Extended Data Fig. 5: PRMT5 is the arginine methyltransferase of GPX4.
From: PRMT5-mediated arginine methylation stabilizes GPX4 to suppress ferroptosis in cancer
a, c IB analysis of the HA immunoprecipitant and WCL derived from HEK-293T cells that ectopically express HA-GPX4 (mitochondrial) and indicated GFP-PRMTs constructs. b, Upper: IB analysis of WCL and the immunocomplexes from anti-HA IPs and derived from HEK-293T cells that ectopically express HA-GPX4 (mitochondrial) and wild-type or catalytically dead (E444Q) GFP-PRMT5. Lower: Sequence alignment of partial human mitochondrial and cytoplasmic GPX4. d, C4-2 cells stably expressing shPRMT5 was transinfected with Flag-empty vector (EV), wild-type or E444Q mutant Flag-PRMT5. WCLs were analyzed by immunoblotting with the indicated antibodies. e, qRT–PCR analysis of the mRNA expression of the indicated genes of 786-O and C4-2 cells stably expressing shScr or shPRMT5. f, 786-O, C4-2, and HeLa cells stably expressing shScramble or shPRMT5 were treated with MG132 (20 μM) or CQ (50 μM) for 12 h, WCLs were analyzed by immunoblotting with the indicated antibodies. g-h, The scores of GPX4 and R152-methylated GPX4 immunohistochemical (IHC) staining in 60 prostate (g) and 140 bladder cancer (h) sections and paired adjacent non-cancer tissues. Characteristics of boxplot in g (minimum, maximum and median): GPX4 in adjacent tissue (0, 6, 1), GPX4 in cancer tissue (1, 9, 4). GPX4 R152-me2s in adjacent tissue (0, 4, 2), GPX4 R152-me2s in cancer tissue: minimum (0, 8, 3). Characteristics of boxplot in h (minimum, maximum and median): GPX4 in adjacent tissue (0, 9, 4), GPX4 in cancer tissue (0, 12, 6). GPX4 R152-me2s in adjacent tissue (1, 8, 4), GPX4 R152-me2s in cancer tissue: minimum (0, 12, 6). The horizontal lines mark the median, and the box limits indicate the 25th and 75th percentiles. i, k, m, Representative images of PRMT5, GPX4, and R152-methylated GPX4 staining were shown in lung cancer sections (i), renal cell cancer sections (k), and bladder cancer sections (m).Scale bar, 100 μm. j, l, n, The correlations between PRMT5 and GPX4 levels, PRMT5 and R152-methylated GPX4 levels in 150 consecutive lung tumor sections (j), 90 consecutive kidney tumor sections (l) and 140 consecutive bladder tumor sections (n) that were semiquantified as high or low and analyzed by Spearman rank correlation test. For e, data are mean ± s.d., For e, g, h, data, P values were calculated using a two-tailed Student’s t-test. All of the experiments, from a to f, were repeated at least twice independently, yielding identical outcomes.
