Fig 2: Evaluation of apoptosis induction: Western blot analysis of Bcl-2 (A), Bax (B) and Cleaved-caspase 3 (C) expression. For the analyses, n = 5 animals from each group were employed. A p value of less than 0.05 was considered significant. ** p < 0.01 vs. vehicle, *** p < 0.001 vs. vehicle.

Fig 3: Overexpression of miR-506 and miR-124 inhibits apoptosis and ROS in H2O2-incubated HCMs. Overexpression of (A) miR-506 and (B) miR-124 had no effect on the mRNA expression levels of KLF4 or KLF5. At 24 h post-transfection with miR-506 and miR-124, the protein expression levels of (C) KLF4 and KLF5, and (D) caspase-3 and caspase-9 were measured using western blot analysis. When (E) miR-506 and (F) miR-124 were overexpressed, intracellular ROS production was measured according to changes in the fluorescence intensity of DCF for 12 h treatment in the presence of H2O2, the oxidized derivative of DCF-DA. Values are expressed as the mean ± standard deviation (n=3 in each group). *P<0.05, vs. control group. miR, microRNA; KLF, krüppel-like factor; ROS, reactive oxygen species; DCF-DA, dichlorodihydrofluorescein diacetate.

Fig 4: XPO1 inhibition by KPT-185 induces selective cytotoxicity, apoptosis and cell cycle arrest in PEL cells.(A) PEL cell lines, BC-1, BCBL-1 and JSC-1, and whole PBMC fraction from normal donors were examined for XPO1 expression by western blot.(B) Cell viability of BC-1, BCBL-1 and JSC-1 treated cells as measured by MTT-assay, as compared to treated PBMCs as measured by annexin V-PI flow cytometry. Error bars represent standard error of mean, n = 2.(C) Detection of apoptosis in BC-1, BCBL-1 and JSC-1 cells as early as 24 h after KPT-185 treatment as measured by annexin V-PI flow cytometry. Error bars represent standard error of mean, n = 2.(D) XPO1 inhibition promotes cell death through a caspase-dependent pathway. PEL cells were treated overnight with KPT-185 in the absence or presence of the general caspase inhibitor Q-VD-OPH. Whole cell extracts were assessed for cleavage of PARP and caspase 3 by western blot analyses.(E) BC-1, BCBL-1 and JSC-1 cells were treated with different concentrations of KPT-185 and stained with PI (BD Cycletest Plus, BD Biosciences) for cell cycle profiles using flow cytometric analysis. Upon treatment with KPT-185 a clear reduction of the G2/M and S populations is observed while the G0/G1 population increased as well as the subG1 population. Error bars represent standard error of mean, n = 2.

Fig 5: The effect of the DCL and DCL–OAO derivative conjugates (4a–4d in 10 and 20 µM concentrations) on the cell cycle distribution (A), apoptosis induction (B), and on the proliferation level (C) in HepG2 cells (after 24 h incubation). (A) Graphs represent the percentage of cells in the subG1 (green color before the G1 phase on plots), G1/G0, S, and G2/M phases measured by flow cytometry after propidium iodide staining. Topotecan was used as a positive control. (B) Graphs represent the percentage of cells in the early and late stages of the apoptosis assessed by flow cytometry measurements based on the signal from Annexin V bound to phosphatidylserine externalized in apoptotic cells. A dead cell marker, 7-AAD, was also included. Topotecan was used as a reference for proapoptotic activity. The protein levels of Bax and Caspase-3 is also presented. (C) Graphs representing the percentage of proliferating (Ki67(+)) and nonproliferating (Ki67(-)) cells measured by flow cytometry. Starved cells (cultured in an FBS-free medium) were used as a reference for the anti-proliferative conditions. Representative plots and immunoblots are presented. ß-actin was used as a loading control for protein analysis. For all panels, the mean values ± SEM from three independent experiments is shown. Asterisks (*) denote statistically significant changes from control—DMSO treated cells, and panel B denotes statistically significant changes in the percentage of either early or late apoptotic cells, while hashes (#) denote statistically significant changes in the percentage of total apoptotic cells, p < 0.05. Statistical significance was assessed by Student’s t-test.