Fig 2: Role of PIM2 knockdown in apoptosis and cell death of HepG2 and Huh-7 liver cancer cell lines. (A) Cells were transfected and stained 48 h later with Annexin V-FITC and PI and underwent FACS analysis. The amount of apoptotic cells (Annexin-V-positive and PI-negative) was determined (n=3). (B) Measurement of the activity of effector-caspases caspase 3 and caspase 7 72 h post-transfection (n=3). (C) Protein expression of full length PARP and cleaved PARP in Huh-7 was examined by western blot analysis. A representative blot is shown (black lines indicate where an irrelevant lane was cut out). Bands were quantitated with ImageJ and the ratio of cleaved to full length PARP was calculated (n=2). (D) Cells were transfected and seeded into ultra-low attachment plates after 48 h for spheroid formation. Spheroids were stained with PI after 7 days (n=3). Images show representative examples. *P<0.05, significant differences compared to the siCtrl; the hash symbol (#) indicates that there were no significant differences. PIM2, proviral integration site for Moloney murine leukemia virus 2; PARP, poly (ADP-ribose) polymerase.

Fig 3: Influence of PIM2 knockdown on the cell cycle of lines HepG2 and Huh-7 liver cancer cells. (A) Cell cycle distribution with nocodazole treatment was analyzed by FACS 72 h following transfection (n=4). Representative plots are shown. (B and C) The expression of cell cycle-related genes was analyzed at the RNA level by RT-qPCR (bar graphs, n=3) and at the protein level by western blot analysis (representative blots are shown), at 48 h or 72 h following transfection, respectively in (B) the HepG2 and (C) the Huh-7 cells. The black lines in the western blots indicate where an irrelevant lane was cut out.*P<0.05, **P<0.01 and ***P<0.001, significant differences compared to the siCtrl; the hash symbol (#) indicates that there were no significant differences. PIM2, proviral integration site for Moloney murine leukemia virus 2.

Fig 4: BBR induces XIAP-mediated apoptosis through PIM-2. (A) Representative western blot analysis of PIM-2 expression in ALL cells EU-4 and EU-6 treated with different concentration of BBR. (B) PIM-overexpressed EU-4 and EU-6 cells were treated with 50 µM BBR for 24 h. The transfection efficiency was determined using western blot. (C) Cell apoptosis was measured by TUNEL assay. (D) PIM-2 overexpressed EU-4 and EU-6 cells were treated with 50 µM BBR in the presence or absence of XIAP antagonist ASTX660 for 24 h followed by treatment with 50 µM BBR for 24 h. Cell apoptosis was detected by TUNEL assay. Results are expressed as mean ± SD.**p<0.01, compared to the vector or control group.

Fig 5: Expression and correlations of XIAP, PIM-2 and miR-24-3p in ALL samples. The relative expressions of (A) miR-24-3p, (B) PIM-2 and (C) XIAP in the bone marrow of ALL patients (n=39) or normal control (n=3) were detected by qRT-PCR. (D, E) Representative western blot and quantification analysis of PIM-2 and XIAP in ALL and normal samples. (F–H) Correlation analysis was performed between miR-24-3p, PIM-2 and XIAP in ALL patients. (I) The signaling pathway of BBR induced ALL cells apoptosis. Results are expressed as mean ± SD, **p<0.01, ***p<0.001, compared to Normal group.