Fig 2: Caspase-3 immunohistochemistryRepresentative examples of microphotographs from 24 hours after hypoxic-ischemia in the hippocampal dentate gyrus (A) and cortex (B). Note that the Caspase-3 immunoreactive signals were significantly increased in the HI group compared to the Sham, and decreased after the RSV post-treatment. Scale = 100 μm.versus the Sham, and decreased after post-treatment with RSV. Scale = 100 μm.

Fig 3: Role of p53 in apoptosis induced by PHD (prolyl hydroxylase domain) inhibition using CoCl2 in cultured rat cardiomyocytes. A, Tp53 expression in cultured cardiomyocytes, treated with vehicle (Veh) and CoCl2 (500 μmol/L) for 24 hours, or transfected with small interfering RNA (siRNA) for control (CTL) (scramble siRNA, CTL) and p53, was quantified by quantitative reverse transcription‐polymerase chain reaction; n=6 in each group. B, Expression of Hif‐1α, p53, and CC‐3 (cleaved caspase 3) in cardiomyocytes treated with Veh and CoCl2 (500 μmol/L) for 24 hours, or transfected with siRNA for CTL (scramble siRNA, CTL) and p53. GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) was used as a loading CTL. C, Quantification of Western blots shown in (B); n=6 in each group. Data are shown as a ratio to the average of Veh+CTL. D, Images of cardiomyocytes treated with Veh and CoCl2 (500 μmol/L), or transfected with siRNA for CTL (scramble siRNA, CTL) and p53. E, Cellular viability using cultured cardiomyocytes treated with Veh and CoCl2 (500 μmol/L), or transfected with siRNA for CTL (scramble siRNA, CTL) and p53 by CellTiterBlue Assay; n=6 in each group. **P<0.01, analyzed by 1‐way ANOVA, followed by Tukey post hoc test. KD indicates knockdown; and N/A, not applicable.

Fig 4: CPX induces apoptosis in bladder cancer cell lines.A T24 and UM-UC-3 cells were treated with CPX stained with Annexin V (FITC) and PI, and analyzed by flow cytometry. CPX treatment induced significant early and late apoptosis in T24 and UM-UC-3 cells. B The flow cytometric quantification of early and late apoptotic cells after CPX treatment over a period of 72 h in T24 and UM-UC-3 cells in Annexin-PI assay. C CPX induces apoptosis in T24 cells. T24 cells treated with CPX (4 µM) for 48 h were stained with Annexin V-FITC/PI solution and studied using immunofluorescence. CPX induced apoptosis in T24 cells. D Lysates from T24 and UM-UC-3 cells treated with CPX were studied by western blot for evaluating the effect on proteins involved in apoptosis. CPX treatment reduced the levels of antiapoptotic marker proteins Bcl2 and Bcl-XL. CPX treatment also increases the PARP cleavage compared to untreated controls. E CPX induces autophagy early which then followed by apoptosis. Lysates from T24 and UM-UC-3 cell treated with CPX (4 µM) for 24–48 h were analyzed by western blot. CPX treatment increased the expression of LC3B at 24 h and cleaved caspase-3 expression at 48 h. The data suggest that at early time points, CPX induces autophagy, which switches to apoptosis in T24 and UM-UC-3 cells.

Fig 5: Bioengineered miR-34a prodrug enhanced the apoptosis of osteosarcoma 143B cells.(A) Comparison of flow cytometry histograms of Annexin V/propidium iodide stained cells, following the treatments with tRNA/mir-34a, control tRNA/MSA and vehicle. (B) Comparison of the percent of apoptotic cells with different treatments. (C) Confocal immunofluorescent analyses of Caspase-3 biomarker in 143B cells. (D) The number of Caspase-3-positive cells were compared for different treatments. Cells were treated with 4 nM tRNA/mir-34a, tRNA/MSA or the vehicle for 72 h, and apoptosis was assessed by Annexin V/propidium iodide flow cytometric analysis. A separate batch of treated cells were fixed with 10% formalin, stained with Caspase-3 antibody, and counterstained with DAPI. Caspase-3 fluorescence and DAPI-stained nuclei images were acquired with a confocal microscope. Values are mean ± SD of triplicate treatments. *P < 0.05, **P < 0.01, and ***P < 0.001 (two- or one-way ANOVA).