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. Author manuscript; available in PMC: 2017 Jan 20.
Published in final edited form as: Oncogene. 2016 Jun 6;36(3):373–386. doi: 10.1038/onc.2016.205

Figure 1. Aberrant PRMT5-MEP50 expression and histone methylation in lung cancer cells is a target for shRNA-mediated knockdown.

Figure 1

A. Dotplot of Wilcoxon rank-sum test p-values for PRMT5 and MEP50 expression in cancers with data collected from the TCGA. Upregulated expression (purple) and downregulated (green) are shown in dots scaled by–log(p). Corresponding cancer cell lines probed in this study are bolded.

B. Heatmap of gene expression of PRMT5 and MEP50 from TCGA lung cancer RNA-Seq data in normal and patients with lung adenocarcinoma (LUAD) or squamous cell carcinoma (LUSC), ranked by sample type (tumor or normal tissue) and then Shedden poor survival markers showing substantially increased expression of PRMT5 and MEP50 relative to normal tissue and poor survival markers.

C. Dot-plot showing distribution of TCGA individual sample data for LUAD or LUSC; mean FPKM expression RNA-Seq value shown by black bar; Comparison between tumor and normal tissue expression p < 0.001 from Mann-Whitney Rank Sum Test.

D. Whole cell lysate immunoblots for PRMTs and MEP50 in the normal and corresponding cancer cell lines. GAPDH is a loading control.

E. Whole cell lysate and chromatin extracted from IMR90 and A549 probed with indicated antibodies. GAPDH and H3 are controls for lysate and chromatin immunoblots, respectively.

F. Immunoblots for PRMT5 or MEP50 from A549 cells expressing shRNA targeted against GFP as a control (GFPkd), against PRMT5 (PRMT5kd) or against MEP50 (MEP50kd).

G. Representative immunofluorescence images for PRMT5 (red) or MEP50 (red) and counterstained with DAPI (blue) from GFPkd, PRMT5kd, and MEP50kd A549 cells demonstrating primarily cytoplasmic localization. Scale bar, 50 μm.

H. Blue-native gel immunoblots for PRMT5 or MEP50 from knockdown A549 cells as in E. Native Molecular mass markers are indicated. Recombinant human PRMT5-MEP50 complex (HsPRMT5-MEP50) or SDS/boiled HsPRMT5-MEP50 with SDS (lanes 1 and 2) are used for identification of intact complex (indicated) or free PRMT5 and MEP50, respectively.