Fig 1: RFC2 activates the MET/PI3K/AKT/mTOR pathway in CRC cells. A Results from GSEA forecast. B Correlation analysis between RFC2 and MET based on TCGA-COAD datasets using Pearson’s correlation analysis. C MET protein expression in cells was measured using western blot. D Representative protein expression in the PI3K/AKT/mTOR pathway was measured using western blot. *p < 0.05 vs. si-NC or Vector group
Fig 2: CREB5 regulates RFC2 transcriptional activity. A A sketch map of RFC2 promoter region. B The ov-CREB5 or ov-NC-transfected cells were transfected with promoter WT or mutant and luciferase activity was detected using dual-luciferase reporter assay. C ChIP assay was performed in cells. D Western blot of CREB5 and RFC2 in cells. E RFC2 protein in cells was measured using western blot. F Cell viability was examined using CCK-8 assay. G Wound healing assay was conducted in cells. H Transwell assay was performed in cells. *p < 0.05 vs. ov-NC + RFC2-luc, lgG, si-NC, or si-NC + vector group. #p < 0.05 vs. si-NC + RFC2 group
Fig 3: Activator or inhibitor partially rescues the effects of RFC2 knockdown or overexpression in CRC cells. A Cell viability was examined using CCK-8 assay. B and C Wound healing and transwell assays were conducted in SW480 cells. D and E Glucose uptake and lactate production were detected in SW480 cells. F Marker proteins of glycolysis were detected using western blot. *p < 0.05 vs. si-NC or Vector group. #p < 0.05 vs. si-RFC2-2 or oe-RFC2 group
Fig 4: Knockdown of RFC2 results in accumulation of DNA damage, increased apoptosis and growth inhibition in CRPC cells. (A) The expression level of RFC2 at the protein level in multiple prostate cell lines. Lysates from five prostate cell lines (RWPE, LNCaP, 22Rv1, DU145 and PC3 cells) were used for Western blot analysis with specific antibodies to replication factor C subunit2 (RFC2). β-Actin was used as a loading control. (B) The expression level of RFC2 at the protein level in LTAD and parental LNCaP cells. Lysates were used for Western blot analysis with specific antibodies to replication factor C subunit2 (RFC2). β-Actin was used as a loading control. (C) Knockdown of RFC2 suppresses cell proliferation of Pca cell lines. LNCaP, DU145, PC3 and 22Rv1 cells were treated with 5 nM siControl or siRFC2 (#1, #2, and #3). MTS assay was carried out at the indicated time points (N = 6). The absorbance quantified in the plate reader is shown. The results are presented as mean and SD. One-way ANOVA and Dunnet’s post-doc tests were performed to obtain P-values. *P < 0.05, **P < 0.01, ***P < 0.001, compared with siControl. (D) Knockdown of RFC2 increases DNA damage and apoptosis in Pca cell lines. PC3, DU145 and 22Rv1 cells were transfected with 5 nM siControl or siRFC2 (#1, #2, and #3) for 48 h. Western blot analysis for RFC2, cleaved PARP and γH2AX was carried out and β-actin was used as a loading control.
Fig 5: Immunohistochemical (IHC) analysis of RFC2 protein expression in Pca tissues. (A–D) Representative IHC images of RFC2 in Pca tissues. (A) Positive (anti-RFC2 antibody) and negative control (non-specific rabbit IgG antibody) using bladder cancer specimens. (B) Anti-RFC2 in benign prostate, intensity score 0. (C) Anti-RFC2 in Pca, intensity score 1 and 2. (D) Anti-RFC2 in CRPC, intensity score 3. Scale bar = 50 μm. (E) Rate of cases in which positive IR was detected by RFC2 IHC in benign (N = 12), Pca (N = 103), and CRPC (N = 15) tissues. Chi-squared test was done to calculate P-value. IR scores of 0‐4 and 5–8 were defined as low and high IR, respectively. (F,G) High immunoreactivity (IR) score of RFC2 is associated with poor prognosis of prostate cancer patients. Progression-free survival (F) and cancer-specific survival (G) of prostate cancer patients are shown (N = 103). Survival curve was obtained by Kaplan–Meier method and P-value was determined by log-rank (Mantel–Cox) test. (H) Evaluation of the time to CRPC diagnosis in CRPC cases (N = 15). The median follow-up time from radical prostatectomy to CRPC diagnosis was summarized in CRPC cases with low and high RFC2 IR. Mann–Whitney test was done to calculate P-value.
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