Journal: PLoS ONE

Article Title: E3 Ubiquitin Ligase Synoviolin Is Involved in Liver Fibrogenesis

doi: 10.1371/journal.pone.0013590

Figure Lengend Snippet: (A) The serum levels of alanine aminotransferase (ALT) were analyzed at 0, 3, 24, 48, and 72 h after CCl 4 administration in the acute hepatic injury model mice. Data are represented as mean ± SEM (n = 4–5 mice per group). Unpaired Student's t -test was used for statistical analysis. *** P <0.001, ** P <0.01. (B) Time-course expression of KLF6, synoviolin, Acta2 (α-SMA), and COL1A1 (collagen I) mRNA in livers inoculated with CCl 4 (black bar) or vehicle (white bar) quantified using real-time RT-PCR. Data are represented as mean ± SEM (n = 4–5 mice per group). The mRNA level was normalized relative to the amount of the transcript of 18S rRNA, a housekeeping gene. Unpaired Student's t -test was used for statistical analysis. *** P <0.001, ** P <0.01, * P <0.05. (C) Immunohistochemical analysis of liver sections of wt mice at 48 h after treatment with CCl 4 (n = 4) or vehicle (n = 5). The expression and localization of synoviolin (arrows, right panel) were analyzed using anti-synoviolin antibodies. Normal IgG antibody was used for the negative control (left panels). Scale bar = 100 µm. (D) Double-labeled fluorescent immunohistochemical analysis of liver sections of wt mice at 48 h after treatment with CCl 4 (n = 4). The nuclei were counterstained with DAPI (Vectashield). The expression and localization of synoviolin (green) and α-SMA (red)—a marker of activated HSCs, were analyzed using anti-synoviolin and α-SMA antibodies. Scale bar = 200 µm. (E) Double-labeled fluorescent immunohistochemical analysis of liver sections of human healthy liver (n = 30) and cirrhotic liver (n = 40) using human tissue array sections. The expression and localization of synoviolin (green label) α-SMA (red label) were analyzed using anti-synoviolin and α-SMA antibodies. Scale bar = 100 µm.

Article Snippet: Pathologically confirmed human liver tissue arrays were obtained from U.S. Biomax (Rockville, MD).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Negative Control, Labeling, Marker

Journal: Molecular Metabolism

Article Title: NIK/MAP3K14 in hepatocytes orchestrates NASH to hepatocellular carcinoma progression via JAK2/STAT5 inhibition

doi: 10.1016/j.molmet.2022.101626

Figure Lengend Snippet: NIK inhibits GH-induced STAT5 activation via JAK2S633 phosphorylation . A) Western blot analysis of pSTAT5, STAT5 and β-ACTIN levels in primary hepatocytes isolated from NIK fl/fl and NIK LKO mice after ex vivo stimulation with GH. B) Western blot analysis of pSTAT5, STAT5 and β-ACTIN levels in primary hepatocytes isolated from NIK fl/fl and NIK LKO mice after ex vivo double stimulation with GH and TWEAK and the respective quantification (n = 3). C) NIKTΔ3 construct used in ex vivo studies. After HTNC-mediated deletion of the loxP-flanked STOP cassette, NIKTΔ3 is expressed under control of the ROSA26 promoter. D) For Co-IP R26- NIKΔT3 fl and NIKΔT3 MEFs were treated with GH for 0 min or 15 min. Cell extracts were co-immunoprecipitated (IP) with an α -Flag antibody and immunoblotted with JAK2 (n = 3) and STAT5. E) Western blot analysis of pJAK2, JAK2 and β-ACTIN levels in liver tissue NASH-fed NIK fl/fl and NIK LKO mice (n = 3/3). F) Western blot analysis of pJAK2, JAK2 and β-ACTIN levels in liver tissue from CDAA-fed NIK fl/fl and NIK LKO mice (n = 3/3) G) Western blot analysis of pJAK2, JAK2 and β-ACTIN levels in non-tumor liver tissue from DEN-injected NIK fl/fl and NIK LKO mice (n = 3/3). H) JAK2 domain structure (human). Positive regulatory phosphorylation sites are labeled green. Negative inhibitory phosphorylation sites are marked in red. FERM: FERM domain, PK: pseudokinase, TK: tyrosine kinase. Single letter amino acid sequence of NIK binding motif in IKK1 and the predicted binding motif in JAK2. Box indicates conserved amino acids. I) Western blot analysis of pSTAT5, STAT5 and β-ACTIN levels in NIK fl/fl and NIK KO MEF transfected with either pCMV- JAK2wt or with pCMV- JAK2S633A and treated with GH for the indicated timepoints (n = 3). Data are presented as mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01, versus control. Statistical analyses were performed using Student's t -test.

Article Snippet: Human Commercial cDNA Array NIK mRNA levels were analyzed in a commercial cDNA array (origene, #LVRT101) prepared from hepatocarcinoma (Grade 1-3) and healthy human liver tissues.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Isolation, Ex Vivo, Construct, Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Injection, Labeling, Sequencing, Binding Assay, Transfection

Journal: Cells

Article Title: Combination Treatment with the Vimentin-Targeting Antibody hzVSF and Tenofovir Suppresses Woodchuck Hepatitis Virus Infection in Woodchucks

doi: 10.3390/cells10092321

Figure Lengend Snippet: The presence of vi-VIM is significantly increased in HBV-infected liver. ( a ) A human liver tissue array was stained with mVSF by IHC and the staining intensity and distribution of antibody-bound vi-VIM were scored on a 0–5 scale. ( b ) Comparison of the average scores between HBV-uninfected and -infected liver tissues. ( c ) Comparison of percentages of HBV-uninfected and -infected liver tissues with a score of ≥3.

Article Snippet: A human liver tissue array was purchased from US Biomax (Derwood, MD, USA; cat. no. LV1601) and subjected to immunohistochemistry (IHC) or immunofluorescence staining.

Techniques: Infection, Staining

Journal: Cells

Article Title: Combination Treatment with the Vimentin-Targeting Antibody hzVSF and Tenofovir Suppresses Woodchuck Hepatitis Virus Infection in Woodchucks

doi: 10.3390/cells10092321

Figure Lengend Snippet: The presence of vi-VIM is strongly increased in HBV-infected liver. A human liver tissue array was used for detecting changes in the presence of intracellular VIM (red color) and vi-VIM (green color) by immunofluorescence staining with D21H3 or mVSF antibodies, respectively. Merging of both stains (yellow color) indicated a perinuclear colocalization of intracellular VIM and vi-VIM in many hepatocytes. Hoechst 33,342 staining was used to detect cell nuclei (blue color). Representative pictures of two HBV-infected and two HBV-uninfected livers are shown. Abbreviation: NAT, nonalcoholic liver tumor.

Article Snippet: A human liver tissue array was purchased from US Biomax (Derwood, MD, USA; cat. no. LV1601) and subjected to immunohistochemistry (IHC) or immunofluorescence staining.

Techniques: Infection, Immunofluorescence, Staining