Supplementary Figure 1: HCC induction and liver damage in mice and hepatocytes. | Nature Cell Biology

Supplementary Figure 1: HCC induction and liver damage in mice and hepatocytes.

From: EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma formation

Supplementary Figure 1

(a) Tumour development was initiated in male mice of the indicated genotypes by DEN injection at 4 weeks of age (black arrow). At 8 weeks of age tumours were promoted by a diet complemented with phenobarbital (PB) until mice were sacrificed. Top: EGFRΔhep mice (EGFRf/f; Alfp-Cre). Middle: EGFRΔMx mice (EGFRf/f; Mx-Cre) that received 3 pIpC injections with 2 days intervals at 7 weeks of age to delete EGFR in the liver (grey arrow). Bottom: EGFRΔMx mice (EGFRf/f; Mx-Cre), that received pIpC injections on day 9, 11, and 13 (grey arrow) after birth. (b) Southern Blot analysis showing EGFR deletion in EGFRΔMx (left) and EGFRΔhep (right, non-recombined EGFR allele from non-parenchymal cells) livers (f: floxed EGFR allele (6 kb), Δ: Cre-deleted EGFR allele (3.9 kb). (c) Ki67-positive (left, EGFRf/f, n = 45 (six mice); EGFRΔMx, n = 48 (7 mice)) and TUNEL-positive cells (right, EGFRf/f, n = 56 (six mice); EGFRΔMx: n = 55 (six mice)) in adjacent non-tumour tissue. n = HPF. (d) Representative H&E staining of livers 0, 36, 48, and 72 h after DEN intoxication in vivo. Black arrows indicate necrotic areas. Scale bar, 500 μm. (e,f) Alanine transaminase (ALT, 0 h and 24 h: n = 5 mice per genotype, 48 h: EGFRf/f and EGFRΔMx: n = 4, EGFRΔhepn = 5 mice) and Aspartate transaminase (AST, 0 h: EGFRf/f: n = 3, EGFRΔhep: n = 4, EGFRΔMx: n = 4, 24 h: EGFRf/f: n = 3, EGFRΔhep, n = 3, EGFRΔMx: n = 4, 48 h: EGFRf/f: n = 4, EGFRΔhep, n = 4, EGFRΔMx: n = 3 mice) measured in serum 0, 24, and 48 h after DEN intoxication. (g) Representative active caspase-3 staining (Alexa 488, green) and nuclei (DAPI, blue) of EGFRf/f and EGFRΔhep liver sections 24 and 96 h after DEN intoxication, showing increased apoptosis in EGFR-deficient livers after 96 h. Scale bar indicates 500 μm. (h) HMGB1 staining of cultured EGFRf/f and EGFRΔMx hepatocytes 12 h after DEN treatment in vitro. Nuclei (DAPI, blue), actin (Phalloidin, red), HMGB1 (Alexa 488, green). Scale bar indicates 100 μm. (i) Active caspase-3 staining of cultured hepatocytes of EGFRf/f and EGFRΔMx mice incubated with TNFα/CHX for 12 h. Nuclei (DAPI, blue), actin (Phalloidin, red), active caspase-3 (Alexa 488, green). Scale bar indicates 50 μm. (c) Data represent mean ± s.e.m. (e,f) Data represent mean ± s.d. Student’s t-test for independent samples and unequal variances was used to assess statistical significance (P < 0.05,P < 0.01,P < 0.001). Original data are provided in Supplementary Table 1.

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