Fig. 2: Citrullination of IGF2BP1 at R167 promotes the aggressive behaviors of RA-FLSs.

A Schematic illustration of IGF2BP1^R167K, IGF2BP1^R168K, IGF2BP1^R174K, IGF2BP1^R575K, and IGF2BP1^4R-K mutants. B The effect of IGF2BP1 knockdown in RA-FLSs was detected by western blot. ShIGF2BP1-1 showed best effect and was used for following experiments. C The citrullination level of IGF2BP1 in RA-FLSs was detected by western blot. The IGF2BP1-KD RA-FLSs were rescued with IGF2BP1 mutants, then the citrullination level of IGF2BP1 was detected using F95 antibody. D Cell proliferation of RA-FLSs was detected using EdU experiment. Total cells were detected by Hoechst staining (blue), and cells with active DNA replication were detected by EdU staining (red). E Cell migration of RA-FLSs detected by scratch assay. Statistical significance was determined by ANOVA of repeated measurements. The wound closure ratio of each group was calculated using the following equation: migrated cellular area/scratched area × 100%. F Cell invasion of RA-FLSs detected by Transwell assay. Images of RA-FLSs on the undersurface of a filter were captured under the microscope (original magnification × 100) and the number of FLSs per field in each group was quantified by ImageJ. G Formation of invasive pseudopodia were detected by F-actin staining with TRITC-Phalloidin. All the experiments were performed in triplicate. The data were presented as mean ± SD. One-way ANOVA (C, D, F, G) followed by Dunnett test was used as parametric test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.