Fig 2: AdStath infection increases hepatocyte STMN1 expression in mice. (A) Levels of Stmn1 mRNA relative to uninjected control liver in mouse livers at the indicated days after AdGFP or AdStath infection (*P < 0.05, **P < 0.01, # P < 0.001, compared to AdGFP-infected mice at the same time point; n = 3-6). (B,C) Immunoblots of total hepatic protein from uninfected mice (day 0) and mice infected with AdGFP or AdStath for the days indicated and probed for STMN1 and ß-actin. (D) Immunofluorescence images of mouse livers infected with AdGFP or AdStath for 3 and 5 days stained for STMN1 (original magnification ×400). Protein bands and molecular weights in kilodaltons are indicated by arrows in (B,C). Abbreviation: C/Con, control.

Fig 3: STMN1 mRNA is a direct target of miR-770. a The three predicted targeting sequence of miR-770 of the STMN1 3' UTR. b Sequence alignment of the predicted miR-770 seed region in the 3' UTR of STMN1 from 5 organisms. c qRT-PCR and western blot assay revealed that overexpression of miR-770 level significantly reduced STMN1 expression in MDA-MB-468 and MDA-MB-231 cells. d The diagram illustrates the construction of the luciferase reporter plasmids. The substitutions of nucleotides within the three binding sites were shown and the three constructs named as mut1, mut2, and mut 3 separately (Wt: wild type; Mut: mutant type.). e Luciferase reporter assay in HEK293T cells revealed miR-770 suppressed STMN1 3' UTR luciferase activity of all wide-type constructs. Firefly luciferase activity was normalized to the internal Renilla luciferase activity. Data represent means ± S.D. of at least three independent experiments. *P < 0.05, **P < 0.01

Fig 4: 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 5: 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.