FIG. 6.

ER is involved in KHDC1A-induced apoptosis. (A) Overexpressed KHDC1A is colocalized with ER. HeLa cells were transfected with KHDC1A or KHDC1B. Immunostaining was performed to determine the expression of KHDC1A, KHDC1B (FITC, green), and ER marker Calnexin (Cy5, red). Scale bar was 16 μm. (B) C-terminus of KHDC1A determines the ER localization. HeLa cells were transfected with Flag-tagged KHDC1A-tr or KHDC1B-fu. Immunostaining was performed to determine the expression of KHDC1A-tr, KHDC1B-fu (FITC, green), and ER marker Calnexin (Cy5, red). Scale bar was 16 μm. (C) The cleavage of caspase-12 is detected in Flag-tagged KHDC1A-transfected Hela cells. HeLa cells were transfected with control vector, Flag-tagged KHDC1A, or KHDC1B plasmids. The cells were harvested, and Western blot analysis was performed with antibodies against Flag, caspase-12, cleaved PARP, and β-tubulin. (D) Caspase-12 specific inhibitor Z-ATAD rescues KHDC1A-induced apoptosis. HeLa cells were transfected with KHDC1A. Transfected cells were treated with 20 μM Z-ATAD, Z-DEVD, or DMSO as control. Twenty-four hours after transfection, the cells were harvested, and apoptosis was assayed by flow cytometry. Data were summarized from three independent experiments. (E) Caspase-12 specific inhibitor Z-ATAD cannot rescue Fas-induced apoptosis. Hela cells were pretreated with mock or 1 μg/mL of anti-Fas/Apo1 antibodies (Biovision) for 6 h and then exposed to 20 μM of caspase-12 inhibitors Z-ATAD FMK for another 48 h. Cells were then harvested, and apoptosis was assayed by flow cytometry. Data were summarized from three independent experiments. ER, endoplasmic reticulum.