Extended Data Fig. 10: PDIA6-scaffolded chaperone condensates reduce ER protein aggregation.
From: A multichaperone condensate enhances protein folding in the endoplasmic reticulum
a, Co-localization analysis by Pearson Correlation Coefficient (PCC) for pixel intensity of proinsulin and endogenous and overexpressed (*) PDIA6 in HeLa cells. Individual data points (PCC per cell) (n = 15/11 cells for PDIA6-proinsulin and PDIA6*-proinsulin respectively) pooled across three independent experiments and standard deviation (for details see Supplementary Table 2). b, Secretion levels of insulin (medium) and expression levels of proinsulin, PDIA6 and α-tubulin (lysate) in proinsulin, proinsulin + PDIA6–GFP WT or proinsulin + PDIA6–GFP 5x linker mutant transfected HeLa cells after 18 h treatment with the proteasome inhibitor MG-132. c, Live-cell imaging of INS-1 cells transfected with PDIA6–GFP WT (left) or PDIA6–GFP 5x linker mutant (right). PDIA6–GFP was visualized by imaging eGFP. Scale bar, 10 μm. d, Aggresomes detected by PROTEOSTAT dye (green) in fixed HeLa cells transfected with PDIA6–GFP WT (left) or PDIA6–GFP 5x linker mutant (right) (pink). Scale bar, 20 μm, insert 5 μm. e,f, FRAP experiments of mApple–Sec61 (grey) in HeLa cells, co-transfected with PDIA6–GFP WT (e) or PDIA6–GFP 5x linker mutant (f) (pink). Due to strong bleaching of the mApple, contrast and brightness were adjusted for each image individually. Scale bar, 20 μm, insets 5 μm. g, Fluorescence recovery curve of photobleached mApple–Sec61 in PDIA6–GFP WT (pink) or PDIA6–GFP 5x linker mutant (grey) co-transfected HeLa cells. Average and standard deviation of experimental triplicates. Bleached areas did not overlap with condensates. h,i, Cell viability of PDIA6–GFP WT (pink) and PDIA6–GFP 5x linker mutant (grey) transfected cells relative to mock-transfected cells by MTT. (***P = 0.0003, two-sided t-test) (n = 9, triplicates of three independent biological experiments; h) and trypan blue assay (***P = 0.0003, two-sided t-test) (n = 9, parallel triplicates from three independent biological experiments; i). For the trypan blue assay a total of 12,760 (PDIA6–GFP WT), 8,519 (PDIA6–GFP 5x) and 16,150 (mock-transfected) cells were counted (i). Mean and standard deviation. j,k, Cell viability of PDIA6-knockdown cells, of PDIA6–GFP WT (pink) and PDIA6–GFP 5x linker mutant (grey) transfected cells relative to mock-transfected cells by MTT (PDIA6-siRNA1: **P = 0.003, PDIA6-siRNA2: ***P = 0.00008, two-sided t-test)(n = 9, triplicates of three independent biological experiments; j) and trypan blue assay (PDIA6-siRNA1: ** p = 1.64·10−8, PDIA6-siRNA2: *** p = 0.0004, two-sided t-test) (n = 9, triplicates of from three independent biological experiments; k). For the trypan blue assay a total of 90,350 (PDIA6–GFP WT, siRNA1), 76,070 (PDIA6–GFP 5x, siRNA1), 75,040 (mock-transfected, siRNA1), 54,850 (PDIA6–GFP WT, siRNA2), 66,210 (PDIA6–GFP 5x, siRNA2) and 41,530 (mock-transfected, siRNA2) PDIA6-knockdown cells were counted (k). Mean and standard deviation. l, Western blot showing expression levels of PDIA6 and α-tubulin and in non-treated and PDIA6-knockdown (siRNA1 and siRNA2) HeLa cells. Unprocessed blots are available.
