Inhibition of PRMT5 attenuates E2F1signaling in MCL. (A) Log2 fold change (FC) of DEGs in the RNA sequencing data set of PDX-AA cells of mice treated in vivo with PRT-382 in comparison with VC. (B) FC in mRNA expression of HALLMARK E2F targets analyzed by RT-qPCR in PDX (DA and AA) cells treated in vivo and in primary patient samples (7949 and 1972) treated for 3 days in vitro. Data are normalized to GAPDH and shown relative to control. PDX data are in biological triplicate (n = 3 per group) apart from VC in PDX-DA, which is a technical duplicate (n = 1 VC mouse). Primary patient samples are in biological duplicates. (C) FC in mRNA expression of HALLMARK E2F targets analyzed by RT-qPCR in Z-138 and Granta-519 treated for 6 days in vitro with the indicated doses of PRT-382. Data are normalized to ACTB and shown relative to DMSO control. (D) E2F1 was immunoprecipitated from protein lysates of Z-138 treated with PRT-382 for 4 days and patient 1972 treated with PRT-382 for 3 days. Lysates were immunoblotted for E2F1, RB, and SDMA. β-Actin was used as a loading control for the western blot (WB) input lysates. E2F1 was used as a loading control for eluted bound antigen IP lysates. (E) Z-138 and Granta-519 were treated with the indicated doses of PRT-382 for 6 days. Protein lysates were immunoblotted for p-RB(s780), CHK1, CCNA2, CDK1, and TK1, with α-tubulin used as a loading control. Protein densitometry values were normalized to α-tubulin and shown relative to 0 nM DMSO control. (F) Immunoblot of CDK1 and CCNA2 in protein lysates from PDX-AA treated with PRT-382 or ibrutinib for 2 weeks in vivo. Protein densitometry values normalized to β-actin and shown relative to VC in lane 1. (G) CSFE (carboxyfluorescein succinimidyl ester) staining of viable cells shows decreased cell replication in PRT-382–treated cells compared with DMSO control after 9 days.