Extended Data Fig. 2: p53R175H suppresses BACH1-mediated transcriptional regulation of SLC7A11 and ferroptosis.
a, SLC7A11 promoter sequences containing the BACH1 consensus binding motif (WT or mutated; -118 to -127bp). Wild-type or mutant SLC7A11 promoters were cloned into the pGL3 luciferase reporter vector. b, Dual-luciferase reporter assay for SLC7A11 transcription in H1299 cells co-transfected with control, p53-R175H, BACH1, p53-R175H + BACH1, or WT p53 plasmid, along with SLC7A11 luciferase reporter plasmid (containing mutant motif) and Renilla plasmid. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. c, Western blot analysis of SLC7A11 expression in HCC1937 cells (p53R306-stop) treated with NC siRNA, p53 siRNA, BACH1 siRNA, or p53 siRNA+BACH1 siRNA for 48 h. The experiment was repeated twice with similar results. d-f, qPCR analysis of SLC7A11 mRNA expression in HuCCT1 cells (d), KLE cells (e) and Cal-33 cells (f) treated with NC siRNA, p53 siRNA, BACH1 siRNA, or p53 siRNA+BACH1 siRNA for 48-72 h. HuCCT1 cells, 48 h; KLE cells, 56 h; Cal-33 cells, 72 h. n = 3 technical replicates. The experiments were repeated twice with similar results. g-j, qPCR analysis of BACH1 targets in Cal-33 cells treated with NC siRNA, p53 siRNA, BACH1 siRNA, or p53 siRNA+BACH1 siRNA for 72 h. HMOX1 (g); GCLM (h); FTH1 (i); FTL (j). n = 3 technical replicates. The experiment was repeated twice with similar results. k, Cell death assay of Cal-33 NC, p53, BACH1, or p53 + BACH1 knockdown cells treated with 1 mM TBH for 8 h in presence or absence of 5 μM ferr-1. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. l, Western blot analysis of SLC7A11, p53 and BACH1 expression in mouse sarcoma cells. p53R172H/R172H sarcoma cell were from spontaneously-developed sarcoma tumors derived from p53R172H/R172H mice and p53R172H-/- and p53R172H/R172HBACH1-/- cells were made from these p53R172H/R172H cells by CRSIPR method. The experiment was repeated twice with similar results. m, Cell death assay for p53R172H/R172H, p53R172H-/- and p53R172H/R172HBACH1-/- sarcoma cells treated with 0.5 μM erastin for 18 h in the presence or absence of 5 μM ferr-1. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. Data represent mean of three technical replicates.
