Fig. 2

Thioridazine induces ER stress and ICD markers in CRC cell lines. Next-generation sequencing (NGS) of total RNA following THD treatment for 6 h was performed in HT29 cells. A Number of upregulated genes. B Autophagy and ER stress markers. C GSEA revealed that THD-induced upregulation of genes associated with the ATF4-activated ER stress response. D Protein levels of p-eIF2α, ATF4, CHOP, and TRIB3 of the ER stress pathway in HT29 and LoVo cells following treatment with varying concentrations of THD for 24 h were analyzed by western blotting (left) and the corresponding quantification of these proteins is shown in the right panel. 4-PBA, an ER stress inhibitor, was treated at the concentration of 5 mM alone or in combination with 15 μM THD. The level of extracellular ICD markers including (E) ATP, (F) HMGB1, and (G) calreticulin (CRT) were examined using an ATP detection kit, ELISA, and flow cytometer, respectively, after 24 h of drug treatment in CRC cells SW480. Data are mean ± SD (N = 3). *: p < 0.05; **: p < 0.01; ***: p < 0.001