Fig 2: PRMT6 expression is increased in the LV of DCM patients. (A Western blot analysis showing the increase in PRMT6 and H3R2Me2a expression in the heart of DCM patients (n = 5) compared to control hearts (n = 4). GATA-4 expression is shown in parallel. H3K4Me3 is decreased in DCM hearts. Total histone H3 and GAPDH were used as loading control. (B) shows the quantitative analysis of panel (A). (C) H3R2Me2a and H3K4Me3 levels were quantitated to show their reciprocal expression. Statistical significance was calculated by multiple unpaired t-tests assuming fewer assumptions without same scatter (SD) using GraphPad prism and data were graphically represented as mean ± SD. ∗p < 0.05. LV: left ventricle; DCM: dilated cardiomyopathy. The full blot images are available as supplementary material.

Fig 3: Model depicting the role of PRMT6 in PE-induced cardiac hypertrophy. In response to PE stimulation, PRMT6 is upregulated in cardiac cells, which increases its signature histone methylation “mark” H3R2Me2a and reduces H3K4Me3. These histone posttranslational modifications may activate hypertrophic genes such as ANP directly or indirectly, resulting in cardiac hypertrophy. Over time, these changes have profound effects on cardiac beating rates and global electrical activity, indicating that PRMT6 is required for the PE-mediated hypertrophic effect and to maintain contractility as well as global electrical activity of cardiac cells.

Fig 4: Indirect immunofluorescence showing PRMT6 expression in NRVM maintained in serum-free condition or in media supplemented with PE for 24 and 48 h PRMT6 (red-TRITC), α-actinin (green-FITC), and nuclei counterstained by DAPI are shown as well as the merged images. For each condition, the inlet shows a higher magnification for each image. The arrow shows a prominent PRMT6 localization in NRVM nuclei after 24 h of PE stimulation. The arrowhead shows cytoplasmic PRMT6 in NRVM at 48 h of PE treatment with residual nuclear staining. Images are representative of four independent cardiomyocytes preparations. NRVM: primary neonatal rat ventricular myocytes, PE: phenylephrine.

Fig 5: p18 is a direct target of PRMT6 and interferes with G1/S phase arrest in LUAD. (A) Reverse transcription-quantitative PCR analysis was used to determine the expression levels of important regulatory genes involved in the G1/S phase in A549 cells infected with Scr, or PRMT6 sh1or sh2. **P <0.01 vs. Scr group. (B) Western blotting was used to analyze p18 and PRMT6 expression levels in A549 cells following PRMT6 knockdown. (C) Representative immunohistochemistry images of p18 expression levels in LUAD tissues and matched NAT from 85 patients with LUAD. Scale bar, 50 µm. (D) Quantitative analysis of immunohistochemistry scores of p18 protein expression levels in LUAD tissues and matched NAT from part (C). (E) Western blotting was used to determine the expression levels of p18 in LUAD tissues and matched NAT (n=7). GAPDH served as the normalization control. (F) Kaplan-Meier survival curves were used to determine the overall survival according to low (n=51) and high (n=34) expression levels of PRMT6 using immunohistochemistry scores from 85 patients with LUAD. (G) Correlation between PRMT6 and p18 expression levels was evaluated using Pearson's correlation analysis. Chromatin immunoprecipitation assays were used to determine the effects of PRMT6 knockdown on (H) H3R2me2a and (I) H3K4me3 enrichment in the p18 promoter of A549 cells. Normalized inputs of A549 chromatin DNA were pulled down by antibodies against H3R2me2a, H3K4me3 or negative IgG. **P <0.01 vs. IgG group. PRMT6, protein arginine methyltransferase 6; LUAD/L, lung adenocarcinoma; NAT/N, matched normal adjacent tissues; sh; short hairpin RNA; Scr, scramble; IgG, immunoglobulin G; CCND1, cyclin D1; CCNE1, cyclin E1; SKP2, S-phase kinase-associated protein 2; CDKN3, CDK inhibitor 3; H3R2me2a, Histone H3 asymmetric demethylation at arginine 2; H3K4me3, histone H3 lysine 4 trimethylation.