Figure 3. Increased HCC in Nlrp12-/- mice is associated with increased cell death and proliferation in the livers.

WT and Nlrp12^-/- were injected with DEN (25 mg/kg i.p.) or PBS (healthy control) at the age of 14 days and sacrificed at 10 months after DEN administration. (A) Liver tissue sections from healthy controls and DEN-treated mice were immunostained with Ki67 antibody and the number of Ki67-positive cells was counted under 20X objective. Data were collected from at least 10 fields per liver section and three mice/group. Data represent means ± SEM (n = 50). Statistical difference was determined by two-tailed unpaired t-test. (B) The expression of Ki67 in tumor tissues was measured by real-time qPCR. Data represent means ± SEM (n = 15; each sample represents individual mouse). Statistical difference was determined by two-tailed unpaired t-test. (C) Apoptosis in the healthy and HCC livers were measured by TUNEL assay. The number of TUNEL-positive cells (green) under 20X objective was counted and plotted as individual values. Data were collected from at least 10 fields per liver section and three mice/group. Data represent means ± SEM. Statistical difference was determined by two-tailed unpaired t-test. (D) Liver sections from DEN-treated mice (n = 3) were immunostained for cleaved caspase-3 (brown). Cleaved caspase-3 positive cells were counted under 20X objective. Data represent means ± SEM (n = 40). Statistical difference was determined by two-tailed unpaired t-test. (E) Liver tumor lysates were immunoblotted with anti-cleaved caspase-3, cytochrome c, and β-actin. The band intensities of caspase-3 and cytochrome c were measured. Data represent means ± SEM (n = 5; each sample represents individual mouse). Statistical difference was determined by two-tailed unpaired t-test.

Figure 3—figure supplement 1. NLRP12 regulates hepatocyte death and proliferation during HCC.

(A) DEN-treated mice at 10 months were injected with BrdU at 2 hr before sacrifice (n = 5/group) via i.p. Formalin-fixed liver sections were immunostained with BrdU antibody. The number of BrdU-positive cells (brown) per HPF was counted. The analysis includes counting from 10 to 15 fields/section (under 20X objective) and three mice/group. Data represent means ± SEM. *p<0.05. **p<0.01. Statistical difference was determined by two-tailed unpaired t-test. (B and C) Primary hepatocytes were grown on cover slip, treated with or without LPS for 24 hr, and stained for TUNEL (green). The number of TUNEL-positive cells were counted from 30 fields. Data are representative of three independent experiments. Data represent means ± SEM. *p<0.05. **p<0.01. Statistical difference was determined by two-tailed unpaired t-test.