Cells were either fed or starved for amino acids (STV), with (+) or without (−) BafilomycinA1 (BafA1).

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