Figure 7.
Inhibition of PRMT5 activates negative regulators of BCR/PI3K/AKT signaling. (A) PTPRO and PIK3IP1 mRNA expression measured by RT-qPCR in MCL samples (n = 10 total samples: cell lines [n = 3], PDX [n = 2], primary patient samples [n = 5], activated normal B cells [n = 5], and resting normal B cells [n=7]). Data show normalized fold expression relative to resting normal B cells (n = 7) using ACTB as internal control. (B) ChIP assay was performed with antibody H3R8me2s crosslinked magnetic beads. Retained DNA was amplified by qPCR with PTPRO and PIK3IP1 TaqMan primers. Fold enrichment was calculated relative to the input sample (PI). (C) Log2FC mRNA expression measured by RT-qPCR in PDX-DA and PDX-AA cells treated in vivo. Data are shown relative to VC treatment with ACTB as internal control. Data are in biological triplicates (n = 3 per group) apart from VC in PDX-DA is a technical duplicate. (D) Log2FC mRNA expression measured by RT-qPCR in primary patients with MCL (7949 and 1972), treated for 3 days with 500 nM PRT-382 in vitro. Data are normalized to GAPDH and shown relative to VC. Data are biological triplicates or duplicates. (E) PTPROt protein expression evaluated by immunohistochemistry (IHC) staining in spleens obtained from PDX-AA animals treated in vivo with PRT-382 or VC. Quantification of IHC PTPROt-stained slides showing the proportion of pixels positive for staining. Each dot represents an individual mouse. (F) Jeko-1 cells were treated with DMSO control or 500 nM PRT-382 for 5 days then evaluated for protein expression of PTPROt by confocal microscopy. The total number of cells counted and the percentage of cells positive for fluorescent immunolabeling of PTPROt is shown. (G) The indicated MCL cell lines were treated with day 9 IC50 values of PRT-382, and protein lysates were collected at day 6 and immunoblotted for PIK3IP1 with β-tubulin as a loading control. Protein expression densitometry values shown normalized to loading control and relative to 0 nM DMSO control–treated cells. (H) Jeko-1 cells were treated with 500 nM PRT-382 and Granta-519 cells were treated with 50 nM PRT-382 out to 8 days. Protein lysates were immunoblotted for PHLDA3, p-AKT(s473), and AKT with α-tubulin and GAPDH as loading controls.

Inhibition of PRMT5 activates negative regulators of BCR/PI3K/AKT signaling. (A) PTPRO and PIK3IP1 mRNA expression measured by RT-qPCR in MCL samples (n = 10 total samples: cell lines [n = 3], PDX [n = 2], primary patient samples [n = 5], activated normal B cells [n = 5], and resting normal B cells [n=7]). Data show normalized fold expression relative to resting normal B cells (n = 7) using ACTB as internal control. (B) ChIP assay was performed with antibody H3R8me2s crosslinked magnetic beads. Retained DNA was amplified by qPCR with PTPRO and PIK3IP1 TaqMan primers. Fold enrichment was calculated relative to the input sample (PI). (C) Log2FC mRNA expression measured by RT-qPCR in PDX-DA and PDX-AA cells treated in vivo. Data are shown relative to VC treatment with ACTB as internal control. Data are in biological triplicates (n = 3 per group) apart from VC in PDX-DA is a technical duplicate. (D) Log2FC mRNA expression measured by RT-qPCR in primary patients with MCL (7949 and 1972), treated for 3 days with 500 nM PRT-382 in vitro. Data are normalized to GAPDH and shown relative to VC. Data are biological triplicates or duplicates. (E) PTPROt protein expression evaluated by immunohistochemistry (IHC) staining in spleens obtained from PDX-AA animals treated in vivo with PRT-382 or VC. Quantification of IHC PTPROt-stained slides showing the proportion of pixels positive for staining. Each dot represents an individual mouse. (F) Jeko-1 cells were treated with DMSO control or 500 nM PRT-382 for 5 days then evaluated for protein expression of PTPROt by confocal microscopy. The total number of cells counted and the percentage of cells positive for fluorescent immunolabeling of PTPROt is shown. (G) The indicated MCL cell lines were treated with day 9 IC50 values of PRT-382, and protein lysates were collected at day 6 and immunoblotted for PIK3IP1 with β-tubulin as a loading control. Protein expression densitometry values shown normalized to loading control and relative to 0 nM DMSO control–treated cells. (H) Jeko-1 cells were treated with 500 nM PRT-382 and Granta-519 cells were treated with 50 nM PRT-382 out to 8 days. Protein lysates were immunoblotted for PHLDA3, p-AKT(s473), and AKT with α-tubulin and GAPDH as loading controls.

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