Fig 1: Effects of MASTL overexpression on the proliferation of liver cancer cell lines HepG2 and SUN387. (A) Overexpression of MASTL in HepG2 and SUN387 cells was confirmed by western blot analysis. Transfection of an empty vector was used as a control, and β-actin was used as a loading control. (B) An MTT assay was performed following overexpression of MASTL at the indicated time points. (C) Staining of WT or kinase-dead mutant MASTL expression with anti-flag in HepG2 cells by immunofluorescence (magnification, ×100). (D) Cell cycle progression of HepG2 cells in overexpressing WT or mutant MASTL was examined using fluorescence-activated cell sorting. (E) The percentages of cells in the respective cell cycle phases. Experiments were repeated three times. Results are displayed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. respective controls. MASTL, microtubule associated serine/threonine kinase-like; WT, wild-type.
Fig 2: Effects on morphology and cell cycle progression of silencing of MASTL in liver cancer cell lines. (A) Knockdown of MASTL in HepG2 and SUN387 cells was confirmed by western blot analysis. (B) Fluorescence-activated cell sorting analysis and (C) quantification confirmed that the silencing of MASTL in HepG2 cells facilitated G2/M phase arrest (37.9%) compared with the control (23.1%). (D) DAPI staining (magnification, ×100) and (E) quantification revealed increased multinuclear cells in HepG2 with silenced MASTL compared with the control. Cells transfected with scrambled siRNA served as a control. (F) An MTT assay was used to determine the relative cellular proliferation of SUN387 and HepG2 cells transfected with siRNA-MASTL, measured at 490 nm. Experiments were repeated three times. Results are displayed as the mean ± standard error of the mean. *P<0.05, **P<0.01, ***P<0.001 vs. respective control. MASTL, microtubule-associated serine/threonine kinase-like; siRNA, small interfering RNA; CTL, control; PI, propidium iodide.
Fig 3: Expression of MASTL in human liver samples and the mechanisms involved in the IL-6 and TNF-α-induced expression of MASTL in liver cancer cell lines. (A) IL-6 and TNF-α quantification in healthy and liver cancer patient sera by ELISA. (B) Detection of MASTL in liver cancer and non-tumor liver tissues by immunofluorescence or immunohistochemistry (magnification, ×50). (C) ChIP assay of H3K4Me3 modification at the MASTL promoter in HepG2 cells treated with IL-6 (right) and TNF-α (left) at indicated time points. (D) MASTL mRNA expression in HepG2 cells pretreated with or without PDTC (30 µmol/l) in response to IL-6 and TNF-α stimulation. (E) ChIP assay of H3K4Me3 modification pretreated with or without PDTC at indicated time points. Experiments were repeated three times. Results are displayed as the mean ± standard error of the mean. **P<0.01 vs. respective controls. MASTL, microtubule associated serine/threonine kinase-like; TNF-α, tumor necrosis factor α; IL-6, interleukin 6; PDTC, pyrrolidine dithiocarbamate; ChIP, chromatin immunoprecipitation; IgG, immunoglobulin G.
Fig 4: Induction of MASTL expression in HepG2 and SUN387 cells. Cells were stimulated with (A) TNFα and (B) IL-6. Examination of relative MASTL mRNA expression in HepG2 cells treated with TNF-α or IL-6 (100 pg/ml) at the indicated time points by reverse transcription-quantitative polymerase chain reaction. The expression of MASTL in HepG2 and SUN387 cells were measured (C and D), and β-actin was used as an internal control. Data are representative of three independent experiments. Results are displayed as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. respective control. MASTL, microtubule associated serine/threonine kinase-like; TNF-α, tumor necrosis factor α; IL-6, interleukin 6.
Fig 5: MASTL depletion sensitized HCC cells to paclitaxel. (A) Dose–response curve of HepG2 and MHCC‐LM3 cells treated with increasing concentrations of paclitaxel (0, 5, 10, 20, 40 nM) for 48 h. Cell viability was assessed by CCK‐8 assay. (B) Histogram showing the cell survival rates from panel A. Asterisks indicate statistical significance as determined by an unpaired Student's t‐test. (*p < 0.05, **p < 0.01, ***p < 0.001).
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