Extended Data Fig. 10: CLIMP63 and p180 regulate autophagic flux.
From: ER proteins decipher the tubulin code to regulate organelle distribution
a, U2OS cells were pre-treated with the indicated compounds for 2 h (except etoposide, which was added for 16 h), then with EBSS plus the same compound for 30 min, and then subjected to western blotting. Compounds used: 1 μM lysosome degradation inhibitor DC661, 1 μM proteosome degradation inhibitor MG132, 6 μg/mL ER translation inhibitor puromycin, 100 μg/mL protein translation inhibitor cycloheximide (CHX), 50 μM etoposide that causes DNA damage and also blocks protein expression, and 1 μm N-linked glycosylation inhibitor tunicamycin. b, Western blot analysis of wild-type, CLIMP63 or p180 knockout U2OS cells (Parental, no stable mEmerald-Sec61β expression). c, Representative images and quantifications for wild-type, CLIMP63 or p180 knockout U2OS cells expressing GFP-mCherry-LC3. The GFP signal is quenched by the acidic environment of lysosomes, so only autophagosomes that have not yet fused with lysosomes have green GFP signal, while autolysosomes only exhibit mCherry signal. n = 40 cells. Scale bar, 10 μm. d, Wild-type or CLIMP63 knockout cells were starved in EBSS for 0 or 8 h, with or without brefeldin A treatment to block lysosomal degradation, and then immunoblotted. Relative amounts of autophagic substrate p62 are shown in the lower panel. n = 4 experiments. e, Cathepsin L activity as determined by substrate reaction. n = 4 experiments. f, Lysosome acidification analysis using Lysosensor Green. n = 200 cells. g, Wild-type or p180 knockout cells were starved in EBSS for 0 or 8 h, with or without brefeldin A treatment to block lysosomal degradation, and western blotted. h, Wild-type or p180 knockout cells were starved in EBSS for 2 h, then re-supplemented with regular medium for 0-30 min. Cells were immunoblotted for phosphorylated S6K (p-S6K) and total S6K to indicate activity of mTOR signaling. n = 4 experiments. i, Western blots reveal the indicated microtubule modifications with or without EBSS starvation for 0.5 and 2 h. Relative intensities of GT335 and polyE immunoreactive signals are quantified at the right. n = 5 experiments. j, Representative images for PLA of p180 and the ribosomal marker RPL3 at 0 or 2 h of EBSS starvation, or else for cells treated with 2 μg/mL puromycin for 2 h. Scale bar, 50 μm. k, Representative images of U2OS cells transfected with mEmerald-Sec61β, p180s-mEmerald, or p180L-mEmerald with or without co-transfection of TTLL7-3×flag. Scale bar, 10 μm. l, Quantifications of microtubule alignments in cells transfected with the indicated constructs; n = 4 experiments, with at least 100 cells counted per experiment. m, Microtubule alignments of p180L-mEmerald in cells treated with EBSS or 2 μg/mL puromycin for 2 h. n = 6 experiments, with at least 100 cells counted per experiment. Data are mean ± s.d. P values are shown along the top, two-tailed t-tests. See Supplementary Information for uncropped western blots.
