Fig 2: FOXD3 hypermethylation suppresses ovarian cancer apoptosis in vitro. a, b, c, f Flow cytometry showed that the percentage of cell apoptosis elevated in control group, but FOXD3 over-expression group and 5-Aza- dC group increased the most. The data from flow cytometry confirmed the results, *p < 0.05. d, e, g, h Expression levels of apoptotic proteins Annexin V, cleaved PARP and caspase-3 in SKOV3 and OV90 cells. The results showed that the expression level of apoptotic proteins in the over-expression FOXD3 group and the demethylated group was higher than that in the control group, **p < 0.01

Fig 3: APIM-peptide increases the effects of docetaxel on apoptosis and cellular signaling(A) Cell cycle analysis and fraction of apoptotic (A) vs necrotic (N) cells (values from contour plots are given). (B) Relative expression of phosphorylated Akt, ERK1, ERK2, S6K, and p38, cleaved-PARP, and caspase 3. For all experiments, PC3 and Du145 cells were treated with APIM-peptide (14 μg/mL, green), docetaxel (2.5 ng/mL: PC3; 1.3 ng/mL: Du145; 5 ng/mL, blue) and the combination of these (black) for 24 hours prior to the analysis. The protein levels (B) were adjusted for loading differences (β-tubulin) and normalized against untreated cells, and additionally for total protein levels for the phosphorylated proteins. Data are from three biological replicas (different symbols represents extracts acquired on different days). Statistically significant differences (*p < 0.05) were calculated by a Kruskal-Wallis H test, and a post-hoc Dunn’s test was used to determine which groups this applied to.

Fig 4: The ARTS-mimetic A4 is a potent inducer of caspase-9- and caspase-3-mediated apoptotic cell death.a (I) Immunofluorescence images of cleaved caspase-3 (cCasp-3, green) after 6 and 24 h of treatment with A4. Magnification ×40, scale bar = 50 µm. (II, III) Quantification of the percentage of cells stained positive for activated caspase-3 (in I) normalized to DMSO-treated cells (n = 3). Dots represent an average of five frames from each independent experiment; bars; SEM, one-way ANOVA, **p < 0.01, ***p < 0.001. b (I, II) Representative Western blot (WB) analysis of XIAP and of cleaved caspase-3 (cCasp-3) (I) or cleaved caspase-9 (cCasp-9) expression in Jurkat cells (I) or in A375 cells (II) upon treatment with A4 (n = 3). c Caspase-3/7 activity in HeLa cells treated with A4. Data were normalized to DMSO-treated cells (n = 3). d cCasp-9 activity measured in A375 cells treated with A4 (n = 3). Dots represent an average of triplicates for each independent experiment; bars; SEM, one-way ANOVA, **p < 0.01. e–g Jurkat cells treated with A4 in the presence or absence of Q-VD-Oph. e Representative dot plots from FACS analysis of Jurkat cells stained with Annexin V/PI. f Quantification of Jurkat cells that were positive for Annexin V staining and negative for PI staining upon treatment with A4 in the absence or presence of Q-VD-Oph (ON) (n = 4). Dots represent an average of triplicate experiments. Bars; SEM, one-way ANOVA, *p < 0.05, **p < 0.01. g Representative WB for the expression of cCasp-9 and cleaved PARP (cPARP) in Jurkat cells treated with A4 in the absence or presence of Q-VD-Oph (n = 3).

Fig 5: ADR+Olaparib induces apoptosis by altering the protein expression of γ-H2AX, PARP1, cleaved-PARP, caspase 3 and cleaved-caspase 3 in K562/ADR cells. (A) mRNA expression of PARP1 and (B) H2AX. (C) Western blotting and (D) statistical analysis of PARP1, cleaved-PARP, Caspase 3 and cleaved-Caspase 3 normalized to β-actin. (E) Western blotting and (F) statistical analysis of γ-H2AX normalized to β-actin. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001, as indicated; ##P<0.01 and ###P<0.001 vs. Control. ADR, Adriamycin; H2AX, H2A histone family member X; PARP, poly (adenosine diphosphate-ribose) polymerase.