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: Effect of PIM2 knockdown on tumor growth in a mouse model of subcutaneous xenografts. (A) Subcutaneous HepG2 tumor xenografts were treated with siRNA-loaded nanoplexes via intraperitoneal injection. Growth of tumors was monitored by measurement of tumor size at the indicated time points. Representative examples of mice are shown. (B) Tumors were resected and PIM2 protein expression was determined by western blot analysis. The representative blot shows PIM2 in representative tumor samples. *P<0.05, **P<0.01 and ***P<0.001, significant differences compared to the siCtrl. PIM2, proviral integration site for Moloney murine leukemia virus 2.

Fig 4: 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.

Fig 5: MiR-24-3p is implicated in the BBR-induced apoptosis by targeting PIM-2. (A) The expression profile of miR-24-3p in ALL cells, KOPN-8, EU-4, NALM-6, EU-6, SEM, and human lymphoma cell H9. (B) Effect of BBR on miR-24-3p expression in EU-4 and EU-6 cells. EU-4 and EU-6 cells were treated by BBR at the indicated concentrations for 24 h. The relative expression of miR-24-3p was measured by qRT-PCR and normalized to U6. EU-4 and EU-6 cells were transfected with siRNA targeting miR-24-3p (simiR-24-3p) or negative control (siControl), and subsequently treated with 50 µM BBR for 24 h. (C) Cell apoptosis was measured by TUNEL assay. (D) Representative western blot analysis showing PIM-2 expression in the cells. (E) Representative images of PIM-2 immunofluorescence staining in the treated cells. The numbers above the western blots band are the quantification of the gray value normalized to control group. Results are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, compared to the indicated group.