Fig. 3

ER accumulation of CSp variants triggers cell death. (A) RNA-seq gene set enrichment analysis of the gene set: Response to ER stress (human gene set Gene Ontology GO:0034976) comparing si-USP39 and si-Ctrl in HeLa and RPE1 cells (n = 3). NES, normalized enrichment score. (B) Immunofluorescence analysis comparing BiP (red) and calreticulin (purple) protein localization and abundance in si-USP39 and si-Ctrl cells (left). BiP abundance is shown in the box plots on the right (n = 50; two-tailed t test; scale bars are 10 ?m). DAPI, 4?,6-diamidino-2-phenylindole; RFU, relative fluorescence units. (C) Volcano plots showing ER proteomic fraction for si-USP39 and si-Ctrl cells (red indicates significantly increased protein abundances in proteins translated from misspliced transcripts in si-USP39 cells; n = 3). (D) Immunoblots against phosphorylated IREI (p-IRE1), ATF6, ATF4, and glyceraldehyde phosphate dehydrogenase (GAPDH) in si-USP39 and si-Ctrl cells (left) and a quantification of p-IRE1, ATF4, and cleaved ATF6 in si-Ctrl and si-USP39 HeLa cells (right) (data are means ± SD; two-tailed t test; n = 3). (E) Heat map representing RNA-seq expression (z-score) of representative UPR target genes for ATF6, IRE1, and PERK UPR pathways in si-USP39 and si-Ctrl HeLa cells (n = 3). (F) Immunofluorescence comparing CHOP protein abundance (green) in si-USP39 and si-Ctrl cells (left). A CHOP quantification is shown in the box plots on the right (n = 50; two-tailed t test; scale bars are 10 ?m). (G) IncuCyte S3 assay quantifying cytotoxicity (green dye penetrates dead cells). Cytotox green levels after USP39 depletion in control and CHOP KO HeLa cells (data are means ± SD; two-tailed t test; n = 3). (H) Schematic model. CS generates proteotoxic isoforms (misfolded and unfolded proteins), which accumulate in the ER, causing ER-stress, UPR activation, and apoptosis. For box-and-whisker plots, the center line represents the median, box limits are upper and lower quartiles, and whiskers are minimum and maximum values.