Fig 1: CDCA5 knockdown induces cell cycle arrest. (A and B) Cell cycle distribution was determined by flow cytometric analysis in (A) C4-2 and (B) PC-3 cells after CDCA5 knockdown. The percentages of cells in the G0/G1, S and G2/M phases were calculated (*P<0.05). (C and D) Western blotting was used to detect the expression levels of cyclin B1 and CDK1 after CDCA5 knockdown in C4-2 and PC-3 cells. GAPDH was used as an internal control. CDCA5, cell division cycle-associated 5; sh, short hairpin; CON, control.
Fig 2: pPeOp affects the expression of cell cycle- and migration-associated genes/proteins in MC-4 cells. Cells were treated with pPeOp (30, 60 or 90 µg/ml), 90 µg/ml PVP (negative control) or 100 µg/ml 5-FU (positive control) for 24 h. In addition, untreated cells were used as the control group. Reverse transcription-quantitative polymerase chain reaction analysis was used to assess cell cycle-associated gene expression levels. All data were normalized to the untreated control. (A) Effects of pPeOp on the mRNA expression levels of the cell cycle-associated genes CDK1, CDK2, CDK4, cyclin A, cyclin B and cyclin D1. (B) Effects of pPeOp on the mRNA expression levels of the migration-associated genes MMP-2 and MMP-9. (C) Western blotting was conducted to examine the effects of pPeOp on the expression of cell cycle- and migration-associated proteins, with β-actin used as a loading control. (D) Quantification of protein expression levels of cell cycle- and migration-associated proteins normalized to β-actin. *P<0.05, **P<0.01. pPeOp, purified Omphalia lapidescens protein; PVP, polyvinylpyrrolidone; 5-FU, 5-fluorouracil; CDK, cyclin-dependent kinase; MMP, matrix metalloproteinase.
Fig 3: Upregulation of CDK1 was observed after the overexpression of TONSL-AS1. The effects of overexpression of TONSL-AS1 and miR-490-3p on the expression of CDK1 mRNA (Fig. 3a) and protein (Fig. 3b) in OVCAR3 cells were analyzed by qPCR and western blot, respectively. Experiments were repeated 3 times and data were expressed as mean values, *, p < 0.05
Fig 4: Combination treatment of BC based on the reciprocal expression of CDK1 and ID2.a BC cells were treated with the indicated concentrations of apigenin and RO-3306, and cell growth was assessed (n = 3). NT indicates the nontreated control group. b, c BC cells were treated with suboptimal concentrations of apigenin (15 µM) and RO-3306 (3 µM) for up to 48 h. Cell death was assessed by (b) immunoblotting of cleaved caspase-3 and PARP and by (c) Annexin-V/PI staining and flow cytometry. The quantitative assessments of apoptotic cells (Annexin-V + /PI− population, %; n = 3) are shown in the panels to the right of the representative flow cytometry plots. Quantitative data are expressed as the mean ± SEM values. Statistical analyses were performed using one-way (c) or two-way (a) ANOVA with the Bonferroni post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the NT control group. d Experimental overview of the orthotopic BC xenograft model generated by transplantation of 1.0 × 106 human HT1376 BC cells through the outer layer of the bladder of nonobese diabetic (NOD) Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunodeficient mice. Treatment with RO-3306 alone or in combination with apigenin was performed by intraperitoneal injection according to the indicated schedules. e Representative images (left panel) and weights (right panel) of bladders bearing tumors after treatment with RO-3306 (4 mg/kg) alone or combined with apigenin (50 mg/kg) in duplicate experiments (five mice in each replicate). The data are shown as dot plots of the mean ± SEM values from ten independent animals in each group. ***p < 0.001 compared with the vehicle control group; ###p < 0.001, one-way ANOVA with the Bonferroni post hoc test. f Representative images of hematoxylin and eosin staining of bladder tissues from the indicated xenograft groups at 100× (upper panel) or 200× (lower panel) magnification. Scale bars = 100 µm. g Immunofluorescence assay for detecting ID2, CDK1, and TFCP2L1 (green) proteins in xenograft tumors. Representative merged images are shown at 200× magnification. Scale bars = 100 µm. Nuclei were stained with DAPI (blue).
Fig 5: Restoration of DLGAP5 reverses the effects of miR-409-5p on related downstream protein levels. SKOV-3 cells were divided into three groups, miR-NC + pcDNA3.1; miR-409-5p mimics + pcDNA3.1 and miR-409-5p mimics + pcDNA3.1-DLGAP5. Western blot analysis was performed to detect the protein expression levels of CDK1, Cyclin B1, Bad and Bcl-2 in the three groups. Data are presented as the mean ± standard deviation (n=3). **P<0.01, ***P<0.001 vs. the miR-NC + pcDNA3.1 group; ##P<0.01, ###P<0.001 vs. the miR-409-5p mimics + pcDNA3.1 group. DLGAP5, discs large-associated protein 5; miR, microRNA; NC, negative control.
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