Fig. 5: Targeted silencing of INTS13 induces apoptosis in cervical cancer cells while sparing cervical epithelial cells.

Primary pCCa-1 cervical cancer cells were transfected with three distinct shRNAs (shINTS13-Sq1, shINTS13-Sq2, and shINTS13-Sq3) or control shRNAs (shC). Cells were further cultivated for the indicated time periods, subsequently, the enzymatic activities of Caspase-3 (A) and Caspase-9 (B) were assessed. Western blot analysis (C) was performed to examine the proteolytic cleavage of PARP and Caspase-3 (Cld-PARP1 and Cld-Caspase-3, respectively). Cytosol cytochrome C levels were also measured using an ELISA kit (D). Mitochondrial depolarization was assessed using JC-1 staining to determine the green monomers intensity (E). The nuclear TUNEL staining assay (F) and Trypan blue staining assay (G) were employed to assess apoptotic cells and overall cell death. The efficacy of the pan-caspase inhibitor z-VAD-fmk (50 μM) and the caspase-3 inhibitor z-DEVD-fmk (50 μM) in inhibiting INTS13 silencing (shINTS13-Sq3)-induced cell death (H) and viability (CCK-8 OD) reduction (I) was evaluated. The apoptotic effects of INTS13 silencing (shINTS13-Sq3) were examined in additional primary cervical cancer cell types (pCCa-2, pCCa-3) and the established HeLa cervical cancer cell line, assessing mitochondrial depolarization (J), Caspase-3 activity (K), prevalence of TUNEL-positive cells (L), and cellular death (M). The effect of shINTS13-Sq3 was investigated in non-cancerous primary human cervical epithelial cells (priCEpi-1 and priCEpi-2). INTS13 mRNA silencing (N) and INTS10 mRNA levels (O) were assessed in these cervical epithelial cells. Apoptosis induction in priCEpi-1 and priCEpi-2 cells was evaluated by TUNEL staining (P), and cell death was tested via Trypan blue staining assays (Q). “Ctrl” stands for the parental control cells. Data are presented as mean ± standard deviation (SD) with n = 5 biological replicates. Asterisks (*) indicate statistically significant differences (P < 0.05) compared to shC group. # P < 0.05 (H, I). “n.s.” denotes no statistically significant difference (P > 0.05). The experiments were independently replicated five times with consistent results. Scale bar represents 100 μm.