Figure 1: Neutrophils are required for experimentally induced HCC.

(a) Graph shows average total number of hepatic neutrophils ( NIMP-R14 ) in DEN-injured WT or nfkb1^−/− mice. Red line denotes basal neutrophil levels in normal WT liver. (b) Average tumour counts and liver/body weight ratio in 30- and 40-week DEN-injured WT and nfkb1^−/− mice. Representative pictures of livers from 40-week DEN-injured WT and nfkb1^−/− mice. (c) Average total number of PCNA+ hepatocytes in DEN-injured WT and nfkb1^−/− mice. (d) Diagram showing the experimental plan to deplete neutrophils (Ly6G antibody) in WT mice from week 32–40 after DEN injury. Average tumour counts, representative pictures of livers and average liver/body weight ratio graph in 40-week DEN-injured WT mice treated with control IgG or Ly6G antibody (Ab) for 8 weeks. n=10 (e) Representative pictures of liver from 20 month (aged under normal conditions) WT and nfkb1^−/− mice. Representative photomicrographs of haematoxylin and eosin (H&E)- and Sirius red/fast green-stained liver sections from 20-month nfkb1^−/− mice (upper panel) revealed steatohepatitis (fat, blue arrows; inflammation, yellow arrows; ballooned hepatocytes with Mallory–Denk bodies, green arrows) and fibrosis (black arrows). Focal dysplasia (yellow dotted line) with focal inflammation (black arrow) and HCC in H&E-stained nfkb1^−/− aged livers (lower panel). Graphs show average liver/body weight ratio and tumour frequency identified histologically using H&E sections from 20-month, aged WT and nfkb1^−/− mice. n=7 WT and ten nfkb1^−/− mice. (f) Diagram showing the neutrophil depletion experimental protocol in nfkb1^−/− mice from week 22–30 after DEN injury. Average tumour counts, representative pictures of livers and graph showing average liver/body weight ratio from 30-week DEN-injured nfkb1^−/− mice treated with control IgG or Ly6G for 8 weeks (n=10). All data are means±s.e.m; scale bars, 200 μm. For a and c, n=6, 4, 6, 7, 15 and 9 (nfkb1^−/−), and 4, 4, 4, 5, 11 and 14 (WT) for the 5- to 40-week time points). Statistical significance was determined using one-way analysis of variance with Tukey’s post-hoc test (a,c) or an unpaired t-test (b,e), *P<0.05, **P<0.01 or ***P<0.001 compared with control.