Deletion of DDB1 in hepatocytes results in hepatic oval cells activation.

<p>(A) H&E staining of liver sections from 4-week old <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> mice. PV, portal vein. (B) IHC staining for DDB1 on liver sections from 4-week old <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> mice. (C) Co-IF staining for DDB1 and A6 on 4-week old <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> liver sections.</p

EpCAM-positive cells from DDB1 mutant liver proliferate <i>in vitro</i> and repopulate the liver <i>in vivo</i>.

<p>(A) Analysis by flow cytometry of EpCAM⁺ F4/80⁻ cells in non-parenchymal fractions prepared from 4-week old <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> mice. The ratio of EpCAM⁺ F4/80⁻ cells from a representative experiment is shown in each panel. (B) <i>In vitro</i> culture of EpCAM⁺ cells sorted by FACS from DDB1 mutant liver and seeded on Matrigel. Cells form colonies after 1, 3, and 9 days of culture (upper panels) and exhibit EpCAM positivity (lower panels). (C) Co-IF staining for A6 and AFP on colonies from cultured EpCAM⁺ cells. (D) Experimental scheme for <i>in vivo</i> differentiation of EpCAM⁺ OCs. Adult <i>DDB1^F/F;Mx1-Cre^+/−;Rosa26-lacZ</i> mice were generated and injected with poly(I:C). EpCAM⁺ cells were isolated by FACS 6 weeks later, and injected intrasplenically to nude mice that had been fed with DDC diet. The recipient nude mice received poly(I:C) injection 2 weeks later and liver collected for analysis on the following day. (E) Isolation of EpCAM⁺ F4/80⁻ cells from donor or control mouse liver. The ratio of EpCAM⁺ F4/80⁻ cells is shown in each panel. (F) IHC staining for ß-gal on liver sections from recipient nude mice. Arrows indicate positively stained hepatocytes.</p

DDB1 mutant mice exhibit minor liver damage compared with DDC-treated mice.

<p>(<b>A</b>): Co-IF staining for A6 and EpCAM on liver sections from DDC-treated mice. (<b>B</b>): Gross appearance of liver from mice fed with DDC diet for 4 weeks. (<b>C</b>): H&E staining of the liver in (<b>B</b>). (<b>D</b>): IHC staining for CD45 (upper panels) and F4/80 (lower panels) on liver sections from <i>DDB1^F/F</i>, <i>DDB1^F/F;Alb-Cre^+/+</i> and DDC-treated mice. (<b>E</b>): Percentage of EpCAM⁺ cells from <i>DDB1^F/F;Alb-Cre^+/+</i> and DDC-diet liver, determined by FACS analysis. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as the means±SEM; n = 3. (<b>F</b>): Serum alanine aminotransferase (ALT) levels. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as means±SEM; n = 3 **P<0.01.</p

Deletion of p21 partially restores proliferation of DDB1-deficient hepatocytes and alleviates OC activation.

<p>(A) Western blot for some substrates of DDB1-Cul4A ubiquitin ligase using lysates of hepatocytes isolated from <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> mice. (B) Western blot for DDB1 and p21 using lysates of cytoplasmic fraction (C) and nuclear fraction (N) prepared from MEFs. (C) IHC staining for DDB1 and Ki67 on liver sections from <i>DDB1^F/F;Alb-Cre^+/−, DDB1^F/F;p21^−/−</i> and <i>DDB1^F/F;Alb-Cre^+/−;p21^−/−</i> mice. (D) Co-IF staining for A6 and DDB1 on <i>DDB1^F/F;p21^−/−</i> and <i>DDB1^F/F;Alb-Cre^+/−;p21^−/−</i> liver sections. Arrows in <i>DDB1^F/F;Alb-Cre^+/−;p21^−/−</i> mice indicate A6 positive oval cells.</p

Expression of oval cell markers in DDB1 mutant mouse liver.

<p>(A) Co-IF staining for Cytokeratin-19 (CK19) and Albumin (Alb) (upper panels), E-cadherin and DDB1 (middle panels), α-fetoprotein (AFP) and CD133 (lower panels). (B–D) Co-IF staining for EpCAM and A6 on liver sections from 4-week old <i>DDB1^F/F;Alb-Cre^+/+</i> (B), 6-week old <i>DDB1^F/F;Alb-Cre^+/−</i> (C), and adult <i>DDB1^F/F;Mx1-Cre^+/−</i> mice at 6 weeks after receiving poly(I:C) injection (D).</p

Characterization of OCs from DDB1 mutant mice and DDC-treated mice.

<p>(<b>A–C</b>): Quantitative real-time PCR analysis for selected genes expressed in hepatocytes isolated from wild type mice, and EpCAM⁺ cells isolated from DDC-treated and <i>DDB1^F/F; Alb-Cre^+/+</i> mice. All data are normalized to <i>18s</i> rRNA level. Data are representative of 4 independent experiments with 3–4 mice per group. *P<0.05 (<b>A</b>): Expression levels of hematopoietic markers <i>Sca1</i>, <i>Thy1</i> and <i>Cd44</i>, and OC markers <i>CD133</i>, <i>Connexin43</i> and <i>Ncam1</i>. (<b>B</b>): Expression levels of cholangiocyte marker <i>Ck19</i> and <i>Spp1</i>, and hepatocyte marker <i>Alb</i>. (<b>C</b>): Expression levels of candidate OC markers, <i>Reelin</i>, <i>EdnrB</i> and <i>Cd206.</i> (<b>D</b>): IHC staining for reelin on liver sections from <i>DDB1^F/F</i>, <i>DDB1^F/F;Alb-Cre^+/+</i> and DDC-treated mice. Arrows in DDC-treated liver indicate non-specific brown deposits.</p