Fig 1: Dose-dependent regulation of matriptase, HAI-1 by cisplatin in endometrial cancer cellsAfter treatment with different concentration of cisplatin for 24 h, down regulation of the matriptase and HAI-1 mRNA were observed in endometrial cancer cells with increasing dose of cisplatin in HEC-1A (A), HEC-1B (B) and RL-952 (C). (D) The western-blot analysis for the protein expression of matriptase and HAI-1 show similarly trend with mRNA expression after cisplatin treatment. (E) The ratio of matriptase/HAI-1 was initially raised and then decreased at the increased cisplatin concentration in HEC-1A and RL-952 cells. (F) After treated RL-952 cell with 2 mg/L DDP cisplatin from 0h to 24 h, there was obvious time-dependent manner between cisplatin and matriptase mRNA. (G) The ratio of matriptase/HAI-1 at specific time point after treated with cisplatin. *P < 0.05, #P > 0.05.
Fig 2: Down regulation of matriptase mediated by Lentivirus siRNA infection(A) Infection with lentivirus mediated siRNA targeted on matriptase, HEC-1A and RL-952 cells were observed by optical microscope and fluorescence microscopic with a magnification of 400. Matriptase mRNA expression was significantly down-regulated after the infection with siRNA in HEC-1A (B) and RL-952 cell (C). (D) The protein levels of matriptase and HAI-1 show consistent situation with expression of mRNA and after the infection with lentivirus. **mean P < 0.05, ##mean P > 0.05.
Fig 3: Downregulation of matriptase results in cell cycle arrest and increased apoptosis in HO-8910PM cells. (A) Flow cytometry analysis demonstrated that the percentage of cells in the G1/G0 phase was significantly higher for matriptase-depleted HO-8910PM-SI2 cells than for HO-8910PM-NC and HO-8910PM cells (42.73±0.39%, P<0.01). Tthe percentage of HO-8910PM-SI2 cells in the G2/M phase was significantly lower (4.16±0.74%) than that of HO-8910PM-NC (17.65±0.63%, P<0.01) and HO-8910PM cells (18.35±0.65%, P<0.01). Conversely, no differences in S phase content were noted among the 3 cell lines. (B) Matriptase depletion significantly decreased the percentage of surviving cells and increased the percentage of early apoptotic cells in HO-8910PM-SI2 cells compared with the negative control and wild type cells. The percentages of late apoptotic and necrotic cells were not significantly different. (C) Relative mRNA and protein expression levels of matriptase and HAI-1 were detected in HO-8910PM, HO-8910PM-SI2 and HO-8910PM-NC cells. The highest silencing efficiency was achieved in HO-8910PM-SI2 cells using matriptase-SI2. The mRNA and protein expression levels of matriptase and HAI-1 were comparable in HO-8910 and HO-8910-NC cells.#compared with HO-8910PM-NC and HO-8910PM cells, significantly fewer HO-8910PM-SI2 cells survived and the number of early apoptotic HO-8910PM-SI2 cells was significantly higher. *P>0.05 vs. HO-8910PM; #P<0.05 vs. HO-8910PM. Q1, cellular debris, Q2, late apoptotic and necrotic cells, Q3, surviving cells, Q4, early apoptotic cells. NC, negative control; SI, small interfering RNA; HAI-1, hepatocyte growth factor activator inhibitor-1.
Fig 4: Inhibition of metastatic ability of endometrial cancer cells via matriptase suppression by cisplatin(A) After treated by different concentrations of cisplatin, migration were measured by scratch assays, magnification, x200. (B) After treated by different concentrations of cisplatin, invasion ability was measured by Transwell assays, magnification, x200. (C) the migration distance of three cell lines shows similar change trend with expressions of matriptase and HAI-1. migration distance of endometrial cancer cell in low concentrations cisplatin group was widened compared with blank control group; and migration distances were significantly reduced with the increase concentration of cisplatin. (D) The number of endometrial cancer cells that migrated through the Transwell membrane was increased in low concentrations cisplatin group compared with blank control group; and transmembrane cell number was significantly reduced with the increase concentration of cisplatin. *mean P < 0.05, #mean P > 0.05.
Fig 5: Downregulation of matriptase expression by RNA interference. (A) Microscopic examination of the infection efficiencies of different lentiviral-mediated SI constructs targeting matriptase in HO-8910PM cells (magnification, ×100). There was no signal observed in HO-8910PM-PBS. The infection efficiencies exceeded 80% at a MOI of 80. (B) Relative matriptase mRNA expression levels in HO-8910PM cells infected with different lentiviral particles at high MOI. Matriptase SI2 achieved the highest inhibition of matriptase mRNA expression, and little inhibition was observed in HO-8910PM-NC cells. The SI inhibited matriptase expression but not HAI-1 expression. The ratio of matriptase/HAI-1 was significantly reduced in the HO-8910PM cells treated with matriptase SI2. (C) Relative matriptase protein expression levels in HO-8910PM cells transfected with different lentiviral particles at high MOI. Protein quantification was normalized using ß-actin. The highest silencing efficiency was achieved in HO-8910PM-SI2 cells using matriptase SI2. *P>0.05 vs. HO-8910PM; #P<0.05 vs. HO-8910PM. MOI, multiplicity of infection; HAI-1, hepatocyte growth factor activator inhibitor-1; NC, negative control; SI, small interfering RNA.
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