Fig 1: CDK1/CDC14B-dependent phosphorylation of USP9X at serine 2563 promotes mitotic survival via WT1 and IL-8.a Mitotic apoptosis in response to CXCL8 versus control knockdown detected by immunoblot in U2OS cells that were treated with the respective siRNA and arrested in mitosis using nocodazole for 8 h. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies. b Quantification of relative amount of cleaved caspase 3 in n = 3 biologically independent experiments conducted as described in a. Ratio paired t-test was applied with **p = 0.0055. c Immunoblot analysis showing increased mitotic apoptosis in U2OS cells in response to treatment with nocodazole (8 h) and the CXCR1/2 inhibitor reparixin (RPX) or DMSO for 48 h. d Quantification of relative amount of cleaved caspase 3 in n = 4 biologically independent experiments conducted as described in c. Ratio paired t-test was applied with **p = 0.0031. e Immunoblot analysis confirming reversal of mitotic apoptosis following exogenous reconstitution of IL-8 in CXCL8-depleted cells. Experiment was performed as in a, with addition of exogenous IL-8 for the last 48 h. Cells were treated with nocodazole for 8 h. f Immunoblot analysis detecting induction of mitotic apoptosis in response to WT1, CXCL8, or WT1 and CXCL8 knockdown compared to control knockdown in U2OS cells that were arrested in mitosis using nocodazole (8 h). g Immunoblot analysis showing increased mitotic apoptosis by WT1 knockdown only in USP9XWT but not in USP9XMut U2OS cells. Analyzed cells were treated with control or WT1 siRNA and then arrested in mitosis using nocodazole. h Induction of mitotic apoptosis in USP9XMut U2OS cells that were kept asynchronous or treated with nocodazole for 32 h, stained with PI and measured by flow cytometry. PI-positive cells were quantified in each sample using FlowJo software. Mean is shown from n = 3 biologically independent experiments. Paired t-test was applied with **p(Mitosis) = 0.00278, p(AS) = 0.0745. i Immunoblot analysis revealing decreased mitotic apoptosis after CDC14B knockdown in USP9XWT but not in USP9XMut U2OS cells. Before lysis cells were transfected with control or CDC14B-directed siRNA, arrested in mitosis, and collected for analysis by western blot. Throughout this figure, mean and standard deviations as error bars are displayed.
Fig 2: WT1 regulates IL-8 transcription and secretion in mitosis.a Heatmap of RNA-Seq analysis depicting differentially regulated genes in mitotic control cells versus WT1 knockdown U2OS cells. Each column displays an independent biological replicate, negatively regulated genes on the top, positively regulated genes on the bottom. b RNA-Seq analysis showing differentially regulated genes in mitotic USP9XWT versus USP9XMut U2OS cells. c Quantitative RT-PCR showing accumulation of IL-8 mRNA in mitotically synchronized versus asynchronous U2OS cells. Mean is shown from n = 4 biologically independent experiments. One sample t-test was applied with *p = 0.0198. d Extracellular IL-8 measured by ELISA is increased in the supernatant of mitotic versus asynchronous U2OS cells. Mean is shown from n = 3 biologically independent experiments. Ratio paired t-test was applied with ***p = 0.0002. e Quantitative RT-PCR from mitotic U2OS cells showing reduced IL-8 mRNA levels in response to USP9X, WT1, or double compared to control knockdown. Mean is shown from n = 3 biologically independent experiments. One-way ANOVA was applied with ****p<0.0001 followed by Dunnett’s test with ****p < 0.0001 (p(USP9X versus WT1) = 0.211; p(USP9X versus USP9X+WT1) > 0.9999; p(WT1 versus USP9X+WT1) = 0.2061). f Secreted IL-8 measured by ELISA in the supernatant of mitotic U2OS cells following USP9X, WT1, or double versus control knockdown. Mean is shown from n = 3 biologically independent experiments. One-way ANOVA was applied with ***p = 0.0001 followed by Dunnett’s test with ***p(Ctrl versus USP9X) = 0.0004, **p(Ctrl versus WT1) = 0.0055, ***p(Ctrl versus USP9X+WT1) = 0.0002; p(WT1 versus USP9X) = 0.3081, p(WT1 versus USP9X+WT1) = 0.0791, p(USP9X versus USP9X+WT1) = 0.6772. g Quantitative RT-PCR showing reduced IL-8 mRNA in USP9XMut versus USP9XWT U2OS cells that were mitotically synchronized using nocodazole. Mean is shown from n = 3 biologically independent experiments. One sample t-test was applied with **p = 0.002. h ELISA showing reduction of secreted IL-8 in the supernatant of mitotically synchronized USP9XMut compared to USP9XWT U2OS cells. Mean is shown from n = 3 biologically independent experiments. Ratio paired t-test was applied with *p = 0.0277. i Chromatin immunoprecipitation (ChIP) showing WT1 occupancy on the CXCL8 promoter in nocodazole-synchronized U2OS cells. ChIP signal was quantified using RT-PCR. WT1 occupancy was normalized to total input DNA. Mean from n = 3 biologically independent experiments is shown. Ratio paired t-test was applied with **p = 0.00277. j CXCL8 reporter assay performed in nocodazole-treated U2OS cells that were transfected with luciferase reporter construct harboring the human CXCL8 promoter or an empty reporter construct and a WT1 overexpressing vector as indicated. Luminescence was normalized to the background luminescence of the empty reporter construct. Mean is from n = 4 biologically independent experiments. Ratio paired t-test was applied with ***p = 0.0007. Throughout this figure standard deviations are displayed as error bars.
Fig 3: Metformin alleviates aortic calcification by activating the PI3K/AKT signaling pathway in a concentration-dependent manner in vitro. a Metformin inhibits inflammatory factor secretion including IL6, IL8, and MCP-1 in cell supernatant, after treatment with phosphate medium (PM) with or without metformin for 72 h as determined by ELISA; b ROS production was detected and quantified after PM treatment with or without metformin for 72 h; c Cell viability was detected by CCK8 after treatment with PM with or without metformin for 72 h; d Immunofluorescence staining images of p-AKT and OPN expression in AVICs after PM treatment with or without 100 µM metformin for 72 h, Scale bar, 50 µm; e The protein expression of p-AMPK (Thr172), p-AKT (Ser473), PI3K, p-eNOS (Ser1177), BMP2, and OPN in AVICs after PM treatment with or without metformin for 72 h as determined by WB; f Calcium deposition were detected by ARS Staining after various treatments for 7 days, Scale bar, 200 µm. n = 6 per group. CTL, control; Met, metformin; *p < 0.05 versus PM group (one-way ANOVA with Bonferroni post hoc test)
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