Journal: Biophysics Reports

Article Title: Establishment of a Fah-LSL mouse model to study BEC-to-hepatocyte conversion

doi: 10.52601/bpr.2023.230034

Figure Lengend Snippet: Strategy of crossing mice for the purpose of experiment. First, Fah-LSL homozygous mice was constructed. Fah-LSL/LSL mice and Fah-LSL/LSL;ACTB-Cre mice were generated through mating. By examing the Fah expression and phenotypes of the mice, the liver injury mouce model was validated. Next, to trace BECs in the liver injury model, CK19-CreER;Fah-LSL/LSL;R26-tdT mice were generated, and their tdT was induced and Fah was restored in BECs after tamoxifen-induced Cre-loxp recombination. Lastly, a Cre-loxp and Dre-rox based dual genetic lineage tracing approach to idelibly label CK19+ HNF4α+ TLPCs was developed to further characterize BEC-derived TLPCs.

Article Snippet: • Weigert's iron hematoxylin kit (Sigma-Aldrich, cat. no. 115973) • Immunostaining antibodies • DAPI (Invitrogen, cat. no. D21490) • tdT (Rockland, cat. no. 600-401-379, 1:500) • p21 (Abcam, cat. no. ab188224; 1:500) • Ki67 (Abcam, cat. no. ab15580; 1:200) • CK19 (Developmental Studies Hybridoma Bank, cat. no. TROMA-III, 1:500) • HNF4α (Cell Signalling, cat. no. 3113s; 1:500) • FAH (Abclonal, cat. no. A13492; 1:500) • GS (Abcam, cat. no. Ab49873; 1:1,000) • E-cadherin (E-cad, cat. no.

Techniques: Construct, Generated, Expressing, Derivative Assay

Journal: Biophysics Reports

Article Title: Establishment of a Fah-LSL mouse model to study BEC-to-hepatocyte conversion

doi: 10.52601/bpr.2023.230034

Figure Lengend Snippet: BEC-derived hepatocytes contribute to liver regeneration. A Schematic diagram showing the experimental design for recovery of Fah gene in BECs and lineage tracing. B Schematic diagram showing the experimental strategy of lineage tracing in CK19-CreER;Fah-LSL/LSL;R26-tdTmice. C Immunostaining for tdT, CK19, and HNF4α on liver sections collected at day 0. Scale bar, 1 mm. Right panel shows quantification of cell labeling efficiency. Data represent mean ± SD; n = 5 mice. D Whole-mount fluorescence liver images. Scale bars, 1 mm. E Immunostaining for tdT on liver sections from mice treated with TAM or oil. Scale bars, 1 mm. F Immunostaining for tdT, HNF4α, and CK19 on the liver sections from mice treated with TAM or oil. Quantification of percentage of tdT+ BECs and tdT+ Heps is shown in the adjacent graph. Data represent mean ± SD; n = 5 mice. Scale bars, 100 µm. G Immunostaining for tdT and FAH on the liver sections from mice treated with TAM or oil. Scale bars, 100 µm. H Serum ALT and AST of mice treated with TAM or oil. Data are the mean ± SD; n = 5 mice. * P < 0.0001; * P < 0.0001. I , J Immunostaining for tdT, HNF4α and p21 ( I ) or Ki67 ( J ) on liver sections. Inserts show green and red fluorescence channels. Quantification of Ki67 or p21 staining in tdT+ and tdT– hepatocytes is shown in the adjacent graph. Data represent mean ± SD; n = 5 mice. * P < 0.0001; Statistical analysis in Panels H and J was performed by two-tailed unpaired Student’s t -test. K Immunostaining for tdT with E-CAD or CYP2E1 on the liver sections from mice treated with TAM or Oil. Scale bars, 100 µm

Article Snippet: • Weigert's iron hematoxylin kit (Sigma-Aldrich, cat. no. 115973) • Immunostaining antibodies • DAPI (Invitrogen, cat. no. D21490) • tdT (Rockland, cat. no. 600-401-379, 1:500) • p21 (Abcam, cat. no. ab188224; 1:500) • Ki67 (Abcam, cat. no. ab15580; 1:200) • CK19 (Developmental Studies Hybridoma Bank, cat. no. TROMA-III, 1:500) • HNF4α (Cell Signalling, cat. no. 3113s; 1:500) • FAH (Abclonal, cat. no. A13492; 1:500) • GS (Abcam, cat. no. Ab49873; 1:1,000) • E-cadherin (E-cad, cat. no.

Techniques: Derivative Assay, Immunostaining, Labeling, Fluorescence, Staining, Two Tailed Test

Journal: Biophysics Reports

Article Title: Establishment of a Fah-LSL mouse model to study BEC-to-hepatocyte conversion

doi: 10.52601/bpr.2023.230034

Figure Lengend Snippet: Biopotent TLPCs generate hepatocytes or adopt BEC fate during liver repair. A Schematic showing the strategy for TLPC lineage tracing. B Schematic showing experimental strategy. C Immunostaining for tdT, HNF4α and CK19 on liver sections collected at day 25 from CK19-CreER;HNF4α-DreER;Fah-LSL/LSL;R26-RL-tdT mice. Scale bars, 50 µm. Right panel shows the percentage of BECs, TLPCs and hepatocytes in tdT+ cells. Data represent mean ± SD; n = 5 mice; total 289 tdT+ cells were counted (BECs: 25; TLPCs: 258; hepatocyte: 6). D Percentage of Ki67+ cells in tdT+ TLPCs. Data represent mean ± SD; n = 5 mice. E Whole-mount tdT fluorescent liver images from indicated mice at 7 weeks after the first NTBC removal. Bright field images are shown as inserts. Arrows indicate tdT+ clones. Scale bars, 1 mm. F Immunostaining for tdT, HNF4α and CK19 on serial sections (20 µm) of livers collected at week 7 from CK19-CreER;HNF4α-DreER;Fah-LSL/LSL;R26-RL-tdT mice. Scale bars, 50 µm. G Immunostaining of tdT, and CK19 on serial liver sections (20 µm) collected at week 7 from CK19-CreER;HNF4α-DreER;Fah-LSL/LSL;R26-RL-tdT mice. Scale bars, 50 µm. White arrows indicate tdT+ BECs. H Percentage of tdT+ clones containing BECs, TLPCs and hepatocytes. Schematic on the right panel showing that TLPCs could give rise to hepatocytes and revert back to BECs. Data represent mean ± SD; n = 5 mice; total 480 tdT+ clones were counted. I Percentage of hybrid clone that consists of BECs and hepatocytes. Schematic showing that TLPCs could not give rise to hepatocytes and BEC simultaneously. Data represent mean ± SD; n = 5 mice

Article Snippet: • Weigert's iron hematoxylin kit (Sigma-Aldrich, cat. no. 115973) • Immunostaining antibodies • DAPI (Invitrogen, cat. no. D21490) • tdT (Rockland, cat. no. 600-401-379, 1:500) • p21 (Abcam, cat. no. ab188224; 1:500) • Ki67 (Abcam, cat. no. ab15580; 1:200) • CK19 (Developmental Studies Hybridoma Bank, cat. no. TROMA-III, 1:500) • HNF4α (Cell Signalling, cat. no. 3113s; 1:500) • FAH (Abclonal, cat. no. A13492; 1:500) • GS (Abcam, cat. no. Ab49873; 1:1,000) • E-cadherin (E-cad, cat. no.

Techniques: Immunostaining, Clone Assay

Journal: Dose-Response

Article Title: TSN Disrupts Fanconi Anemia Pathway Activation Through JAK/STAT1-Mediated Transcriptional Repression of FA Core Subunits in Bladder Cancer

doi: 10.1177/15593258251406039

Figure Lengend Snippet: Toosendanin down-regulates FA subunits. (A-C) Western blot shows the expression of FANCL, FANCB, FAAP100, FANCA, FANCC, FANCF, FANCG, FANCM and FAAP24 in bladder cancer T24 (A), RT4 (B) and J82 (C) cell lines induced by UVC and treated with TSN. (D-F) Histogram shows the RNA levels of FANCL, FANCB, FAAP100, FANCA, FANCC, FANCF, FANCG, FANCM and FAAP24 in bladder cancer T24 (D), RT4 (E) and J82 (F) cell lines induced by UVC and treated with TSN by qPCR. Numerical P -values indicate the statistical significance compared with cells treated with UVC

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against FANCI (20789-1-AP, Proteintech, China, 1: 2000), FANCD2 (NB100-316, Novus Biologicals, USA, 1: 2000), β-Actin (AC026, ABclonal, China, 1: 2000), FANCL (A6812, ABclonal, 1: 2000), FANCB (A2435, ABclonal, 1: 2000), FAAP100 (A8591, ABclonal, 1: 2000), FANCA (A9529, ABclonal, 1: 2000), FANCC (A1812, ABclonal, 1: 2000), FANCF (18060-1-AP, Proteintech, 1: 2000), FANCG (A6206, ABclonal, 1: 2000), FANCM (A7602, ABclonal, 1: 2000), FAAP24 (26431-1-AP, Proteintech, 1: 2000), p-SMAD2 (80427-2-RR, Proteintech, 1: 2000), SMAD2 (67343-1-Ig, Proteintech, 1: 2000), p-STAT1 (82674-10-RR, Proteintech, 1: 2000), STAT1 (66545-1-Ig, Proteintech, 1: 2000), p-ERK1/2 (80031-1-RR, Proteintech, 1: 2000), ERK1/2 (11257-1-AP, Proteintech, 1: 2000).

Techniques: Western Blot, Expressing

Journal: Dose-Response

Article Title: TSN Disrupts Fanconi Anemia Pathway Activation Through JAK/STAT1-Mediated Transcriptional Repression of FA Core Subunits in Bladder Cancer

doi: 10.1177/15593258251406039

Figure Lengend Snippet: Toosendanin inhibits JAK/STAT1 pathway. (A-C) Western blot shows the phosphorylation of SMAD2, STAT1 and ERK in bladder cancer T24 (A), RT4 (B) and J82 (C) cell lines induced by UVC and treated with TSN. (D-F) Immunofluorescence shows the nuclear translocation of p-SMAD2 (D), p-STAT1 (E) and p-ERK (F) in bladder cancer T24 cells induced by UVC and treated with TSN. (G-I) Western blot shows the phosphorylation of STAT1 and the expression of FANCA, FANCC, FANCF and FANCM in bladder cancer T24 (G), RT4 (H) and J82 (I) cell lines induced by UVC and treated with TSN, JAK/STAT1 agonist or inhibitor. (J-L) Histogram shows the RNA levels of FANCA, FANCC, FANCF and FANCM in bladder cancer T24 (J), RT4 (K) and J82 (L) cell lines induced by UVC and treated with TSN as well as JAK/STAT1 agonist and inhibitor by qPCR. (M-O) ChIP-qPCR shows the binding capacity of STAT1 on promoters of FANCA, FANCC, FANCF and FANCM in bladder cancer T24 (M), RT4 (N) and J82 (O) cell lines induced by UVC and treated with TSN and JAK/STAT1 agonist. Numerical P -values indicate the statistical significance compared with cells treated with UVC and TSN. Abbreviation: JI, JAK/STAT1 inhibitor Ruxolitinib; JA, JAK/STAT1 agonist IFN-γ

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against FANCI (20789-1-AP, Proteintech, China, 1: 2000), FANCD2 (NB100-316, Novus Biologicals, USA, 1: 2000), β-Actin (AC026, ABclonal, China, 1: 2000), FANCL (A6812, ABclonal, 1: 2000), FANCB (A2435, ABclonal, 1: 2000), FAAP100 (A8591, ABclonal, 1: 2000), FANCA (A9529, ABclonal, 1: 2000), FANCC (A1812, ABclonal, 1: 2000), FANCF (18060-1-AP, Proteintech, 1: 2000), FANCG (A6206, ABclonal, 1: 2000), FANCM (A7602, ABclonal, 1: 2000), FAAP24 (26431-1-AP, Proteintech, 1: 2000), p-SMAD2 (80427-2-RR, Proteintech, 1: 2000), SMAD2 (67343-1-Ig, Proteintech, 1: 2000), p-STAT1 (82674-10-RR, Proteintech, 1: 2000), STAT1 (66545-1-Ig, Proteintech, 1: 2000), p-ERK1/2 (80031-1-RR, Proteintech, 1: 2000), ERK1/2 (11257-1-AP, Proteintech, 1: 2000).

Techniques: Western Blot, Phospho-proteomics, Immunofluorescence, Translocation Assay, Expressing, ChIP-qPCR, Binding Assay

Journal: Dose-Response

Article Title: TSN Disrupts Fanconi Anemia Pathway Activation Through JAK/STAT1-Mediated Transcriptional Repression of FA Core Subunits in Bladder Cancer

doi: 10.1177/15593258251406039

Figure Lengend Snippet: Toosendanin enhances the cisplatin-induced cytotoxic effect on bladder cells in vivo. (A) Comparison of tumor sizes among groups of regular T24 cells, T24 cells treated with cisplatin, as well as T24 cells treated with cisplatin and TSN (top). One-way ANOVA shows the significant differences compared to NC and UV indicated by numerical P -values (bottom). (B) Western blot shows the expression of FANCI, FANCD2, FANCA, FANCC, FANCF and FANCM as well as the phosphorylation of STAT1 in xenograft tumor of regular T24 cells, T24 cells treated with cisplatin, as well as T24 cells treated with cisplatin and TSN. Abbreviation: Cis, cisplatin; T, TSN; mub, mono-ubiquitination; Tyr701, 701 st tyrosine phosphorylation

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against FANCI (20789-1-AP, Proteintech, China, 1: 2000), FANCD2 (NB100-316, Novus Biologicals, USA, 1: 2000), β-Actin (AC026, ABclonal, China, 1: 2000), FANCL (A6812, ABclonal, 1: 2000), FANCB (A2435, ABclonal, 1: 2000), FAAP100 (A8591, ABclonal, 1: 2000), FANCA (A9529, ABclonal, 1: 2000), FANCC (A1812, ABclonal, 1: 2000), FANCF (18060-1-AP, Proteintech, 1: 2000), FANCG (A6206, ABclonal, 1: 2000), FANCM (A7602, ABclonal, 1: 2000), FAAP24 (26431-1-AP, Proteintech, 1: 2000), p-SMAD2 (80427-2-RR, Proteintech, 1: 2000), SMAD2 (67343-1-Ig, Proteintech, 1: 2000), p-STAT1 (82674-10-RR, Proteintech, 1: 2000), STAT1 (66545-1-Ig, Proteintech, 1: 2000), p-ERK1/2 (80031-1-RR, Proteintech, 1: 2000), ERK1/2 (11257-1-AP, Proteintech, 1: 2000).

Techniques: In Vivo, Comparison, Western Blot, Expressing, Phospho-proteomics, Ubiquitin Proteomics

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Urinary proteome analysis of acute hypercoagulable state in rat model induced by ε-aminocaproic acid.

doi: 10.1016/j.biopha.2018.11.148

Figure Lengend Snippet: Fig. 8. Western blot analysis of the differentially expressed urinary proteins fumarylacetoacetase (FAH) and albumin (ALB). C-#1, C-#2, and C-#3, re- present 3 independent biological replicates originated from normal group with intragastric administration of normal saline; Ex-#1, Ex-#2, and Ex-#3, three independent biological replicates originated from experimental group with in- tragastric administration of EACA.

Article Snippet: After blocking in 5% (w/v) skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies for fumarylacetoacetase (A02072, Boster, Pleasanton, USA) and albumin (ab106582, Abcam, Cambridge, UK) overnight at 4 °C.

Techniques: Western Blot, Saline