Extended Data Fig. 7: p53^R175H regulates BACH1 targets through recruiting LSD2.

a, ChIP analysis of the recruitment of p53^R175H to SLC7A11 promoter (-128 to -114) in A549 p53^-/- cells transfected with p53^R175H alone or p53^R175H + BACH1 plasmids. n = 3 technical replicates. The experiment was repeated twice with similar results. b, Coomassie blue staining of SDS–PAGE gel loaded with SFB-p53^R175H protein complex purified from H1299 cells stably expressing SFB-p53^R175H by double IP (SBP IP + S-protein IP). The experiment was repeated three times with similar results. c, LSD2/KDM1B peptides sequences identified from Mass-Spec of SFB-p53^R175H complex. d, Co-IP of SFB-tagged p53^R175H with Flag-HA (FH)-tagged BACH1 or its deletion mutant ∆466-515 in H1299 cells. The experiment was repeated three times with similar results. e, ChIP analysis with LSD2 antibody in Cal-33 p53^R175H-/-BACH1^-/- cells. Cal-33 p53^R175H-/-BACH1^-/- cells were transfected with LSD2, LSD2 + BACH1, LSD2 + BACH1∆466-515, LSD2 + BACH1 + p53^R175H, or LSD2 + BACH1∆466-515 + p53^R175H plasmids and then cells lysates were ChIPed with LSD2 antibody, followed by qPCR analysis of the recruitment of LSD2 to SLC7A11 promoter. n = 3 technical replicates. The experiment was repeated twice with similar results. f, Western blot analysis of SLC7A11 expression in TOV-112D cells transfected with NC, p53, or LSD2 siRNAs for 48 h. The experiment was repeated twice with similar results. g, ChIP analysis of the recruitment of LSD2 to CEMIP promoter (-2036 to -2022) in Cal-33 native cells treated with NC, BACH1, or p53 siRNA for 48 h. n = 3 technical replicates. The experiment was repeated twice with similar results. h, ChIP analysis with LSD2 antibody in Cal-33 p53^R175H-/-BACH1^-/- cells. Cal-33 p53^R175H-/-BACH1^-/- cells were transfected with LSD2, LSD2 + BACH1, LSD2 + BACH1∆466-515, LSD2 + BACH1 + p53^R175H, or LSD2 + BACH1∆466-515 + p53^R175H plasmids and then cells lysates were ChIPed with LSD2 antibody, followed by qPCR analysis of the recruitment of LSD2 to CEMIP promoter. n = 3 technical replicates. The experiment was repeated twice with similar results. Data represent mean of three technical replicates.

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