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Figure 1. M1 virus induces ER stress selectively in MIBC cells and activates the IRE1α pathway. (A) Cell viability of bladder cancer cell lines and normal urothelial cells after M1 infection (MOI = 10, 48 h); (B) Time course of viral yield in four representative cell lines following M1 virus infection (MOI = 10); (C) Pathway enrichment heatmap based on RNA-seq data comparing M1-treated vs. control tumor cells; ER stress and UPR-related pathways are upregulated (red arrows); (D) TEM of ER morphology in T24, UM-UC-3, and EJ cells treated with control or M1. ER: endoplasmic reticulum; N: nucleus; (E) Quantification of ER width based on TEM images; (F-H) Relative mRNA expression of UPR pathway genes IRE1α, PERK, and ATF6 at 0, 12, 24, and 48 h post-infection in T24, UM-UC-3, and EJ cells; (I) Immunoblot analysis of ER stress proteins (IRE1α, PERK, ATF6), antiviral protein ZAP, and M1 viral proteins (E1, NS3) in the same cells over 48 h post-infection. GAPDH was used as a loading control. The expression levels of IRE1α and PERK in T24, UM-UC-3, and EJ cells were further quantified by densitometric analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; N.S.: not significant. MIBC: Muscle-invasive bladder cancer; IRE1α: inositol-requiring enzyme 1 alpha; MOI: multiplicity of infection; TEM: transmission electron microscopy; UPR: unfolded protein response; ZAP: zinc finger antiviral protein; PERK: protein kinase RNA-like ER kinase.