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Figure 2. ER stress induction enhances M1 virus-mediated cytotoxicity through the IRE1α pathway in MIBC cells. (A) Western blot of PERK and IRE1α expression across normal and bladder cancer cell lines; (B and C) Cell viability of T24, UM-UC-3, and EJ cells treated with M1 alone or M1 + STF (10 µM) for 48 h; (D) T24 and UM-UC-3 cells were treated with STF (10 µM), M1 (MOI = 0.01), or a combination of both. Protein levels of IRE1α, sXBP1, and M1 viral proteins E1 and NS3 were analyzed by Western blot, followed by quantification of E1 and NS3 expression; (E) Morphological changes in T24 and UM-UC-3 cells treated with control (CTL), STF (10 μM), M1 (MOI = 0.01), or STF + M1 (scale bar = 100 μm); (F) Western blot analysis of ER stress markers (IRE1α, PERK, ATF6, and p-eIF2α) in T24 and UM-UC-3 cells over time following co-treatment with STF (10 μM) and M1 (MOI = 0.01). Densitometric analysis of IRE1α and PERK expression in UM-UC-3 cells is also shown; (G and H) Phase-contrast images and viability of HBSMCs treated with M1 (MOI = 10) and/or STF (scale bar = 100 μm); no significant cytotoxicity observed; (I) Cell viability in UM-UC-3 cells treated with M1 (MOI = 0.01) alone or M1 + GSK (10 μM) shows no enhancement of cytotoxicity; (J) Western blot of PERK and IRE1α over time in UM-UC-3 cells treated as in (I). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; N.S.: not significant. ER: Endoplasmic reticulum; IRE1α: inositol-requiring enzyme 1 alpha; MIBC: muscle-invasive bladder cancer; PERK: protein kinase RNA-like ER kinase; MOI: multiplicity of infection; sXBP1: spliced XBP1; HBSMCs: human primary normal bladder epithelial cells.