Fig. 2: CH004 selectively inhibits hCBS, but not hCSE, in cells. | Cell Death & Disease

Fig. 2: CH004 selectively inhibits hCBS, but not hCSE, in cells.

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

Fig. 2

a, b CH004 significantly reduces the activity of hCBS but not of hCSE in HEK293T cells. HEK293T cells were transiently transfected with empty vector (EV, i and ii), hCBS-FL (iii and iv) or hCSE (v and vi) plasmids, followed by incubation with DMSO (i–iii and v) or 10 μM CH004 (iv and vi) for 8 h before staining with 50 μM AzMC, a H2S-specific probe (“Materials and Methods” section). For larger fluorescent and bright-field images, please see Supplementary Figure 4. The expression levels of hCBS or hCSE in HEK293T cells after transient transfections were determined by western blotting using an anti-CBS antibody (3E1, 1:2000; Abnova) or anti-CSE antibody (1:500, GeneTex). The densities of protein bands for hCBS or hCSE in the blots were quantified with the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and expressed as ratios of respective control (EV group). All immunoblots are representative of at least two independent experiments using GAPDH as the loading control. The fluorescent images of the H2S (blue) were accordingly quantified (“Materials and Methods” section) with ImageJ. The quantified levels for H2S (blue) were showed in (b). The DMSO groups of the controls (CTR), which are expressing of hCBS or hCSE, were normalized to 100% and the levels of H2S in CH004-treated or EV group are shown as relative percentages. c, d CH004 dose-dependently suppresses the activity of hCBS WT without affecting the activity of CH004-insensitive mutant. HEK293T cells stably expressing EV (i and ii), hCBS-FL wild type (293T-CBS WT; iii–v) or CBS-FL mutant (293T-CBS Q222A, vi–viii) were treated with DMSO (i–iii and vi), 5 μM CH004 (iv, vii) or 10 μM CH004 (v, viii) for 8 h before staining with the AzMC. For larger fluorescent and bright-field images, please see Supplementary Figures 5–6. The quantified levels for H2S (blue) were showed in (d). The DMSO groups of the controls (CTR), which are expressing of hCBS-FL wt or Q222A mutant, were normalized to 100% and the levels of H2S in CH004-treated or EV group are shown as relative percentages. A representative fluorescence image from three independent experiments is shown. Means ± SDs (n = 3). Statistical analyses were performed on the raw data for each group by one-way (b) or two-way (d) ANOVA with Bonferroni post-tests. ***p < 0.001. Bars, 50 μm

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