Fig. 4: CH004 arrests the cell cycle at the S phase. | Cell Death & Disease

Fig. 4: CH004 arrests the cell cycle at the S phase.

From: A pharmacological probe identifies cystathionine β-synthase as a new negative regulator for ferroptosis

Fig. 4

a CH004 reduces the cell viability of HEK293T and HepG2 cells. HEK293T or HepG2 cells were treated with indicated concentrations of CH004 for 24 h before the cell number was analyzed using the CellTiter96® Aqueous One Solution Cell Proliferation Assay (Promega, Materials and Methods). The means at each concentration of one representative experiment are shown as percentages of DMSO (control, 100%). Means ± SDs (n = 3). b Overexpression of hCBS or knock-down of hCBS counteracts the growth inhibition exerted by CH004 in HEK293T cells. Indicated HEK293T stable cell lines were treated with the CH004 or DMSO for 24 h. Then, the cell viability was quantified with the CellTiter 96® Aqueous One Solution Cell Proliferation Assay under the standard protocol (Promega). The means at each concentration are shown as percentages of their respective DMSO controls (100%). Means ± SDs (n = 3). The efficacy for CBS overexpression and knock-down in HEK293T cells was determined by Western blotting using an anti-CBS antibody (3E1, 1:2000; Abnova). c CH004 induces cell death in HEK293T and HepG2 cells. HEK293T or HepG2 cells were incubated with the indicated concentrations of CH004 or DMSO for 12 h before analysis using a FITC Annexin V Apoptosis Detection Kit (BD bioscience, 556547) on an LSR Fortessa flow cytometer. The means of percentages of dead cells (DMSO, 100%) from three independent samples are shown in d. Means ± SDs (n = 3). e, f Pharmacological inhibition or genetic knock-down of hCBS in HEK293T cells arrests the cell cycle at S phase. HEK293T cells were incubated with indicated compounds for 12 h, and the DNA content was quantified with propidium iodide to analyze the cell cycle distribution by flow cytometry (e). The cell cycle distributions for HEK293T-shNT and HEK293T-shCBS cells were accordingly analyzed and are shown in f. One representative diagram is shown for each condition. The mean percentages of various cell phases from three independent samples are shown in the respective right panels. Means ± SDs (n = 3). Statistical analyses were performed by two-way ANOVA with Bonferroni post-tests. *p < 0.05; **p < 0.01; ***p < 0.001. All the experiments were independently repeated twice and one representative result is present

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