Fig 2: 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.

Fig 3: Knockdown of PRMT6 suppresses LUAD cell growth by activating p18 expression levels in vitro. (A) Transfection efficiency of PRMT6 knockdown was analyzed at the mRNA expression level using reverse transcription-quantitative PCR. **P<0.01 vs. Scr group. (B) Transfection efficiency of PRMT6 knockdown was detected at the protein level using western blotting. (C) Western blotting analysis of p18 protein expression levels following PRMT6 knockdown or the double knockdown of PRMT6 and p18 in A549 cells. (D) Flow cytometric analysis was used to determine the cell cycle distribution of PRMT6 knockdown or the double knockdown of PRMT6 and p18 in LUAD cells. (E) Quantification of the cell cycle distribution of PRMT6 knockdown or double knockdown of PRMT6 and p18 in LUAD cells from part (D). (F) Cell Counting Kit-8 assay was used to determine the effects on proliferation of PRMT6 knockdown or double knockdown of PRMT6 and p18 in A549 cells. (G) Colony formation assay was used to evaluate the effect on proliferation of PRMT6 knockdown or double knockdown of PRMT6 and p18 in A549 cells. (H) Number of colonies formed in part (G) was counted using Image J software. *P<0.05, **P< 0.01. PRMT6, protein arginine methyltransferase 6; LUAD, lung adenocarcinoma; sh; short hairpin RNA; Scr, scramble; OD, optical density.