Fig. 8. ASO-mediated BCL2L12 exon skipping induces apoptosis of ovarian cancer cells.

a Schematic diagram of ASO target sites on BCL2L12 based on the BUD31-binding region on BCL2L12 as determined from the CLIP-seq data. b RT-PCR analysis of the BCL2L12 AS pattern in response to ASOs. c Western blot analysis of the BCL2L12 protein level in A2780 cells transfected with ASOs. Semi-quantitative RT-PCR analysis of the BCL2L12 AS pattern in HEYA8 (d) and A2780 (e) cells transfected with ASO2. Dose-dependence curve of ASO2-treated HEYA8 and A2780 cells showing increased skipping of exon 3 [(exon 3 skipped/exon 3 skipped + full-length) × 100] in relation to the log of the dose. The EC₅₀ was calculated in HEYA8 (63.77 nM) and A2780 (52.91 nM) cells. BCL2L12 and cleaved-caspase-3 were measured by western blot in A2780 cells treated with 200 nM ASO2 for different times (0, 24, 48, and 72 h) (f) and with different concentrations (0, 25, 50, 100, 200 nM) of ASO2 for 72 h (g). h Apoptotic cells were detected by flow cytometry after staining with Annexin V/7-AAD in A2780 and HEYA8 cells treated with ASO2 (200 nM). i, j EdU assay was performed in ovarian cancer cell lines treated with 200 nM ASO2. The IC₅₀ was calculated based on the MTT assay. k–n ASO2 intratumoral injection to subcutaneous tumor xenografts using HEYA8 cells. The xenograft model showed the inhibitory effect of ASO2 on tumor growth (n = 6 mice per group) (k). The tumor weight (l) and volume (m) were measured for each group. The BCL2L12 and Ki-67 expression levels were evaluated with immunohistochemical staining, and the apoptosis level was determined by a TUNEL assay (n). All functional experiments were conducted with n = 3 biological repeats. The p values were obtained by two-tailed unpaired Student’s t test (h, i, l, m), and the results are presented as the mean ± SD. *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.