Annexin 5 fitc pi staining

When cells were treated with ART or IM, cells were collected for Annexin V-FITC/PI staining as the instructions from the manufacturer (MultiSciences Biotech Co., Ltd, Hangzhou, China) and subjected to analysis on a BD flow cytometer as described previously^{23 (link)}.

When cells were treated with WP1130 for 12 h or cells were knocked down of c-Maf or USP5 for 48–72 h, cells were collected for Annexin V-FITC/PI staining as the instructions from the manufacturer (MultiSciences Biotech Co., Ltd, Hangzhou, China) and subjected to analysis on a BD flow cytometer as described previously.^{10 (link)}

For flow cytometric analysis, the cells were treated with SFI003 (10, 20, 50 μM) for 24 h, 48 h, or 72 h or were transfected with 100 nM SRSF3 siRNA or DHCR24 siRNA for 72 h. Then, the cells were collected for Annexin V-FITC/PI staining according to the instructions from the manufacturer (MultiSciences Biotech, Hangzhou, China) and subjected to analysis on a flow cytometer (BD Biosciences, CA, USA). Three independent measurements were performed.
For Hoechst staining, after treatment with SFI003 (10, 20, 50 μM) for 72 h, the cells were fixed, washed three times with PBS, and stained with Hoechst 33258 staining solution according to the manufacturer’s instructions (Beyotime, Shanghai, China). Hoechst 33258-stained nuclei of cells were imaged by inverted fluorescence microscopy (Olympus, Tokyo, Japan). The nuclei in 8 random fields were analyzed at ×400 magnification.