Fig 2: STMN1 expression increases hepatocyte proliferation. (A) Images of BrdU-stained cells from AdGFP-infected (shown as GFP) and AdStath-infected (shown as Stath) mice at the indicated days after infection (magnification ×200). (B) Numbers of BrdU-positive cells per HPF (200×) in uninjected control mice and mice at the indicated days after AdGFP or AdStath injection (*P < 0.04, **P < 0.001, compared to AdGFP-infected mice at the same time point; n = 4-8). (C) Numbers of mitoses per HPF (400×) by histology in the same mice (*P < 0.03, **P < 0.002, compared to AdGFP-infected mice at the same time point; n = 3-6). (D) Representative hematoxylin and eosin images of adenovirus-infected livers (arrows indicate mitotic hepatocytes; scale bars, 50 µm). (E) Immunoblots of liver nuclear protein from an uninjected liver (day 0) and adenovirus-injected livers at the indicated days. Lamin A/C is a loading control. Arrows indicate the protein molecular weights in kilodaltons. Abbreviations: adv, adenovirus; C/Con, control; D, days; HPF, high-power field.

Fig 3: Hepatic STMN1 mRNA and protein expression are increased in mouse and human liver injury. (A) Hepatic Stmn1 mRNA levels relative to uninjected control mice in mice treated for the indicated number of hours with CCl4 by qRT‐PCR (*P < 0.01, **P < 0.0002, compared to control; n = 3‐6). (B) Immunoblots of total hepatic protein from an untreated control mouse and CCl4‐treated mice probed for total STMN1, Ser‐16‐ and Ser‐38‐phospho‐STMN1, and the loading control tubulin. (C) Relative levels of STMN1 mRNA in normal human livers and livers from patients with fulminant hepatic failure (*P < 0.007, compared to normal; n = 4). (D) Total human liver protein from the same livers immunoblotted for total and phosphorylated STMN1, Fos‐related antigen 1, and β‐actin as a loading control. Arrows in (B) and (D) indicate the respective protein bands and molecular weights in kilodaltons. Abbreviations: Con, control; FHF, fulminant hepatic failure; FRA1, Fos‐related antigen 1; h, hours; NL, normal liver; P16‐stathmin, serine‐16 phosphorylated stathmin 1; P38‐stathmin, serine‐38 phosphorylated stathmin 1.

Fig 4: Hepatocyte apoptosis and liver injury occur after hepatocyte proliferation. (A) Representative hematoxylin and eosin images of adenovirus-infected livers at day 5 400×; (black arrows indicate apoptotic hepatocytes, white arrow an area of inflammation). (B) Numbers of apoptotic cells per HPF (400×) by histology in uninjected control mice and AdGFP- or AdStath-injected mice at the indicated days (*P < 0.05, **P < 0.01, compared to AdGFP-infected mice at the same time; n = 3-4). (C) Immunoblots of total hepatic protein from uninfected mice (day 0) and mice infected with AdGFP or AdStath for the days indicated probed for caspase 3, caspase 7, and tubulin. Arrows indicate the procaspase and cleaved active forms. (D) Serum ALT levels in the same mice (*P < 0.01, **P < 0.001, compared to AdGFP-infected mice at the same time; n = 4-7). (E) Inflammation grade in the livers (*P < 0.05, **P < 0.01, compared to AdGFP-infected mice at the same time; n = 3-4). Abbreviations: C/Con, control; Casp, caspase; HFP, high-power field; IU, international unit; p17, cleaved form of caspase 3; p19, cleaved form of caspase 7; Pro, procaspase.

Fig 5: Adenoviral STMN1 expression increases liver mass. (A) Gross images of representative mouse livers 9 days after injection with the adenovirus AdGFP or AdStath. Scale bar, 1 cm. (B) Liver weights and (C) liver to body weight ratios in uninjected control mice and mice at the indicated days after injection with AdGFP or AdStath (*P < 0.01, **P < 0.001, compared to AdGFP-infected mice at the same time point; n = 4-8). (D) Liver to body weight ratios at later times (*P < 0.01 compared to AdGFP-infected mice at the same time point; n = 4). (E) Relative hepatocyte cell size in adenovirus-infected cells (*P < 0.000001 compared to AdGFP-infected mice at the same time point; n = 340-890). Abbreviation: C/Con, control.