Figure S2.

Scheme of mRFP-eGFP-Galectin8 engulfment assay, additional effect of ERdj8 knockdown on mRFP-eGFP-Galectin8 1-µm bead assay, and data on dnj-8 knockdown on C. elegans. (A) Scheme of mRFP-eGFP-Galectin8 engulfment assay. (B and C) HeLa subjected to the indicated siRNA treatment or ATG16L-knockout stably expressing mRFP-eGFP-Galectin8 was treated with 1-µm beads for 24 h and imaged on a DeltaVision system. Insets, enlargements of framed regions. Scale bar, 10 µm. (C) GFP and RFP signal intensities associated with beads were measured; the GFP/RFP ratio of 92 beads is shown. **, P < 0.005. Median: line; upper and lower quartiles: boxes; 1.5-interquartile range: whiskers. (D) The cells were treated with CCCP or DMSO, immunostained with ERdj8 and Tomm20 antibodies, and imaged on a DeltaVision system. Scale bar, 10 µm. The number is the percentage and SD of ERdj8–positive structures associating with Tomm20 per cell (n = 10). White arrows show ERdj8–positive structures associating with Tomm20. Scale bar, 10 µm. (E and F) C. elegans expressing GFP::lgg-1 with or without dnj-8 RNAi treatment was grown to the adult stage, and GFP::LGG-1 in oocytes was imaged on an FV1000. Insets, enlargements of framed regions. (F) Length of the most distal point in each of the eGFP-positive puncta. The average number of eGFP-positive puncta longer or shorter than 1 µm in a total of 29 cells is shown. Error bars indicate SEM. **, P < 0.005. (G and H) GFP::mito mRNA levels (G) and protein levels (H) in mock, dnj-8–knockdown, or atg-5–knockdown. C. elegans embryos from worms expressing GFP::mito around the 1.5-fold stage were measured by RT-PCR or by Western blotting.