Fig 2: Effects of CTT treatment on the expression of IAP family proteins in A549 and H460 cells. (A,C) After treatment with 0, 5, or 10 µM of CTT or 20 µM GF (clinical anticancer drug) for 20 h, the protein levels of cIAP-1, cIAP-2, and survivin were determined through western blotting. (B,D) The calculations of the results were normalized against ß-actin. * p < 0.05 compared to the 0 µM of CTT group. The data and images represent each of the three independent experiments. Significant differences for the treated groups were determined by Duncan’s test for multiple comparisons. Values are represented as the mean ± SD from each experiment.

Fig 3: Effects on apoptosis-associated genes expression in sh-FZD7-transfected HepG2 and Huh-7 cells. (A) Activation of caspase-3 and caspase-9 by silencing of FZD7. (B) Effects of knockdown of FZD7 on the levels of the IAP family members: a, protein expression levels of XIAP, cIAP-1 and cIAP-2 were determined by western blot analysis; b, mRNA levels of XIAP, cIAP-1 and cIAP-2 were evaluated by RT-qPCR. (C) Effects of knockdown of FZD7 on the levels of the Bcl-2 family members: a, protein expression levels of Bcl-2, Bcl-XL, Bax were determined by western blot analysis; b, mRNA levels of Bcl-2, Bcl-XL, Bax were evaluated by RT-qPCR. ß-actin was probed as a loading control in western blot analysis; GAPDH was used as the loading control for RT-qPCR. Data are presented as the mean ± standard deviation of three independent experiments (n=3; *P<0.05, compared with blank and sh-NC). FZD7, Frizzled-7; sh, short hairpin; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; XIAP, X-linked inhibitor of apoptosis; cIAP, cellular inhibitor of apoptosis; Bcl-XL, apoptosis regulator Bcl-XL; Bax, BCL2 associated X apoptosis regulator; Bcl-2, apoptosis regulator Bcl-2.

Fig 4: Relative protein and mRNA expression of IAPs in untreated and BV6 treated MCF7 and MDA-MB-231cells determined by Real-time PCR and western blotting, respectively. (a) Western Blot images showing the protein bands of untreated control and treated breast cancer cells with 0.5 µM and 1 µM of BV6. Histogram displaying the fold change in the XIAP cIAP1, cIAP2 and survivin protein expression levels normalized by Actin. (b) Fold change in XIAP, cIAP1, cIAP2, survivin and caspase3 mRNA expression levels normalized by Actin in both breast cancer cell lines. The experiments are carried out in triplicates displayed as mean ± SEM, and the data represented is statistically significant (*), (**), and (***) show p < 0.05, p < 0.01 and p < 0.001, respectively.

Fig 5: Dapagliflozin inhibits cFLIPL and cFLIPS protein expression in Caki-1 cells. (A) Cells were treated with various concentrations of dapagliflozin. At 24 h after treatment, protein expression levels of cFLIPL, cFLIPS, Bcl-2, Bcl-xL, Bax, Mcl-1, XIAP, cIAP1, cIAP2 and ß-actin were analyzed using western blotting. ß-actin served as the protein loading control. (B) Caki-1 cells were treated with different concentrations dapagliflozin. After 24 h, the levels of cFLIPL, cFLIPS and GAPDH mRNA (upper panel) were determined using RT-PCR. GAPDH was used as a loading control. The density of cFLIPL, cFLIPS and GAPDH was analyzed using ImageJ software. Data obtained from RT-PCR of cFLIPL, cFLIPS and GAPDH were used to evaluate the effect of dapagliflozin on the cFLIPL/GAPDH ratio (middle panel) and cFLIPS/GAPDH ratio (lower panel). Data were acquired in three independent experiments. PARP, poly (ADP-ribose) polymerase; cFLIP, cellular Fas-associated death domain-like interleukin-1-converting enzyme-inhibitory protein Mcl-1, myeloid cell leukemia-1; XIAP, X-linked inhibitor of apoptosis protein; cIAP, cellular inhibitor of apoptosis protein; RT-PCR, reverse transcription PCR; M, marker of RT-PCR.