Journal: Oncogene

Article Title: Identification of low-frequency variants of UGT1A3 associated with bladder cancer risk by next-generation sequencing

doi: 10.1038/s41388-021-01672-1

Figure Lengend Snippet: The UGT1A3 expression plasmid was transfected into UGT1A3-KO cells (UGT-KO), which were designated UGT-KO + UGT over, and then the LAMC2 knockdown plasmid was transfected into UGT-KO + UGT over cells, which were designated UGT-KO + UGT over+ si-LAM. A Differential expression levels of genes between UGT-KO and NC cell lines are displayed by a heat map. B Volcano plots of differentially expressed gene expression. The green or red plots are the DEGs (fold change >4 and Padj < 0.05). C DEGs were analyzed by KEGG pathway enrichment analysis. D The expression of LAMC2 at the mRNA and protein levels was detected by qRT-PCR and Western blot in the UGT-KO and NC cell lines. E The expression of LAMC2 at the mRNA and protein levels was detected by qRT-PCR and Western blot in the UGT-KO, UGT-KO + UGT over and UGT-KO + UGT over+ si-LAM cell lines. F Cell viability was detected using a CCK-8 assay (OD450 absorbance). G The colony formation ability was measured using a colony formation assay. The colonies were counted and captured. H Representative images of invasion (upper) and migration assays (lower). The number of cells was counted. Scale bar, 180 μm. I The cell cycle was measured by flow cytometry. *P < 0.05 according to Student’s t test.

Article Snippet: H&E staining was used to select representative areas, and IHC was applied to confirm the expression of the proliferation markers Ki67 (anti-Ki67, ab15580, Abcam), AR (anti-AR, 22089-1-AP, Proteintech), and LAMC2 (anti-LAMC2, USB-144830, US Biological).

Techniques: Expressing, Plasmid Preparation, Transfection, Knockdown, Quantitative Proteomics, Gene Expression, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Colony Assay, Migration, Flow Cytometry

Journal: Oncogene

Article Title: Identification of low-frequency variants of UGT1A3 associated with bladder cancer risk by next-generation sequencing

doi: 10.1038/s41388-021-01672-1

Figure Lengend Snippet: UGT1A3 knockout (UGT1A3-KO) or negative control (NC) cell lines were injected into humanized NCG mice. A The eight burdened NCG mice in the UGT1A3-KO and NC groups. Red arrows show the position of tumors. B The harvested tumor tissues in the UGT1A3-KO and NC groups. The mean weight of excised tumor tissues (C) and the average volume of xenografts (D) were detected in the UGT1A3-KO and NC groups. The expression of UGT1A3 (E), and LAMC2 (F) in tumors was detected using qRT-PCR. G Images of H&E staining and IHC staining for Ki67, AR, and LAMC2 expression in tumor tissues. Scale bar, 120 μm. H The graphic illustration of our findings. *P < 0.05 according to Student’s t test.

Article Snippet: H&E staining was used to select representative areas, and IHC was applied to confirm the expression of the proliferation markers Ki67 (anti-Ki67, ab15580, Abcam), AR (anti-AR, 22089-1-AP, Proteintech), and LAMC2 (anti-LAMC2, USB-144830, US Biological).

Techniques: Knock-Out, Negative Control, Injection, Expressing, Quantitative RT-PCR, Staining, Immunohistochemistry

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: LAMC2 is significantly overexpressed in the liver tissues of cirrhotic mice. (A) Heatmap of differentially expressed genes in the GSE77627 dataset. (B) Heatmap of differentially expressed genes in the GSE33258 dataset. (C) The intersection of differentially expressed genes in the two GEO datasets and hub genes related to liver cirrhosis in the GeneCards database. (D) KEGG enrichment analysis of 373 intersecting genes. The expression of the profibrotic genes ACTA2 (E), TIMP1 (F), and PDGFRA (G) in the liver tissues of mice treated with DMSO (normal) or CCl4 (model), as examined by RT‐qPCR ( n = 3). (H–J) Western blot analysis and relative protein expression levels of LRAT and GFAP in the liver tissues of mice ( n = 3). (K) Analysis of fibrosis in the mouse liver lobule by HE staining ( n = 5). (L, M) Analysis of fibrosis in the mouse liver lobule by Masson's trichrome staining ( n = 5). (N, O) The LAMC2‐positive area in mouse liver samples was assessed by IHC ( n = 5). (P) LAMC2 mRNA expression in mouse liver samples was assessed by RT‐qPCR ( n = 3). Graphical data are presented as means ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. An unpaired t ‐test was used for statistical analysis. DMSO, dimethyl sulfoxide; IHC, immunohistochemistry; LAMC2, laminin subunit gamma 2; PDGFRA, platelet‐derived growth factor receptor alpha; RT‐qPCR, reverse transcription quantitative PCR; TIMP1, TIMP metallopeptidase inhibitor 1.

Article Snippet: Tissues were incubated with antibodies against LAMC2 (1:100, PK08311, Abmart Shanghai Co., Ltd.), α‐smooth muscle actin (α‐SMA, 1:500, GTX100034, GeneTex, Inc.), and collagen I (1:100, GTX20292, GeneTex) overnight at 4°C and with goat anti‐rabbit IgG (1:2500, ab97051, Abcam) at 37°C for 1 h. Then, immunoassays were performed using a DAB horseradish peroxidase color development kit (P0202, Beyotime Biotechnology Co., Ltd.).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: Poor expression of LAMC2 inhibits liver fibrosis and suppresses HSC activation in vivo. (A) Analysis of fibrosis in mouse liver tissues by HE staining ( n = 5). (B, C) Analysis of fibrosis in mouse liver tissues by Masson's trichrome staining ( n = 5). (D–F) The collagen I‐ and α‐SMA‐positive areas in mouse liver tissues were identified by IHC staining ( n = 5). (G) The mRNA expression of LAMC2 in the liver tissues of mice injected with sh‐NC or sh‐LAMC2 was examined via RT‐qPCR ( n = 3). The levels of the inflammatory factors TNF‐α (H), IL‐1β (I), and IL‐6 (J) in serum were measured using ELISAs ( n = 5). (K‒M) The protein expression of LRAT and GFAP, which are markers of HSC activation, in mouse liver tissues was analyzed by Western blot ( n = 3). Graphical data are presented as means ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. An unpaired t ‐test was used for statistical analysis. ELISAs, enzyme‐linked immunosorbent assays; HSC, hepatic stellate cell; IHC, immunohistochemistry; LAMC2, laminin subunit gamma 2; RT‐qPCR, reverse transcription quantitative PCR.

Article Snippet: Tissues were incubated with antibodies against LAMC2 (1:100, PK08311, Abmart Shanghai Co., Ltd.), α‐smooth muscle actin (α‐SMA, 1:500, GTX100034, GeneTex, Inc.), and collagen I (1:100, GTX20292, GeneTex) overnight at 4°C and with goat anti‐rabbit IgG (1:2500, ab97051, Abcam) at 37°C for 1 h. Then, immunoassays were performed using a DAB horseradish peroxidase color development kit (P0202, Beyotime Biotechnology Co., Ltd.).

Techniques: Expressing, Activation Assay, In Vivo, Staining, Immunohistochemistry, Injection, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: PI3K/Akt signaling activation exacerbates liver fibrosis in mice with sh‐LAMC2. (A–C) The total protein content and phosphorylation of PI3K and Akt in the liver tissues of normal and model mice and mice treated with sh‐NC or sh‐LAMC2 were examined using Western blot assays ( n = 3). (D–H) The extent of PI3K and Akt phosphorylation and the protein expression of LRAT and GFAP in the liver tissues of mice treated with sh‐LAMC2 + DMSO or sh‐LAMC2 + recilisib were examined using Western blot assays ( n = 3). (I) Analysis of fibrosis in mouse liver tissues via HE staining ( n = 5). (J–L) The collagen I‐ and α‐SMA‐positive areas in mouse liver tissues were identified by IHC staining ( n = 5). (M, N) Fibrosis in mouse liver tissues was analyzed via Masson's trichrome staining ( n = 5). The levels of the inflammatory factors TNF‐α (O), IL‐1β (P), and IL‐6 (Q) in serum were measured using ELISAs ( n = 5). Graphical data are presented as means ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. An unpaired t ‐test or one‐way ANOVA was used for statistical analysis. ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; ELISAs, enzyme‐linked immunosorbent assays; IHC, immunohistochemistry.

Article Snippet: Tissues were incubated with antibodies against LAMC2 (1:100, PK08311, Abmart Shanghai Co., Ltd.), α‐smooth muscle actin (α‐SMA, 1:500, GTX100034, GeneTex, Inc.), and collagen I (1:100, GTX20292, GeneTex) overnight at 4°C and with goat anti‐rabbit IgG (1:2500, ab97051, Abcam) at 37°C for 1 h. Then, immunoassays were performed using a DAB horseradish peroxidase color development kit (P0202, Beyotime Biotechnology Co., Ltd.).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Expressing, Staining, Immunohistochemistry

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: The transcription factor SMC1A promotes LAMC2 expression to induce HSC activation in vitro. (A) The transcription factors regulating LAMC2 in the hTFtarget database were intersected with differentially expressed genes in the two GEO datasets. (B) The binding peaks of SMC1A in the promoter region of LAMC2 were analyzed using the ChIP‐seq database. (C, D) The mRNA expression of SMC1A and LAMC2 in TGF‐β1‐stimulated LX‐2 cells and those preinfected with sh‐NC or sh‐SMC1A was examined via RT‐qPCR ( n = 3). The expression of the profibrotic genes PDGFRA (E), TIMP1 (F), and ACTA2 (G) in LX‐2 cells was examined via RT‐qPCR ( n = 3). The localization (H) and expression (I) of SMC1A in LX‐2 cells were examined via immunofluorescence experiments ( n = 3). (J) The enrichment of SMC1A in the LAMC2 promoter region in LX‐2 cells was examined via ChIP‐PCR; immunoprecipitation was performed using an anti‐SMC1A antibody ( n = 3). (K) Dual‐luciferase assay of the promoter transcriptional activity of LAMC2 in LX‐2 cells in the presence of sh‐SMC1A ( n = 3). Representative immunofluorescence images and quantification of collagen I (L, M) and α‐SMA (N, O) in LX‐2 cells ( n = 3). Graphical data are presented as means ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. An unpaired t ‐test or one‐way ANOVA was used for statistical analysis. ACTA2, actin alpha 2, smooth muscle; ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; HSC, hepatic stellate cell; LAMC2, laminin subunit gamma 2; PDGFRA, platelet‐derived growth factor receptor alpha; RT‐qPCR, reverse transcription quantitative PCR; SMC1A, structural maintenance of chromosome protein 1A; TIMP1, TIMP metallopeptidase inhibitor 1.

Article Snippet: Tissues were incubated with antibodies against LAMC2 (1:100, PK08311, Abmart Shanghai Co., Ltd.), α‐smooth muscle actin (α‐SMA, 1:500, GTX100034, GeneTex, Inc.), and collagen I (1:100, GTX20292, GeneTex) overnight at 4°C and with goat anti‐rabbit IgG (1:2500, ab97051, Abcam) at 37°C for 1 h. Then, immunoassays were performed using a DAB horseradish peroxidase color development kit (P0202, Beyotime Biotechnology Co., Ltd.).

Techniques: Expressing, Activation Assay, In Vitro, Binding Assay, ChIP-sequencing, Quantitative RT-PCR, Immunofluorescence, Immunoprecipitation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: LAMC2 overexpression supports the profibrotic phenotype of HSCs in the presence of sh‐SMC1A. (A) The mRNA expression of LAMC2 in LX‐2 cells infected with sh‐SMC1A + NC‐oe or sh‐SMC1A + LAMC2‐oe was examined via RT‐qPCR ( n = 3). The expression of the profibrotic genes ACTA2 (B), TIMP1 (C), and PDGFRA (D) in LX‐2 cells was examined via RT‐qPCR ( n = 3). (E–G) The total protein content and phosphorylation of PI3K and Akt in LX‐2 cells were examined using Western blot assays ( n = 3). (H–J) Representative immunofluorescence images (H) and quantification of collagen I (I) and α‐SMA (J) in LX‐2 cells ( n = 3). (K‒M) The protein expression of LRAT and GFAP, which are markers of HSC activation, in LX‐2 cells was analyzed by Western blot assays ( n = 3). Graphical data are presented as means ± SDs. * p < 0.05, ** p < 0.01, and *** p < 0.001. An unpaired t ‐test or one‐way ANOVA was used for statistical analysis. ACTA2, actin alpha 2, smooth muscle; ANOVA, analysis of variance; HSCs, hepatic stellate cells; LAMC2, laminin subunit gamma 2; PDGFRA, platelet‐derived growth factor receptor alpha; RT‐qPCR, reverse transcription quantitative PCR; SMC1A, structural maintenance of chromosome protein 1A; TIMP1, TIMP metallopeptidase inhibitor 1.

Article Snippet: Tissues were incubated with antibodies against LAMC2 (1:100, PK08311, Abmart Shanghai Co., Ltd.), α‐smooth muscle actin (α‐SMA, 1:500, GTX100034, GeneTex, Inc.), and collagen I (1:100, GTX20292, GeneTex) overnight at 4°C and with goat anti‐rabbit IgG (1:2500, ab97051, Abcam) at 37°C for 1 h. Then, immunoassays were performed using a DAB horseradish peroxidase color development kit (P0202, Beyotime Biotechnology Co., Ltd.).

Techniques: Over Expression, Expressing, Infection, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Immunofluorescence, Activation Assay, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: LAMC2 overexpression accentuates liver fibrosis in mice treated with sh‐SMC1A. (A) HE analysis of fibrosis in liver tissues from mice treated with sh‐SMC1A alone or sh‐SMC1A + NC‐oe/LAMC2‐oe ( n = 5). (B, C) Analysis of fibrosis in mouse liver tissues via Masson's trichrome staining ( n = 5). (D–F) The collagen I‐ and α‐SMA‐positive areas in mouse liver tissues were identified by IHC staining ( n = 5). The levels of the inflammatory factors TNF‐α (G), IL‐1β (H), and IL‐6 (I) in serum were measured by ELISAs ( n = 5). The mRNA expression of SMC1A (J) and LAMC2 (K) in liver tissues was examined via RT‐qPCR ( n = 3). (L–O) The protein expression of SMC1A and LAMC2 in activated HSCs (α‐SMA + ) was examined via immunofluorescence staining ( n = 3). (P–R) The protein expression of LRAT and GFAP, which are markers of HSC activation, in mouse liver tissues was analyzed by Western blot ( n = 3). Graphical data are presented as means ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. A one‐way ANOVA was used for statistical analysis. ANOVA, analysis of variance; ELISAs, enzyme‐linked immunosorbent assays; HSC, hepatic stellate cell; IHC, immunohistochemistry; LAMC2, laminin subunit gamma 2; RT‐qPCR, reverse transcription quantitative PCR; SMC1A, structural maintenance of chromosome protein 1A.

Article Snippet: Tissues were incubated with antibodies against LAMC2 (1:100, PK08311, Abmart Shanghai Co., Ltd.), α‐smooth muscle actin (α‐SMA, 1:500, GTX100034, GeneTex, Inc.), and collagen I (1:100, GTX20292, GeneTex) overnight at 4°C and with goat anti‐rabbit IgG (1:2500, ab97051, Abcam) at 37°C for 1 h. Then, immunoassays were performed using a DAB horseradish peroxidase color development kit (P0202, Beyotime Biotechnology Co., Ltd.).

Techniques: Over Expression, Staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, Immunofluorescence, Activation Assay, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: Overview diagram of the mechanism. SMC1A activates the PI3K/Akt signaling pathway by promoting LAMC2 transcription, which facilitates HSC activation and ECM deposition, leading to liver fibrosis progression. This figure was drawn using Figdraw ( https://www.figdraw.com/#/ ). ECM, extracellular matrix; HSC, hepatic stellate cell; LAMC2, laminin subunit gamma 2; SMC1A, structural maintenance of chromosome protein 1A.

Article Snippet: Tissues were incubated with antibodies against LAMC2 (1:100, PK08311, Abmart Shanghai Co., Ltd.), α‐smooth muscle actin (α‐SMA, 1:500, GTX100034, GeneTex, Inc.), and collagen I (1:100, GTX20292, GeneTex) overnight at 4°C and with goat anti‐rabbit IgG (1:2500, ab97051, Abcam) at 37°C for 1 h. Then, immunoassays were performed using a DAB horseradish peroxidase color development kit (P0202, Beyotime Biotechnology Co., Ltd.).

Techniques: Activation Assay