Fig 2: Arginine methylation at SHANK2 R240 promoted metastasis through activates FAK signalling.(A) Equal numbers of MDA-MB-231 cells expressing SHANK2 shRNA with or without reconstituted expression of WT SHANK2, SHANK2 R240K or SHANK2 R240F mutant. SHANK2 R240F group was treated with GSK2256098 or DMSO. Cells were tail-vein injected into BALB/c nude mice, and lung metastasis was determined. The gross appearance of the lungs and the tumour nodules on the lungs were examined. Tumours were paraffin embedded and stained with H and E at Day12. n = 5 tumours in each group. IHC analyses of tumour tissues were performed with anti-p-FAK or anti-p-cortactin antibody. Scale bars,100 µm. (B) The number of visible surface metastatic lesions in lungs was counted. (n = 5 mice for each group, **p<0.01, ***p<0.001, ns = not significant, one-way ANOVA test). (C, D) p-FAK and p-cortactin -positive cells were quantified in 10 microscope fields. **p<0.01, ***p<0.001, ns = not significant, one-way ANOVA test. (E, F) Typical pictures of the immunohistochemical staining of PRMT7, p-FAK and p-cortactin in breast cancer samples. Scale bars,100 µm. n = 27, **p<0.01, Student’s t test.

Fig 3: PRMT7 methylated SHANK2 at R240.(A) The purified SHANK2 from MDA-MB-231 cells transfected with HA-SHANK2 was analyzed for methylation by mass spectrometry. The SHANK2 R240 residue in fragmentation of KAARMRN was methylated. The Mascot score was 27.28, and the expectation value was 3.74E-04. (B) Alignment of the consensus SHANK2 sequences between different species near the R240 methylation site. (C) MDA-MB-231 cells with or without expressing SHANK2 shRNA and with or without reconstituted expression of WT SHANK2 or SHANK2 R240K mutant. Immunoprecipitation of HA with anti-HA antibody was performed. (D) Flag-tagged SHANK2 WT or SHANK2 R240K was transfected into MDA-MB-231 cells. HA-purified SHANK2 WT or SHANK2 R240K proteins were incubated with purified bacterially expressed PRMT7 and SAM. Methylation of SHANK2 protein was determined by western blot. (E) HEK293T cells were co-transfected with increasing amounts of Flag-PRMT7 and SHANK2 WT or SHANK2 R240K expression plasmids. Immunoprecipitation of SHANK2 with anti-SHANK2 antibody was performed.

Fig 4: Effects of PRMT7 deletion on gene expression in T-ALL cells. Volcano plots displaying the differentially expressed genes (log2FC = ±1.5, FDR < 0.05) in (a) J-KO3 and (b) J-KO5 cell lines. (c) A volcano plot displaying gene expression changes (log2FC = ±1.5, FDR < 0.05) in genes known to be associated with T-ALL pathogenesis and altered in all six PRMT7 knockout cell lines labeled with different symbols. Red color indicates genes with increased and blue with decreased expression compared to unmodified cells. Three biological replicates were combined for the analysis for each cell line. J stands for Jurkat and M for Molt4 cell line, EV for empty vector and WT for wild type.

Fig 5: SHANK2 R240 methylation promotes FAK/dynamin2/talin complex co-localized with endosome.(A) SHANK2 associated proteins from HA-SHANK2 expressing stable MDA-MB-231 cells were immunopurified with anti-HA (a-HA) affinity resins. The protein bands were analyzed by mass spectrometry. (B) Representative peptide fragments of SHANK2 associated proteins and peptide coverage of the indicated proteins are shown. (C) Endogenous talin, FAK, dynamin2, EEA1 and SHANK2 were IP from MDA-MB-231 cells, with indicated antibodies, and the binding of talin, FAK, dynamin2 and SHANK2 was examined by western blot. (D) MCF7 cells with expression of the indicated Flag-Vector or Flag-tagged PRMT7. Immunoprecipitation of cortactin with anti-cortactin antibody was performed. (E) Confocal images of PRMT7, dynamin2, SHANK2, EEA1, talin, Rab5, FAK, cortactin and DAPI staining in MCF7 cells with expression of the indicated Flag-Vector or Flag-tagged PRMT7. Scale bars, 10 µm. (F) MDA-MB-231cells with or without stable expression of the indicated PRMT7 shRNA or a control shRNA. Immunoprecipitation of cortactin with anti-cortactin antibody was performed. (G) confocal images of PRMT7, dynamin2, SHANK2, EEA1, talin, Rab5, FAK, cortactin and DAPI staining in MDA-MB-231cells with or without stable expression of the indicated PRMT7 shRNA or a control shRNA. Scale bars, 10 µm. (H) MDA-MB-231cells expressing SHANK2 shRNA with or without reconstituted expression of WT SHANK2 or SHANK2 R240K mutant. Immunoprecipitation of cortactin with anti-cortactin antibody was performed. (I) Confocal images of PRMT7, dynamin2, SHANK2, EEA1, talin, Rab5, FAK, cortactin and DAPI staining in MDA-MB-231cells expressing SHANK2 shRNA with or without reconstituted expression of WT SHANK2 or SHANK2 R240K mutant. Scale bars, 10 µm. (J) MCF10A cells with reconstituted expression of SHANK2 WT, SHANK2 R240K or SHANK2 R240F (arginine to phenylalanine) mutant. Immunoprecipitation of SHANK2 with anti-SHANK2 antibody was performed. (K) Confocal images of PRMT7, dynamin2, SHANK2, EEA1, talin, Rab5, FAK, cortactin in MCF10A cells with reconstituted expression of SHANK2 WT, SHANK2 R240K or SHANK2 R240F mutant. Scale bars, 10 µm.