Fig. 5: Knockdown of FliI causes ER Ca²⁺ release through RyRs, which induces ER stress-mediated apoptosis in CT26 cells.

a Ctrl- and FliI-KD cells were pretreated for 2 h with 2-APB (50 μM) and dantrolene (50 μM) and tetracaine (50 μM) and then incubated in Fura-2AM for 30 min. Ratiometric Ca²⁺ imaging was performed in 0 mM Ca²⁺ Tyrode’s solution with or without 5 μM ionomycin, and Ca²⁺ influx was monitored based on the Fura-2 fluorescence ratio. b After 24 h transfection of ctrl- and FliI-KD cells with pcDNA-D1ER, dantrolene (50 μM) and tetracaine (50 μM) were treated for 2 h, and the FRET signal was measured. Data are shown as the mean ± SEM. ***p < 0.001, shRNA-Ctrl cells vs. shRNA-FliI cells. Dan dantrolene, Tet tetracaine. c Ctrl- and FliI-KD cells were treated with 50 nM TG for 6 h following pretreatment with 50 μM dantrolene. Cell lysates were analyzed by western blotting for phospho-PERK, phospho-IREα, GRP78/BiP, CHOP, FliI and tubulin. d, e Ctrl- and FliI-KD cells were pretreated for 2 h with dantrolene (20 μM) and then with TG (100 nM) for 48 h. Apoptosis was detected by annexin V-PI flow cytometry assay (d) and western blotting for PARP-1, cleaved caspase-3, FliI, and tubulin (e). TG thapsigargin, Dan dantrolene.