Fig 1: Overexpression and knockdown of RASAL2 expression in ACHN cells. (A) RASAL2 expression was evaluated in ACHN cells using reverse transcription-quantitative PCR, (B) western blotting and (C) immunofluorescence staining following transfection with plasmids expressing either the RASAL2 protein or shRASAL2. Magnification, x200. *P<0.05 vs. Control group. #P<0.05 vs. scramble group. Blank, un-transfected control; Control, control pcDNA.31 plasmid; sh, short hairpin RNA; scramble, scrambled shRNA; RASAL2, ras protein activator like 2; shRASAL2, RASAL2 shRNA.
Fig 2: Ectopic expression of RASAL2 promotes CRC tumorigenesis. a Ectopic stable expression of RASAL2 protein in SW480 confirmed by western blot. b-d Ectopic expression of RASAL2 significantly enhanced cell viability b, anchorage-dependent growth (c) as well as cell invasion and migration (d) by MTT, colony formation and transwell cell invasion and migration assays in SW480 cells stably overexpressing RASAL2. e Representative image of tumors in xenograft mouse model (Left: pLPCX; Right: pLPCX-RASAL2) and that of tumor formed in nude mice inoculated subcutaneously with RASAL2 plasmids or empty vectors (Upper panel); The tumor growth curve of SW480 stably transduced with RASAL2 in nude mice was significantly dampened compared with SW480 transduced control (Middle panel); The mean tumor weights in the pLPCX-RASAL2 and pLPCX vector groups were summarized in histogram (Lower panel). f IHC assay of RASAL2 and Ki-67 in xenograft tumor tissues. (*, P < 0.05; **, P < 0.01, Student’s t-test, n = 3 independent experiments)
Fig 3: Outline of RASAL2-mediated hippo signaling pathway in CRC. RASAL2 showed high expression in CRC and targeted LATS2/YAP1 axis. This led to YAP1 dephosphorylation and nuclear translocation, thus preventing YAP1 from ubiquitination in the cytoplasm and functioning as a transcriptional co-activator to stimulate expression of pro-proliferation genes like CCND1 in CRC
Fig 4: RASAL2 silencing exhibits inhibitory effect in CRC. a RASAL2 endogenous expression in CRC cell lines by western blot. ß-actin was used as a loading control. b siControl or siRASAL2 was transfected into DLD-1, HCT 116 and SW620 and Caco2 cells. The downregulated RASAL2 was confirmed by western blot. c siRASAL2 significantly reduced the cell proliferation and anchorage-dependent growth with MTT assays and colony formation assays in KRAS/NRAS wild-type and mutant CRC cells. d Reduced anchorage-independent growth in RASAL2 knockdown cells using soft agar assays. e Flow cytometry analysis on cell cycle in RASAL2 knockdown cells. Representative figure (Left) and percentage of cells in each of the G1, S and G2 phases (Right). f Knockdown of RASAL2 by two different siRNAs suppressed cell invasion and migration in DLD-1 and HCT 116 cell lines by cell invasion and migration assays. g Tumors isolated from nude mice at the end of investigation (Upper: siControl; Lower: siRASAL2); Tumor growth was summarized using a line chart, while mean tumor weights in the siControl and siRASAL2 groups were shown in the histogram. (*, P < 0.05; **, P < 0.01, Student’s t-test, n = 3 independent experiments)
Fig 5: Upregulation of RASAL2 is associated with poor prognosis and metastasis of CRC. a Representative IHC images showing positive (H-score > 100) and negative (H-score = 100) RASAL2 expression in CRC. b The distribution of RASAL2 histoscore in RAS wild-type and mutant CRC samples. c RASAL2 overexpression had significantly shorter disease-free survival and overall survival in a cohort of 208 CRC patients. d-e Copy number gain (d) and upregulated mRNA levels (e) of RASAL2 showed aggressive disease-free survival and overall survival in TCGA CRC cohort
Supplier Page from Abcam for Anti-RASAL2 antibody