Fig 2: Induction of cell cycle arrest and apoptosis by the acetonitrile extract of Salvia miltiorrhiza Radix is dependent on the generation of intracellular ROS. (A) PC-3 cells were exposed to the acetonitrile extract of S. miltiorrhiza Radix for 24 h and stained with 2′,7′-dichlorofluorescin diacetate. The generation of intracellular ROS was monitored by fluorescence-activated cell sorting analysis. (B) PC-3 cells were exposed to the acetonitrile extract of S. miltiorrhiza Radix alone or in combination with NAC. Western blot analysis was performed using polyclonal antibodies against G1/S cell cycle regulator proteins (p21, CDK4 and cyclin D). β-Actin was used as a loading control. (C) PC-3 cells were exposed to the acetonitrile extract of S. miltiorrhiza Radix alone or in combination with NAC and an MTT assay was performed. *P<0.05, **P<0.01, ***P<0.001 vs. control; ##P<0.01, ###P<0.001 vs. treatment with NAC. (D) PC-3 cells were exposed to the acetonitrile extract of S. miltiorrhiza Radix alone or in combination with NAC, and western blot analysis was performed using polyclonal antibodies against apoptosis-related proteins (cleaved caspase-3 and cleaved PARP). β-Actin was used as a loading control. (E) PC-3 cells, grown on a coverslip were exposed to the acetonitrile extract of S. miltiorrhiza Radix alone or in combination with NAC, and hybridized using primary antibody against 8-OH-G. Representative confocal microscopy images of the immunocytochemistry are presented. ROS, reactive oxygen species; NAC, N-acetylcysteine; CDK4, cyclin-dependent kinase; PARP, poly (ADP-ribose) polymerase; 8-OH-G, 8′-hydroxyguanosine.

Fig 3: Acetonitrile extract of Salvia miltiorrhiza Radix induces cell cycle arrest and apoptosis in PC-3 cells. (A) PC-3 cells were exposed to the acetonitrile extract of S. miltiorrhiza Radix and the resulting cell cycle changes were monitored using fluorescence-activated cell sorting analysis (left panels). The experiments were performed in triplicate and the percentage of G1/S cell cycle arrest was analyzed and depicted (right panel). ***P<0.001 vs. control. (B) PC-3 cells were exposed to the acetonitrile extract of S. miltiorrhiza Radix and western blot analysis was performed using polyclonal antibodies against G1/S cell cycle regulator proteins (p21, CDK2, CDK4 and cyclin D). β-Actin was used as a loading control. (C) PC-3 cells, grown on a coverslip, were exposed to the acetonitrile extract of S. miltiorrhiza Radix and a TUNEL assay was performed. Representative confocal microscopy images are presented. (D) PC-3 cells were exposed to the acetonitrile extract of S. miltiorrhiza Radix and western blot analysis was performed using polyclonal antibodies against apoptosis-related proteins (Bcl-2, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3 and cleaved PARP). β-actin was used as a loading control. CDK, cyclin-dependent kinase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; FITC, fluorescein isothiocyanate; Bcl-2, apoptosis regulator; PARP, poly (ADP-ribose) polymerase.

Fig 4: Lin28A promoted the proliferation and invasion of OC cells by up-regulating RAN. (A) Transiently transfected siRNA knocked down the expression of RAN at the protein level (right) and the RNA level (left) in A2780 Lin28A cells. (B) The role of knockdown RAN on the expression of the stem cell marker molecules CD44, CD133, ABCG2, OCT4, Nanog and SOX2 were measured with qRT-PCR. (C) CCK-8 analysis showed that siRAN reduced the proliferation rate of A2780 Lin28A (top) and PA-1 (bottom) cells compared to control siNC. (D) Knockdown of RAN by siRNA promoted the apoptosis of A2780 Lin28A and PA-1 cells by flow cytometry analysis. (E) Knockdown of RAN by siRNA promoted the expression of cleavage caspase-7, and reduced the expression of PARP with western blot, actin as an internal reference. (F) Knockdown of RAN decreased the invasion of A2780 Lin28A (top) and PA-1 (bottom) cells. (G) The role of knockdown RAN in the expression of invasion-related molecules was examined with western blot, actin as an internal reference.

Fig 5: BSGLWE induces caspase-dependent apoptosis of OS cell in vitro. (A, B) Hoechst 33342 staining showed that the percentage of apoptotic cells in the BSGLWE group was higher than in the NC group. The red arrows mean apoptotic bodies and nuclear condensation. (C, D) After staining with an Annexin V-APC/7-AAD kit, the differently treated cells were analyzed by flow cytometry. The results indicated that the percent of total apoptotic cells significantly increased in the BSGLWE-treated group. (E) The expression of the following apoptosis-related proteins was determined by Western blot analysis: cleaved PARP, caspase-3, −7, and Bcl-2 family members Bcl-2, Bcl-xl. (F, G) The HOS and U2 osteosarcoma cell lines were incubated with BSGLWE (4mg/mL) or control for 24 hrs. The expression level of the protein cleaved-caspase 3 was significantly increased in HOS and U2 cell lines after BSGLWE treatment detected using immunofluorescence assays. Each assay was repeated 3 times. Magnification, ×200 (A), ×200 (F). Scale bar, 100 μm (A), 100 μm (F). Error bar = mean ± SD of at least triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control, #P< 0.05 versus BSGLWE treatment.