(A) Flow cytometry analysis of intracellular PRMT5 of human CML CD34⁺ cells relative to NBM CD34⁺ cells (left). Median fluorescence intensity (MFI) of intracellular PRMT5 protein level in CML (n = 3) and NBM (n = 3) CD34⁺ cells (right). (B) The protein levels of PRMT5 and its histone methylation marks H4R3SDM and H3R8SDM were examined by Western blot analysis in CML (n = 6) and NBM (n = 3) CD34⁺ cells. (C) qRT-PCR of PRMT5 mRNA level in CML (n = 10) and NBM (n = 5) CD34⁺ cells. (D) The mRNA level of PRMT5 was higher in CML CD34⁺CD38^– cells than CD34⁺CD38⁺ cells (n = 3). (E) Western blot analysis of the levels of PRMT5 and its histone methylation mark H2AR3SDM and BCR-ABL and its downstream targets STATs and CRKL in NBM CD34⁺ cells (n = 3) transduced with retroviral constructs encoding BCR-ABL (p210) or empty vector (V). (F) Fusion BCR-ABL gene activated PRMT5 expression at the protein level. (G and H) Inactivation of BCR-ABL activity decreased PRMT5 expression. Uveal melanoma Mel270 cells not harboring BCR-ABL served as a control. (I) The mRNA level of PRMT5 was decreased in CD34⁺CD38^– cells and CD34⁺CD38⁺ cells (n = 3) from CML patients after IM (5.0 μM) treatment for 12 hours. (J and K) BCR-ABL knockdown by shRNA for 72 hours diminished PRMT5 expression in K562 (J) or primary CML CD34⁺ cells (n = 3) (K). Scr, Scramble shRNA. (L–N) BCR-ABL activated transcription of the PRMT5 gene via STAT5. (L) Inactivation of BCR-ABL decreased PRMT5 mRNA levels. (M) ChIP assay of STAT5 binding to the PRMT5 gene promoter in K562 cells. (N) Protein levels of PRMT5, STAT5A/B and its target protein BCL-X_L were determined in K562 cells transduced with lentiviral shRNA against STAT5A or STAT5B for 72 hours. Blot images were derived from samples run on parallel gels. *P < 0.05, **P < 0.001, ***P < 0.0001, 2-tailed Student’s t test.