Fig 2: PRMT5 inhibits STC-1 transcription. (A) Total cell extracts of the shPRMT5 stable cells, wild type cells (WT) and its corresponding control cells (shCON) were analyzed by immunoblotting with the indicated antibodies. (B) qPCR analysis of STC-1 mRNA in the shPRMT5-stable cells, WT and shCON groups *P<0.05. (C-D) STC-1 protein and mRNA levels in MCF-7 cells CPAG co-transfected with pWPI or with PRMT5. (E) Dual-luciferase reporter assay analysis of luciferase activity in MCF-7 cells, which were transiently co-transfected with pCMV-C-FLAG-CAPG or pCMV-C-FLAG and pcDNA3.0-HA-PRMT5 or pcDNA3.0-HA, Renilla luciferase and the pGL3-basic STC-1 -174 bp to 225 bp promoter vector. (F) qPCR-ChIP analysis of PRMT5 binding to the STC-1 ChIP-2 promoter in MDA-MB-231 HM cells. (G) Re-ChIP analysis of CAPG-PRMT5 corecruitment to the STC-1 promoter region was carried out in MDA-MB-231 BO cells. Re-ChIP analysis of CPRMT5 (column 1) and rabbit IgG (column 2) recruitment in the cross-linking protein-DNA complex eluted from CAPG. Re-ChIP analysis of CAPG (column 3) and mouse IgG (column 4) recruitment in the cross-linking protein-DNA complex eluted from PRMT5. * p<0.05, ** p<0.01, *** p<0.001.

Fig 3: Screening for CAPG target genes. (A-C) PCR analysis of 5 genes from the gene expression array in MCF-7-CAPG, MDA-MB-231 HM-shCAPG and MDA-MB-231 BO-shCAPG cells with control cells. (D) PCR analysis of CXCR4, VEGFA and STC-1 in control and CAPG transiently transfected BT474 cells. (E) Pearson analysis of gene expression data from breast cancer patients (GSE2990) was used for depicting the correlation between CAPG and STC-1. (F) MCF-7 cells were transfected with control or CAPG, as shown. STC-1 expression was determined by Western blot. GAPDH was used as loading control. (G-H) MDA-MB231 HM and BO cells were transfected with control or shCAPG, as shown. STC-1 expression was determined by Western blot. GAPDH was used as loading control. (I) Migration and (J) invasion assays for the analysis of control and STC-1 knockdown treatments in MCF-7 CAPG cells. The scale bars is 50µm. * p<0.05, *** p<0.001.

Fig 4: CAPG competes with PRMT5 to bind the exact location of the STC-1 promoter. (A-B) qPCR-ChIP analysis of CAPG binding to the STC-1 ChIP-2 promoter in MCF-7-CAPG cells and MDA-BM-231 BO transfected with pWPI or with pWPI-PRMT5 separately. (C) qPCR-ChIP analysis of PRMT5 binding to the STC-1 ChIP-2 promoter in MCF-7 cells transfected with pCDH (column 3) or with pCDH-CAPG (column 4). (D) ChIP analyses of PRMT5 recruitment to the STC-1 promoter region was carried out in CAPG knocked down MDA-MB-231 BO cells. (E) ChIP analysis of CAPG recruitment to the STC-1 promoter region was carried out in PRMT5 knocked down MDA-MB-231 BO cells. (F) Purified GST and full-length GST-PRMT5 were incubated with core histone and His or His-CAPG for 2 h. Proteins were confirmed by Coomassie staining (input). An anti-histone H4 antibody was used to probe PRMT5-associated histone H4 via Western blotting (IP). (G) In vitro histone methylation (HMT) assays for histone H4. GST (lane 1), GST-PRMT5 (lane 2), GST-PRMT5 and His (lane 3), or GST-PRMT5 and His-CAPG (lane 4) were incubated with AdoMet SAM in HMT buffer and detected by Western blot analysis. (H-I) Kaplan-Meier analysis of DFS (p=0.034) and DDFS (p=0.026) associated with different CAPG plus PRMT5 statuses in 241 patients. CAPG-/PRMT5- patients: n=33; CAPG-/PRMT5+ patients: n=15; CAPG+/PRMT5- patients: n=117; CAPG+/PRMT5+ patients: n=76. (J) The schematic diagram shows the mechanism by which CAPG and PRMT5 regulate STC-1 transcription. * p<0.05, *** p<0.001.

Fig 5: Increased CAPG correlates with poor prognosis in BC. (A) The human gene expression array showed that CAPG is higher in MDA-MB-231 HM cells than in MDA-MB-231 cells. Fold change represents the fold change of CAPG expression in MDA-MB231 HM cells, which was normalized by CAPG expression in MDA-MB231. (B) CAPG protein detection data from iTRAQ-nano-HPLC-MS/MS. (C) iTRAQ-nano-HPLC-MS/MS analysis of the CAPG fold change ratio in MDA-MB-231 and MDA-MB-231 HM cells. (D) qPCR analysis of CAPG mRNA in MDA-MB231 and MDA-MB231 HM cells to validate the human gene expression array result. (E) CAPG expression in seven BC cell lines, with their classification and metastatic ability (Bo: bone only; Bo*: predominantly bone; L*: predominantly lung; BaB: basal B; Lu: luminal). (F) Tissue array from 250 BC patients using immunohistochemistry to determine CAPG expression. The scale bars is 50µm. (G-I) Kaplan-Meier analysis of DFS (p=0.028), DDFS (p=0.064) and OS (p=0.170), respectively, in BC patients with differential CAPG expression levels (CAPG-: n=48, blue line; CAPG+: n=196, green line) according to the tissue microarray. *** p<0.001.