Journal: Nature Communications
Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis
Figure Lengend Snippet: RAF1 is expressed at low levels in human HCC and suppresses the growth of both HCC xenografts and chemically induced tumours. ( a ) RAF1 expression in a cohort of 31 HCC patients. Left panel, representative IHC image (T, tumour; NT, non-tumour). Scale bar, 50 μm. Middle panel, RAF1 expression in matched tumour and non-tumour tissue (a.u.=arbitrary units). Right panel, RAF1 expression in tumours correlates inversely with tumour grade (ratio: protein expression in tumour/non-tumour tissue). ( b ) Inducible shRNA-mediated RAF1 silencing does not impact A- or BRAF expression (top panel) but increases the proliferation of Hep3B cells in culture (bottom panel; n =6). ( c ) Inducible shRNA-mediated RAF1 silencing strongly promotes the growth of Hep3B xenografts. ( d,e ) Ablation of RAF1 in liver parenchymal cells promotes chemically induced hepatocarcinogenesis. Top panels, experimental protocols. Left bottom panel, macroscopic appearance of F/F and Δhep ( d ) or Δp/np ( e ) tumour-bearing livers 30 weeks (w) after DEN injection; arrows indicate tumours. Scale bars, 0.5 cm. Middle panels, liver:body weight ratio of untreated or DEN/Pb-treated mice. Right panels, tumour numbers and % of tumour-occupied area in control, Δhep ( d ) and Δp/np ( e ) livers. In ( d ), no DEN: F/F n =4, Δhep=6; DEN-treated: F/F n =7, Δhep=8. In ( e ), DEN-treated: F/F n =10, Δp/np n =11. ( f , g ) Quantification of Ki67+ cells: ( f ) and inflammatory cells ( g ; F4/80+ cells, granulocytes and CD3+ cells) in tumour-bearing F/F and Δhep livers. Np=non-parenchymal cells. ( h ) Chemokine levels in the serum of F/F and Δhep mice. ( i ) Chemo-/cytokine levels in tumour-bearing livers. Data are presented as mean ± SEM, * P ≤0.05, ** P
Article Snippet: The following antibodies were used for immunohistochemistry: α-mouse, CD44 (550538, BD Biosciences, 1:50), Ki67 (Novocastra, 1:1,000), YAP1 (4912, Cell Signaling, 1:200), F4/80 (MCA497G, AbD Serotec, 1:50), FSP1 (27957, Abcam, 1:300), CD3 (A0452, DAKO, 1:1,000), βcatenin (32572, Abcam,1:500); α-human YAP1 (12395, Cell Signaling, 1:1,000), RAF1 (154754, Abcam, 1:1,500) and STAT3 (9139, Cell Signaling, 1:800).
Techniques: Expressing, Immunohistochemistry, shRNA, Injection, Mouse Assay