Journal: Cell Proliferation

Article Title: Deletion of Tgm2 suppresses BMP ‐mediated hepatocyte‐to‐cholangiocyte metaplasia in ductular reaction

doi: 10.1111/cpr.13646

Figure Lengend Snippet: The deletion of Tgm2 has a significant impact on the structure and function of proliferated cholangiocytes during DR. (A) Schematic diagram of WT and Tgm2 −/− mouse model after 4‐week DDC injury. (B) Plasma ALP, γ‐GT, ALT, and AST were measured in WT and Tgm2 −/− mice after 4‐week DDC injury ( n = 4/group). (C) H&E staining and Masson's staining in WT and Tgm2 −/− mice ( n = 4/group). Scale bar, 50 μM. Black arrow indicated porphyrin. (D) Quantification of the percentage of collagen‐positive staining areas in Masson's staining ( n = 4/group). (E) Hepatic expression levels of OPN and SSTR2 were determined in WT and Tgm2 −/− mice ( n ≥ 3/group). (F) Co‐staining of OPN and SSTR2 with the cholangiocyte marker CK19 and co‐staining of acTUB with the cholangiocyte marker CK7 were observed after 4‐week DDC injury ( n = 4/group). Scale bar, 20 μM. (G) Immunohistochemical staining of TGR5 was observed in WT and Tgm2 −/− mice after 4‐week DDC injury ( n = 4/group). Scale bar, 50 μM. Comparisons between two groups were performed using two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 represent four different levels of significant difference, respectively.

Article Snippet: In some experiments, the specific Tgm2 inhibitors, MDC (MCE, HY‐D1027, 30 or 50 μM) or rhBMP7 (PeproTech, 120‐03P, 200 ng/mL) or ZM39923 hydrochloride (MCE, HY‐12589, 50 or 100 μM), or a BMP signalling inhibitor DMH1 (MCE, HY‐12273, 100 μM) was added to the medium.

Techniques: Staining, Expressing, Marker, Immunohistochemical staining, Two Tailed Test

Journal: Cell Proliferation

Article Title: Deletion of Tgm2 suppresses BMP ‐mediated hepatocyte‐to‐cholangiocyte metaplasia in ductular reaction

doi: 10.1111/cpr.13646

Figure Lengend Snippet: Hepatocytes expressing Tgm2 play a crucial role in the regulation of DR through metaplasia. (A) Two‐week DDC liver injury led to an intermediate phenotype characterized by co‐expression of BEC marker OPN and hepatocyte marker HNF4α ( n = 4/group). White arrows denoted co‐stained cells. Scale bar, 20 μM. (B) The percentage of OPN + HNF4α + cells were determined in HNF4α + hepatocytes ( n = 4/group). (C) Co‐staining of the CK19, HNF4α, and Gs with the GFP lineage label was observed in Tgm2‐CreERT2 and Tgm2 CreERT2 ‐R26T/G f/f mouse livers ( n = 3/group). Scale bar, 10 μM. (D) Robust staining of the cholangiocyte marker CK19 and primary cilia marker acTUB was observed in GFP + cells after 4‐week DDC injury ( n = 3/group). White arrows denoted co‐stained cells. Scale bar, 20 μM. (E) The percentage of marker + cells' (i.e., cells that stained positive for CK19 or acTUB) fluorescence intensity was determined in GFP lineage label by fluorescence colocalization analysis ( n = 4/group). Comparisons between two groups were performed using two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 represent four different levels of significant difference, respectively.

Article Snippet: In some experiments, the specific Tgm2 inhibitors, MDC (MCE, HY‐D1027, 30 or 50 μM) or rhBMP7 (PeproTech, 120‐03P, 200 ng/mL) or ZM39923 hydrochloride (MCE, HY‐12589, 50 or 100 μM), or a BMP signalling inhibitor DMH1 (MCE, HY‐12273, 100 μM) was added to the medium.

Techniques: Expressing, Marker, Staining, Fluorescence, Two Tailed Test

Journal: Cell Proliferation

Article Title: Deletion of Tgm2 suppresses BMP ‐mediated hepatocyte‐to‐cholangiocyte metaplasia in ductular reaction

doi: 10.1111/cpr.13646

Figure Lengend Snippet: Tgm2 regulates H3K4me3Q5ser via serotonin, thus activating BMP signalling to regulate DR. (A) Heat map of BMP2, BMP6, BMP7, and BMP8a in WT and Tgm2 −/− mouse livers by RNA‐seq analysis ( n = 3/group). (B) Hepatic expression levels of BMP2, BMP6, and BMP7 were determined by RT‐qPCR ( n = 5/group). (C) Western blot assay of pSMAD1/5/8, SMAD1, H3K4me3Q5ser, and β‐actin in liver extracts from WT and Tgm2 −/− mice ( n = 3/group). (D) Quantification of pSMAD1/5/8 and H3K4me3Q5ser WB signals measured as integrated density using ImageJ ( n = 3/group). (E) Content of 5‐HT in Tgm2 −/− mouse after DDC‐2 W or DDC‐4 W injury ( n = 3 ≥ group). (F) ChIP experiments on WT and Tgm2 −/− mouse liver tissues ( n = 3/group). Comparisons between two groups were performed using two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 represent four different levels of significant difference, respectively.

Article Snippet: In some experiments, the specific Tgm2 inhibitors, MDC (MCE, HY‐D1027, 30 or 50 μM) or rhBMP7 (PeproTech, 120‐03P, 200 ng/mL) or ZM39923 hydrochloride (MCE, HY‐12589, 50 or 100 μM), or a BMP signalling inhibitor DMH1 (MCE, HY‐12273, 100 μM) was added to the medium.

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Cell Proliferation

Article Title: Deletion of Tgm2 suppresses BMP ‐mediated hepatocyte‐to‐cholangiocyte metaplasia in ductular reaction

doi: 10.1111/cpr.13646

Figure Lengend Snippet: Activation of BMP signalling promotes the development and maturation of BECs to alleviate cholestasis in Tgm2 −/− mice. (A) Schematic diagram of the use of rhBMP7 in Tgm2 −/− mouse after 4‐week DDC injury. (B) Western blot assay of pSMAD1/5/8, SMAD1, BMP7, and β‐actin in liver extracts from WT and Tgm2 −/− mice ( n = 3/group). (C) Quantification of pSMAD1/5/8 and BMP7 WB signals measured as integrated density using ImageJ ( n = 3/group). (D) Co‐staining of CK19 and OPN or SSTR2 and co‐staining of CK7 and acTUB in mouse liver samples ( n = 4/group). Scale bar, 20 μM. (E) Immunohistochemical staining of TGR5 was observed in WT, Tgm2 −/− , and Tgm2 −/− + rhBMP7 groups after 4‐week DDC injury ( n = 4/group). Scale bar, 50 μM. (F) Plasma ALP, γ‐GT, TBIL, and ALT levels were measured in WT and Tgm2 −/− mice ( n ≥ 4/group). Comparisons between multiple groups were performed using ordinary one‐way ANOVA with Dunnett's multiple comparison test. Significant difference was presented at the levels of * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: In some experiments, the specific Tgm2 inhibitors, MDC (MCE, HY‐D1027, 30 or 50 μM) or rhBMP7 (PeproTech, 120‐03P, 200 ng/mL) or ZM39923 hydrochloride (MCE, HY‐12589, 50 or 100 μM), or a BMP signalling inhibitor DMH1 (MCE, HY‐12273, 100 μM) was added to the medium.

Techniques: Activation Assay, Western Blot, Staining, Immunohistochemical staining, Comparison

Journal: Cell Proliferation

Article Title: Deletion of Tgm2 suppresses BMP ‐mediated hepatocyte‐to‐cholangiocyte metaplasia in ductular reaction

doi: 10.1111/cpr.13646

Figure Lengend Snippet: Inhibition of BMP signalling suppresses the metaplasia of hepatocytes, thereby affecting the development and maturation of BECs in DR. (A) Schematic diagram of hepatocyte fate tracing after 4‐week DDC diet and DMH1 injection in Tgm2 CreERT2 ‐R26T/G f/f mice. (B) Hepatic expression levels of ID1, OPN, and SSTR2 in Tgm2 CreERT2 ‐R26T/G f/f mouse liver ( n ≥ 4/group). (C) ALT and TBIL levels were measured in Tgm2 CreERT2 ‐R26T/G f/f mice ( n ≥ 3/group). (D) Immunofluorescence co‐staining of CK19, acTUB, and SSTR2 with the GFP lineage label was observed after 4‐week DDC treatment, but lesser co‐stained cells were observed after DMH1 injection ( n = 4/group). White arrows indicated co‐stained cells. Scale bar, 20 μM. (E) The percentage of marker + cells' (i.e., cells that stained positive for CK19 or SSTR2 or acTUB) fluorescence intensity was determined in GFP lineage label by fluorescence colocalization analysis ( n = 4/group). Comparisons between multiple groups were performed using ordinary one‐way ANOVA with Dunnett's multiple comparison test. Significant difference was presented at the levels of * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: In some experiments, the specific Tgm2 inhibitors, MDC (MCE, HY‐D1027, 30 or 50 μM) or rhBMP7 (PeproTech, 120‐03P, 200 ng/mL) or ZM39923 hydrochloride (MCE, HY‐12589, 50 or 100 μM), or a BMP signalling inhibitor DMH1 (MCE, HY‐12273, 100 μM) was added to the medium.

Techniques: Inhibition, Injection, Expressing, Immunofluorescence, Staining, Marker, Fluorescence, Comparison

Journal: Cell Proliferation

Article Title: Deletion of Tgm2 suppresses BMP ‐mediated hepatocyte‐to‐cholangiocyte metaplasia in ductular reaction

doi: 10.1111/cpr.13646

Figure Lengend Snippet: Inhibition of Tgm2 represses the transdifferentiation of hepatocytes, thus affecting the development and function of BECs in vitro. (A) Biliary transdifferentiation of adult hepatocytes in 3D culture. (B) Phase‐contrast micrographs of hepatocytic spheroids after being embedded in a collagen gel. The WT or Tgm2 −/− hepatocytes were cultured for 21 days in the absence or presence of rhBMP7 (200 ng/mL) ( n = 3/group). Scale bar, 50 μM. (C) RT‐qPCR analysis was conducted to measure the mRNA expression of CK19, OPN, and HNF4α in the following groups: hepatocyte, spheroids, WT, Tgm2 −/− , and Tgm2 −/− + rhBMP7 ( n = 3/group). (D) H&E staining and immunohistochemical staining of CK7 and Sox9 showed ductular structures produced by hepatocytic spheroids after 21‐day collagen gel culture in the absence or presence of 30 μM MDC ( n = 3/group). Scale bar, 20 μM. (E) Images of uptake and excretion of FDA by ductular structures after 28‐day hepatocytic spheroid collagen gel culture ( n = 3/group). Scale bar, 20 μM. Comparisons between multiple groups were performed using ordinary one‐way ANOVA with Tukey's multiple comparison test. Significant difference was presented at the levels of * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: In some experiments, the specific Tgm2 inhibitors, MDC (MCE, HY‐D1027, 30 or 50 μM) or rhBMP7 (PeproTech, 120‐03P, 200 ng/mL) or ZM39923 hydrochloride (MCE, HY‐12589, 50 or 100 μM), or a BMP signalling inhibitor DMH1 (MCE, HY‐12273, 100 μM) was added to the medium.

Techniques: Inhibition, In Vitro, Cell Culture, Quantitative RT-PCR, Expressing, Staining, Immunohistochemical staining, Produced, Comparison