Figure 3. DNA damage is an early event of albNS^cko, followed by apoptosis and hepatic regeneration.

(A) Alb-Cre-driven NSKO (albNS^cko) leads to a sharp increase of γ-H2AX⁺ hepatocytes that peaks at 2 weeks and declines between 3 and 8 weeks of age. Y-axis shows the number of cells per 0.01mm² (mean ± SEM). (B) AlbNS^cko increases the number of apoptotic hepatocytes (green, TUNEL staining) at a later time point (3-4 weeks old). Y-axis represents cell number per 0.01mm². (C) At 2 weeks old, albNS^cko mice show a significant increase in their serum levels of AST and total bilirubin (T. Bil) compared to age-matched NS^flx/flx mice. The ALT levels are the same and the direct (conjugated) bilirubin levels are undetectable in both groups. ** and *** represent p values < 0.001 and 0.0001, respectively. (D1) At 2-3 weeks of age, small basophilic nodules begin to appear in the albNS^cko liver parenchyma (H&E). Most hepatocytes inside these nodules (demarcated by dash lines) express NS, remain mitotically active (BrdU-labeled and Ki67⁺), and are not fully differentiated, as indicated by their α-fetoprotein⁺ (AFP⁺) expression. The PAS⁻ staining pattern reveals loss of glycogen from the nodules. Inset shows an enlarged image of the NS signal. (D2) These newly generated hepatocytes within the nodules show basophilic cytoplasm and nuclear hyperchromasia. In contrast, the hepatocytes outside the nodules (peri-nodule) exhibit acidophilic cytoplasm, normal chromatin, and a single large nucleolus. (E) Surviving hepatocytes in 1-year-old albNS^cko livers display mild nuclear pleomorphism. (F) At 1 year, NS^flx/flx livers show scattered NS signals in a few hepatocytes but not in CK-19⁺ bile duct epithelial cells (BECs) (F1), whereas albNS^cko livers contain regions of mostly NS-negative or low hepatocytes (F2, left upper panel) and restricted areas of strong NS-positive hepatocytes intermixed with NS-negative or low cells (F2, bottom panel). At this age, BECs of albNS^cko livers still show NS-positive signals (F3, right upper panel). Bars, 50um in (A, B, D1), 20um in (D2), 100um in (E top panel), and 40um in (E bottom panel, F).