Figure 5.

Salinomycin treatment increases cytosolic Ca²⁺ and induces ER stress. (A) Acute salinomycin treatment induced Ca²⁺ release from ER. JIMT-1 cells were labeled with Fluo-4 AM and imaged with confocal microscopy. Salinomycin or salinomycin methyl ester 2 was added at a 2 μM concentration in Ca²⁺-containing or Ca²⁺-free medium. Ca²⁺ release channels in the ER membrane were blocked with 100 μM ryanodine and 50 μM 2-APB before the addition of salinomycin. Data shown are mean ± SE (n = 4). (B) Salinomycin or salinomycin 20-ethyl carbonate 3 treatment increased cytosolic Ca²⁺. After 72 h of treatment at indicated concentrations, cells were stained with Fluo-3 AM and analyzed with flow cytometry. The relative fluorescence intensity representing the Ca²⁺ level in the cytosol was calculated. Data shown are mean ± SE (n = 4). (C) Increased PKC activity in cells treated with 0.5 μM salinomycin for 72 h. Data shown are mean ± SE (n ≥ 4). (D) Supervised hierarchical clustering of differentially expressed genes in JIMT-1 cells treated with 50 nM salinomycin or salinomycin 20-ethyl carbonate 3 for 72 h (n = 6). SAM analysis was performed to identify differentially expressed genes between the groups. Genes with q-value ≤1 and an absolute fold change ≥2 were considered to be significantly differentially expressed. Red represents relative up-regulation, and green represents relative down-regulation. (E) Top significantly enriched biological processes up-regulated in carbonate 3-treated cells. Gene ontology (GO) enrichment analysis was performed using the AmiGO database. (F) Activation of the ATF6α pathway by salinomycin or inactive salinomycin methyl ester 2 treatment in JIMT-1 and MCF-7 cells. Cells were treated with the indicated concentrations for 72 h. Representative Western blots (n = 4) used for densitometric scanning showing the expression of UPR-related proteins. SA, salinomycin (1). (G) CHOP-siRNA-treated JIMT-1 cells reduce the level of active β-catenin upon salinomycin treatment. Cells were transfected with CHOP-siRNA and scramble siRNA for 72 h, followed by treatment with salinomycin (5 μM) for 72 h. Representative Western blots (n ≥ 4) used for densitometric scanning showing the expression of active β-catenin and CHOP. SA, salinomycin (1). *P < 0.05; **P < 0.01; ***P < 0.001.