Fig 1: Inhibition of migration activity in Sam68-knockdown OSCC cells. (A) Heat map representation of 150 DEGs identified in mRNA sequencing between Sam68 siRNA #1-transfected HO-1-N-1 cells (n=3) and control siRNA-transfected cells (n=3). (B) GO enrichment analysis of 130 downregulated DEGs in Sam68-knockdown HO-1-N-1 cells. Bar graph of enriched GO terms (biological process) across input gene lists, colored by P-values. (C) HO-1-N-1 cells transfected with control siRNA (control) or Sam68 siRNA (Sam68 #1 or #2) were analyzed for wound closure at 72 h after wounding. Scale bar, 200 µm. Data are expressed as the mean ± SD. Dunnett's test compared with the control. **P<0.05. (D) HO-1-N-1 cells transfected with control siRNA (control) or Sam68 siRNA (Sam68 #1 or #2) were cultured in a Transwell chamber for 48 h. Migrated cells were stained and counted. Scale bar, 200 µm. Data are expressed as the mean ± SD. Dunnett's test compared with the control. *P<0.01. Sam68, Src-associated in mitosis 68 kDa; OSCC, oral squamous cell carcinoma; DEGs, differentially expressed genes; GO, Gene Ontology; siRNA, small interfering RNA.
Fig 2: AXL KO, SAM68 KO and AXL inhibition increase cholesterol biosynthesis.a Venn diagram depicting the number of genes upregulated in HeyA8 AXL KO and SAM68 KO cell lines and their common genes upregulated. b Top 10 upregulated pathways of common differentially expressed genes in AXL KO and SAM68 KO cell lines. Red indicates candidate pathways chosen for validation. c Relative fold change in expression of cholesterol biosynthesis genes in SKOV3 and d HeyA8; *p < 0.05, **p < 0.01, ***p < 0.001, determined by two-tailed t-test with Welch’s correction. e Filipin III staining in SKOV3 and HeyA8. f Immunoblot of cholesterol biosynthesis protein expression levels peaking at 24 h upon AXL inhibition in SKOV3 and HeyA8. The numbers below blots reflect protein band intensity normalised against DMSO. g Quantification of free cholesterol levels and h cholesteryl esters levels upon treatment with AXL inhibition using LC–MS; *p < 0.05, **p < 0.01, ***p < 0.001, determined by two-tailed t-test with Welch’s correction.
Fig 3: Depletion of SAM or AXL increases DNA damage and pADPr.a CRISPR-Cas9 mediated AXL knockout in SKOV3 and HeyA8. b CRISPR-Cas9 mediated SAM68 knockout in HeyA8. c Immunoblot of HeyA8 SAM68 KO treated with R428 and Olaparib. d Immunoblot of AXL nuclear localisation upon AXL inhibition in HeyA8 SAM68 KO. Numbers below blots reflect protein band intensity normalised against respective control. e Combination treatment of R428 with ATR inhibitor BAY1895344 in pooled SAM68 KO cell lines and f single clone SAM68 KO cell lines. g Combination index of R428 and BAY1895344. h Dose reduction index of BAY1895344. Fa Fraction affected.
Fig 4: Expression of Sam68 in oral cancer cell lines. (A) Western blot analysis of Sam68 in oral cancer cells (HSC2, HSC3, HO-1-N-1, and HOC313). (B) Sam68 siRNA (#1 or #2) or control siRNA (control) was transfected into HO-1-N-1 cells, and the proteins were estimated at 48 or 144 h by western blotting. Sam68, Src-associated in mitosis 68 kDa.
Fig 5: Immunohistochemistry of vimentin and Sam68 in the same tumor. (A) Representative image of the broad sheet of tumor cells without vimentin expression at the superficial area. (B) Representative image of the small cords of tumor cells exhibiting positive vimentin expression and higher Sam68 immunoreactivity at the central area. (C) Representative image of the small cords and clusters of tumor cells exhibiting positive vimentin expression and higher Sam68 immunoreactivity at the invasive front. (magnification, ×100; scale bar, 50 µm). Sam68, Src-associated in mitosis 68 kDa.
Supplier Page from Abcam for Anti-SAM68 antibody [EPR3231]