USP5-mediated ORP8 accumulation contributes to brigatinib-induced ER stress in CRC cells. A, Immunoblotting of ORP8 in CRC cells treated with the indicated concentrations of brigatinib for 24 hours. B, Immunofluorescence analysis of ORP8 in CRC cells treated with 1 μM brigatinib for 12 hours. Scale bar, 10 μm. C, Immunoblotting of ORP8 in CRC cells treated with or without 1 μM brigatinib in the presence or absence of 2 mM 4-PBA for 24 hours. D, Immunoblotting of total and phosphorylated PERK, IRE1α in CRC cells transfected with siORP8 or siScramble followed by treatment with or without 1 μM brigatinib for 24 hours. E-F, Immunoblotting (E) of ORP8 in CRC cells pretreated with 1 μM brigatinib for 12 hours followed by exposure to 50 μg CHX combined with or without 1 μM brigatinib for the indicated time. Quantification of relative ORP8 expression (F) was shown. **, P < 0.01; ***, P < 0.001. G, Coimmunoprecipitation showing the interaction among ORP8, USP5 and ubiquitin after brigatinib treatment. H, Colony formation assay of CRC cells transfected with siORP8 or siScramble followed by treatment with or without 1 μM brigatinib. Representative images (Top) and quantification of colonies (Bottom) were shown. **, P < 0.01. I, Cell growth of CRC cells transfected with siORP8 or siScramble followed by treatment with or without 1 μM brigatinib for 24 hours. *, P < 0.05.