Fig. 5: Short-term methionine starvation enhances CHAC1 expression and promotes ferroptosis onset in vitro and in vivo. | Nature Communications

Fig. 5: Short-term methionine starvation enhances CHAC1 expression and promotes ferroptosis onset in vitro and in vivo.

From: Intermittent dietary methionine deprivation facilitates tumoral ferroptosis and synergizes with checkpoint blockade

Fig. 5

a, b, B16F10 cells were pre-starved in methionine-free medium (St-Met-) for 8 h, followed by cystine deprivation or 0.5 μM IKE treatment for another 18 h. scale bar,100 μm (a), 20 h (b, left), or 24 h (b, right). Representative images show the induction of cell death that is visualized by PI staining (a). Percentages of dead cells were also quantified by flow cytometry (b). c Relative mRNA (left) or protein expression (right) of mouse CHAC1 in B16F10 cells treated with methionine deprivation for 1–8 h. d Immunoblots of CHAC1 in B16F10 cells pre-treated by St-Met− for 4 h followed by cystine deprivation for another 8 h. e Total GSH abundance in B16F10 cells pre-treated by St-Met− for 8 h followed by cystine deprivation for another 8 h. f Cell death of sgControl or sgCHAC1 expressing B16F10 cells pretreated by St-Met− for 8 h followed by cystine deprivation for another 19 h. g–o Effect of dietary methionine intermittent deprivation on IKE-induced tumoral ferroptosis. Experimental design of B16F10 tumor in C57BL/6 mice (g). Tumor-bearing mice were fed with a methionine-free (Met−) diet or Met+ diet and treated with Met re-supplementation (300 mg/kg) plus IKE (40 mg/kg) at indicated days. Methionine concentrations in serum from tail bleeding samples at indicated days were quantified (h). Tumor volumes were monitored over time (i). Subcutaneous tumors were surgically removed and presented (j), and their weights were measured at the endpoint (k). MDA content (l), PTGS2, HMXO1, and TFRC mRNA expressions (m), total GSH abundance (n), and CHAC1 mRNA expression (o) in isolated tumor tissues were quantified. n = 3 biological replicates (b, e, f ) and P values are determined using one-way ANOVA (b, e) or two-way ANOVA (f); n = 6 serum samples and P values are determined by one-way ANOVA (h). n = 7 (IKE, St-Met-) or 8 (Control, St-Met− + IKE) mice per group, and data are presented as mean ± s.e.m. and P values are determined by two-way ANOVA (i); n = 7 (IKE, St-Met–) or 8 (Control, St-Met– + IKE) tumors and P values are determined by one-way ANOVA (k–o). Source data are provided as a Source Data file.

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