Fig 2: PCR array analysis of cancer-related gene expression levels in ECA109 cells with PADI4 overexpression. A, The Human Cancer PathwayFinder PCR array revealed at least a three-fold change in the expression level of tumor-related genes. B, Genes with a more than a three-fold change in expression level are depicted in one map. The expression levels of ANGPT1, CA9, and TEK were upregulated in PADI4-overexpressing ECA109 cells compared with those in cells transfected with an RFP-alone expressing plasmid (OE-RFP)

Fig 3: Role of PADI4 overexpression on the migration of GC cells. SGC-7901 and MGC80-3 cells were transfected with plasmid. The protein expression of PADI4, IL-8, E-cadherin, vimentin and Slug was determined by western blot (A–C). Cell migration was determined by wound healing assay (D, E) (Original magnification: 100×) and Transwell assay (F, G) (Original magnification: 400×). All results were obtained from three independent experiments

Fig 4: Analysis of Tal1 and PADI4 binding to the IL6ST 5'-region and influence of Tal1. (a) Schematic representation of the analysed IL6ST genomic locus including the 5'-region. The genomic position is given and the position of primer pairs used for ChIP analysis is indicated. The first non-coding exon is marked in dark green and the first intron in light green. (b) Mapping of Tal1 binding to the IL6ST 5'-region. Upon ChIP with an anti-Tal1 antibody qPCR with primers at different positions of the IL6ST 5'-region was performed. (c) ChIP shows binding of PADI4 to the IL6ST 5'-region. Upon ChIP with an anti-PADI4 antibody qPCR with primers at different positions of the IL6ST 5'-region was performed. The P-value gives the statistical significant difference between the values gathered with primer E compared with primer A. (d–f) Analysis of concomitant Tal1 and PADI4 binding to the IL6ST 5'-region by ChIP-ReChIP. (d) Concomitant Tal1 and PADI4 binding is detected at a region close to the first exon of IL6ST (primer E). (e) No Tal1/PADI4 is detected with an upstream primer (primer A). (f) Analysis of the ChIP-ReChIP qPCR products by gel electrophoresis at the end point of the qPCR reaction confirms qPCR results. Antibody combinations and primer pairs are given. The P-value (*P<0.05) gives the statistical significant enrichment of the ChIP-ReChIP compared with the IgG controls according to Student’s t-test. (g) ChIP demonstrates decreased Tal1 binding to the IL6ST promoter upon Tal1 knockdown. (h) ChIP shows decreased PADI4 binding to the IL6ST promoter upon Tal1 knockdown. (i) The histone modification marks H3K9ac and H3K4me3 are decreased upon Tal1 knockdown at the IL6ST promoter. (j) The histone modification H3R2me2a remains at a low level upon Tal1 knockdown and H3R17me2a is increased at the IL6ST promoter. (k) PRMT6 and PRMT4 binding are decreased upon Tal1 knockdown at the IL6ST promoter. Values are shown as percent enrichment compared with the input. Error bars represent the s.d. from at least four determinations. The P-values were calculated using the t-test, *P<0.05, **P<0.01.

Fig 5: PADI4 promotes the proliferation of RA-FLS through hypoxia. (A) RA-FLS and N-FLS were incubated under normoxia (20% O2) or hypoxia (1% O2) for 5 days. (B) Cell viability under normoxia and hypoxia was measured using an MTT assay. Data are presented as mean ± standard deviation from three separate experiments. (C) PADI4 expression was determined by immunofluorescence and qPCR in N-FLS under normoxia and hypoxia for 3 days. (D) PADI4 expression was determined by immunofluorescence and qPCR in RA-FLS under normoxia and hypoxia for 3 days. (E) LC3 and Beclin1 expression were determined by qPCR in RA-FLS under normoxia and hypoxia for 3 days. N-FLS were obtained from control rats. Data represent three independent experiments with presented as mean ± standard deviation. **P<0.001. FLS, fibroblast-like synoviocytes; PADI4, peptidyl arginine deiminase type IV; qPCR, quantitative polymerase chain reaction; RA, rheumatoid arthritis.