Fig. 7: PRDM9 maintains cholesterol homeostasis under chemotherapy stress.
From: Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma
a Simplified scheme of cholesterol biosynthesis. b Representative immunoblots of HMGCS1, MVD and DHCR24 in RKI1 cells treated with CMPD1 (10 µM, 3 days) ± MRK-740 (3 µM; left) or transduced with PRDM9 sgRNA (PRDM9 KO, right). (n = 2 biological replicates for MVD; n = 3 biological replicates for HMGCS1 and DHCR24; quantification provided in the Supplementary Fig. 6a). c Representative immunoblots of HMGCS1, MVD and DHCR24 in FPW1 cells treated with CMPD1 (10 µM, 6 days) ± MRK-740 (5 µM). Quantification of n = 4 biological replicates is provided in the Supplementary Fig. 6b. d Representative immunoblots of HMGCS1, MVD and DHCR24 in MMK1 cells treated with CMPD1 (10 µM, 3 days) ± MRK-740 (5 µM). Quantification of n = 4 biological replicates is provided in the Supplementary Fig. 6c. e Cholesterol in RKI1 cells treated with CMPD1 (10 µM, 3 days) ± MRK-740 (3 µM)/MRK-740-NC (3 µM). Data are mean ± SD (n = 4 biological replicates). One sample t-test between treatment vs Parent: ** indicates p (two tailed) = 0.0067; *** indicates p (two tailed) = 0.0005, p (two tailed) = 0.0009 for CMPD1 and CMPD1 + MRK-740, respectively. f Cholesterol in CMPD1 (10 µM, 3 days) treated RKI1 cells transduced with NTC sgRNA (NTC) or PRDM9 sgRNA (PRDM9 KO). Data are mean ± SD (n = 3 biological replicates for PRDM9 (1) KO; n = 4 biological replicates for all other conditions). One samplet-test between treatment vs NTC: ** indicates p (two tailed) = 0.0086, *** indicates p (two tailed) = 0.0002, **** indicates p (two tailed) <0.0001. Unpaired t-test between NTC + CMPD1 vs PRDM9 (1) KO + CMPD1: * indicates p (two tailed) = 0.033. g Cholesterol in FPW1 cells treated with CMPD1 (10 µM, 6 days) ± MRK-740 (5 µM). Data are mean ± SD (n = 4 biological replicates). One sample t-test between treatment vs Parent: ** indicates p(two tailed) = 0.0018; **** indicates p (two tailed) <0.0001. h DAPI-stained images and quantification of DTPs in RKI1 cells treated with CMPD1 (25 µM, 14 days) ± MRK-740 (3 µM) ± cholesterol (5 μg/mL). Bottom two images are of RKI1 cells transduced with PRDM9 sgRNA (PRDM9 KO). Data are mean ± SD (n = 3 biological replicates). One sample t-test between treatment vs CMPD1: * indicates p (two tailed) = 0.0473, ** indicates p (two tailed) = 0.0071. Unpaired t-test between PRDM9 KO vs PRDM9 KO+Chol: * indicates p (two tailed) = 0.0215. Scale bar = 100 µm. i DAPI stained images and quantification of DTPs in FPW1 and MMK1 cells treated with CMPD1 (25 µM, 14 days) ± MRK-740 (5 µM) ± cholesterol (5 μg/mL). Data are mean ± SD (n = 3 biological replicates for FPW1 cells, n = 4 biological replicates for MMK1 cells). One sample t-test between treatment vs CMPD1: ** indicatep (two tailed) = 0.0057, p (two tailed) = 0.0014 for FPW1 and MMK1 cells, respectively. Unpaired t-test between CMPD1 + MRK-740 vs CMPD1 + MRK-740+Chol: * indicates p (two tailed) = 0.0244; **** indicates p (two tailed) <0.0001. Scale bar = 100 µm. j Quantification of reactive oxygen species (ROS), lipid peroxidation and total lipids in RKI1 cells treated with CMPD1 (10 μM, 3 days) ± MRK-740 (3 μM). Data are mean ± SD (n = 3 biological replicates). One sample t-test between treatment vs Parent: * indicates p (two tailed) = 0.0159, *** indicates p (two tailed) = 0.0002, **** indicates p < 0.0001. Unpaired t-test between CMPD1 vs CMPD1 + MRK-740 for lipid peroxidation: *** indicates p (two tailed) = 0.0005. k Working model of PRDM9-dependent cholesterol biosynthesis and persister survival. Source data are provided as a Source Data file.
