Journal: BMC Pharmacology & Toxicology

Article Title: A novel nitidine chloride nanoparticle overcomes the stemness of CD133 + EPCAM + Huh7 hepatocellular carcinoma cells for liver cancer therapy

doi: 10.1186/s40360-022-00589-z

Figure Lengend Snippet: a . Representative organ images showing specific tumor targeting of rhodamine B isothiocyanate labeled TPGS-FA/NC nanoparticles 8 h post-injection into mice bearing Huh7 xenograft (T: tumor, Li: liver, H: heart, L: lung, K:kidney, S: spleen, and B:brain; Color scale: radiant efficiency, [p s − 1 cm − 2 sr − 1 ] [μWcm − 2 ] − 1 ). V b . Quantitative analysis of biodistribution in tumors and normal organs, quantified from the organ images. Intravenous treatment of nude mice bearing orthotopic Huh7 xenografts with TPGS-FA/NC nanoparticles (red) and control groups (turquoise: NC, fuchsia:5-Fu, blue: PBS) every other day for a total of five injections (4 mg kg − 1,NC per body weight, indicated by arrows). c . Mice body weight was monitored during the time course of treatments ( n = 5 biologically independent animals, statistics was calculated by two-tailed unpaired t-test presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, p = 4.3 × 10 − 3 ,3.4 × 10 − 3 and 5.0 × 10 − 4 comparing TPGS-FA/NC to NC,5-Fu and PBS, respectively). d . Representative images of liver cancer tumors harvested from mice after treatments * p < 0.05, ** p < 0.01, *** p < 0.001; p = 0.01, 8 × 10 − 4,and 2 × 10 − 4 comparing TPGS-FA/NC to NC, 5-Fu, and PBS, respectively. Source data are provided as a

Article Snippet: 5-fluorouracil (5-Fu) was purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA).

Techniques: Labeling, Injection, Control, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: UBA1 inhibition sensitizes cancer cells to PARP inhibitors

doi: 10.1016/j.xcrm.2024.101834

Figure Lengend Snippet: Genome-wide CRISPR KO screen identifies UBA1 as a druggable target to enhance PARP inhibitor efficacy (A) Experimental outline of genome-wide CRISPR KO screen in OVCAR8 ovarian cancer cells (created with biorender.com ). See Figure S1 . (B) DrugZ scores comparing relative enrichment/depletion sgRNAs targeting given genes in OVCAR8 cells treated with PARPi olaparib compared to DMSO across genome-wide screen. (C) DrugZ scores from (B) restricted to only druggable genes. (D–F) Depletion of UBA1 induces sensitivity to PARP inhibition. Cells were transfected with control siRNA, UBA1 siRNA1.1, and UBA1 siRNA1.2, treated with indicated doses of olaparib for 10 days, and then stained with crystal violet. Representative image of OVCAR8 cells shown (D). Quantification of relative cell viability by extraction of crystal violet for OVCAR8 (E) and HCC1806 (F) cells. Graphs represent mean +/− standard deviation. Two-way ANOVA with Holm-Sidak post hoc test. ∗∗∗∗ p < 0.0001. n = 3. See Figure S2 . (G) Relative cell viability following treatment with varying concentrations of olaparib or TAK243 alone or the combination for 96 h in various ovarian cancer cell lines (OVCAR8, SKOV3, FUOV1, HOC1, and HOC8) and breast cancer cell lines (MDA-MB-468, HCC1806, BT549, and MCF7). The combination index (CI) was calculated with the Chou-Talalay equation. Experiments were performed in triplicates with two independent biological repeats ( n = 2). Graphs represent the mean with standard deviation.

Article Snippet: FUOV1 , DSMZ , ACC-444.

Techniques: Genome Wide, CRISPR, Inhibition, Transfection, Control, Staining, Extraction, Standard Deviation

Journal: Cell Reports Medicine

Article Title: UBA1 inhibition sensitizes cancer cells to PARP inhibitors

doi: 10.1016/j.xcrm.2024.101834

Figure Lengend Snippet: Validation of TAK243 and PARP inhibition in extended 2D and 3D assays (A and B) Representative images (A) and quantification (B) of ovarian cancer cell lines (OVCAR8, SKOV3, FUOV1, HOC8, and COV362 PARPi-R, see Figures S3 A and S3B), triple-negative breast cancer cell lines (BT549 and HCC1806), and non-malignant mammary epithelial cells (MCF-10A) treated with olaparib, TAK243, or the combination for 10 days. Concentrations given in Table S2 . Graphs represent mean +/− standard deviation. ANOVA with Holm-Sidak post hoc test. ∗∗∗∗ p < 0.0001. n = 3. See also Figure S4 . (C and D) Representative image (C) and quantification (D) of patient-derived ovarian cancer organoids treated either with 1 μM olaparib, 10 nM TAK243, combination thereof, or DMSO vehicle control for 9 days. Scale bar, 1 mm. Representative time-lapsed quantification shown in Figure S3 C. ANOVA with Holm-Sidak post hoc test. ∗∗ p < 0.01, ∗ p < 0.05. n = 3.

Article Snippet: FUOV1 , DSMZ , ACC-444.

Techniques: Biomarker Discovery, Inhibition, Standard Deviation, Derivative Assay, Control

Journal: Cell Reports Medicine

Article Title: UBA1 inhibition sensitizes cancer cells to PARP inhibitors

doi: 10.1016/j.xcrm.2024.101834

Figure Lengend Snippet: TAK243 treatment downregulates DNA damage repair pathway-related proteins (A) Experimental outline of reverse-phase protein array (RPPA) experiment (created with biorender.com ). (B) Heatmap showing changes in protein expression determined by RPPA following 48 h treatment with TAK243 in 10 different ovarian, breast, and colorectal cell lines. Red color indicates upregulated proteins and blue color indicates downregulated proteins. One-sample t test using average change in protein expression for each cell line ( n = 10), with Benjamini-Hochberg procedure to correct for multiple comparisons. TAK243 concentrations given in Table S3 . See Table S4 . (C) Western blot validation of RPPA results in HCC1806, OVCAR8, and FUOV1 cell lines, and in OVCAR8 and HCC1806 following treatment with indicated doses of TAK243.

Article Snippet: FUOV1 , DSMZ , ACC-444.

Techniques: Protein Array, Expressing, Western Blot, Biomarker Discovery

Journal: Cell Reports Medicine

Article Title: UBA1 inhibition sensitizes cancer cells to PARP inhibitors

doi: 10.1016/j.xcrm.2024.101834

Figure Lengend Snippet:

Article Snippet: FUOV1 , DSMZ , ACC-444.

Techniques: Recombinant, CRISPR, Negative Control, Genome Wide, Software

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Primary mouse microglia can be immortalized through HRAS and CMYC transcriptional factors. ( a ) Timeline depicting the transduction of primary CD1 E17.5 microglial cells at DIV4, followed by doxycycline induction of CMYC T58A and HRAS G12V at DIV5. ( b ) Immunocytochemistry results showing IBA1 + (Green) non-transduced control microglia, indicating a high-purity microglial culture. ( c ) Live imaging of cell confluence using IncuCyte for microglial cells transduced with TET-O-CMYC T58A-HRAS G12V with doxycycline (DOX), compared to non-transduced primary microglia at DIV16-23. ( d ) FACS gating strategy for isolating CD11B + LY6C – cells for microglial single-cell colony expansion. ( e ) Relative expression levels of microglial genes C1qa and P2ry12 , as well as human oncogenes CMYC and HRAS, normalized to Gadph. This was assessed using qRT-PCR on CD11b + LY6C − microglial cells, comparing control and transduced TET-O-CMYC T58A-HRAS G12V with DOX microglial cells. Statistical significance was evaluated using an unpaired t-test ( N = 3 replicates), with bars representing mean ± SEM. ( f ) Normalized counts per million (norm CPM) of murine proliferation genes Myc , Hras , Mki67 , Mcm2 and Pcna in primary MG and 2E11 clonal line without doxycycline (2E11 CTRL) and with doxycycline (2E11 DOX) from RNA-sequencing of total mRNA.

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Transduction, Immunocytochemistry, Control, Imaging, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Comparison of commonly used microglia cell lines examining microglial markers and function in vitro. Table modified from Stansley, Post, and Hensley (2012).

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Comparison, In Vitro, Modification, Transformation Assay

Journal: Scientific Reports

Article Title: Development and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies

doi: 10.1038/s41598-025-87543-1

Figure Lengend Snippet: Experimental conditions tested for immortalization of microglia of human and mouse.

Article Snippet: A mutant form of HRAS G12V, containing a P2A sequence, was synthesized by GenScript based on the published HRAS G12V sequence . pUC57 plasmid containing HRAS G12V was subsequently cloned into a lentiviral vector with a tetracycline response element with CMYC T58A (19775, Addgene) using BsaBI and Xba1 restriction enzymes.

Techniques: Concentration Assay

Journal: Science Advances

Article Title: p63 establishes epithelial enhancers at critical craniofacial development genes

doi: 10.1126/sciadv.aaw0946

Figure Lengend Snippet: ( A ) Experimental setup showing inducible expression of p63 and downstream epigenomic analyses. Dox, doxycycline. ( B ) Heatmap of p63 ChIP-seq, ATAC-seq, and H3K27ac ChIP-seq (±2.5 kb from peak center) in control fibroblasts expressing an empty vector (ctrl) and in fibroblasts expressing p63 for 72 hours. ( C ) Graph of called p63 peaks partitioned into open and closed chromatin according to ATAC-seq results in fibroblasts ctrl and fibroblasts + p63. ( D ) Pie chart depicting differences in chromatin landscape at p63 called peaks in ctrl and after ectopic expression of p63 showing clear increase in chromatin accessibility (blue, open) and H3K27ac (patterned lines). ( E ) Distance to nearest TSS for all p63 peaks. ( F ) Partitioning of p63 peaks into different genomic features. ( G ) Heatmap of z -scored RNA sequencing (RNA-seq) results for 1960 genes up-regulated (fold change > 1.5; FDR < 0.05) upon ectopic expression of p63 and top Gene Ontology (GO) categories showing enrichment of epithelial and inflammation categories among up-regulated genes. ( H and I ) Boxplots showing increased chromatin accessibility and H3K27ac at p63/KLF4 sites ±250 bp near up-regulated genes (* P < 10 × 10 −10 ). ( J ) UCSC genome browser tracks showing transcriptional activation of IRF6 (interferon regulatory factor 6) and de novo H3K27ac and ATAC-seq signal at an enhancer upstream of IRF6 in fibroblasts with ectopic expression of p63 (gray highlighted box). Chr1, chromosome 1. ( K ) UCSC genome browser tracks showing transcriptional activation of F11R and de novo H3K27ac and ATAC-seq signal within introns of F11R in fibroblasts with ectopic expression of p63 (gray highlighted boxes).

Article Snippet: We then cloned ∆NP63α-Flag (plasmid #26979, Addgene) and ∆NP63α-Flag cloned with a T2A self-cleaving peptide linker to human KLF4 (plasmid #19777) into a retroviral construct pRetroX-TRE3G-puro construct.

Techniques: Expressing, ChIP-sequencing, Control, Plasmid Preparation, RNA Sequencing, Activation Assay

Journal: Science Advances

Article Title: p63 establishes epithelial enhancers at critical craniofacial development genes

doi: 10.1126/sciadv.aaw0946

Figure Lengend Snippet: ( A ) Immunofluorescence of p63 and KRT14 in fibroblasts + p63 and fibroblasts + p63 + KLF4 showing that KRT14 is only up-regulated when both p63 and KLF4 are expressed. ( B ) Heatmap of z -scored RNA-seq results for 2213 genes up-regulated (fold change > 1.5; FDR < 0.05) upon ectopic expression of p63 + KLF4 showing high enrichment of epidermal and skin-related GO categories. ( C ) Comparison of transcriptional regulation of the top 15 genes up-regulated in fibroblasts + p63 + KLF4 showing that both factors are required for up-regulation of keratinocyte-specific genes. ( D ) Venn diagram showing that p63 and KLF4 are cobound in 13,488 loci in the genome. ( E ) Graph of p63/KLF4 peaks partitioned into open and closed chromatin according to peaks called from ATAC-seq showing increased chromatin accessibility after 72 hours at cobound sites. Fblsts, fibroblasts. ( F ) Pie charts showing stark increase in chromatin accessibility and H3K27ac at p63/KLF4 peaks. ( G ) Heatmap showing H3K27ac enrichment flanking p63/KLF4 peaks (±2.5 kb from peak center) shared in p63 ChIP-seq of basal keratinocytes, comparing fibroblasts, fibroblasts + p63, and fibroblasts + p63 + KLF4, and reanalyzed basal keratinocyte data from . ( H and I ) Boxplots showing increased chromatin accessibility and H3K27ac at p63/KLF4 sites near up-regulated genes (* P < 10 × 10 −10 ). ( J and K ) UCSC genome browser tracks showing de novo H3K27ac and ATAC-seq signal at enhancer and promoter regions close to keratinocyte genes KRT14 and IRF6 , as well as transcriptional activation (gray highlighted box).

Article Snippet: We then cloned ∆NP63α-Flag (plasmid #26979, Addgene) and ∆NP63α-Flag cloned with a T2A self-cleaving peptide linker to human KLF4 (plasmid #19777) into a retroviral construct pRetroX-TRE3G-puro construct.

Techniques: Immunofluorescence, RNA Sequencing, Expressing, Comparison, ChIP-sequencing, Activation Assay

Journal: Science Advances

Article Title: p63 establishes epithelial enhancers at critical craniofacial development genes

doi: 10.1126/sciadv.aaw0946

Figure Lengend Snippet: ( A ) Linear schematic of p63, showing domain structure and selected patient-derived mutations. TA, transactivation; OD, oligomerization domain; TID, terminal inhibitory domain. ( B ) Immunofluorescence of p63 and KRT14 in fibroblasts + fibroblasts + KLF4 and +WT p63, mtDBD, or mtSAM showing that KRT14 is not up-regulated when the DBD is mutated and induction of KRT14 is lower in mtSAM. ( C ) Heatmap of z -scored 3367 differentially expressed genes (fold change > 1.5; FDR < 0.05) between fibroblasts ctrl and fibroblasts + p63 + KLF4 showing that mtDBD has almost no change in transcriptional profile and mtSAM shows a transcriptional profile in between fibroblasts ctrl and fibroblast + WT p63 + KLF4. ( D ) Heatmap showing a stark reduction in chromatin accessibility (ATAC-seq signal) in mtSAM compared to WT p63, decreased H3K27ac, and retained p63 and KLF4 binding. ( E ) Boxplots showing no increase in chromatin accessibility for mtSAM, as well as reduced enrichment of H3K27ac flanking p63/KLF4 peaks. AUC, area under the curve; N.S., not significant. ( F ) Venn diagram showing that more than 1000 genes are no longer up-regulated in mtSAM + KLF4 and about 200 genes are up-regulated de novo by this mutant. ( G ) Boxplots showing that the RNA-seq signal of genes close to p63/KLF4 retained peaks separated into peaks at preestablished enhancers in fibroblasts and newly established enhancers after ectopic expression of p63/KLF4. ( H and I ) UCSC genome browser tracks showing defects (red boxes) in establishing open chromatin and inducing gene expression at FOXN1 and IRF6 .

Article Snippet: We then cloned ∆NP63α-Flag (plasmid #26979, Addgene) and ∆NP63α-Flag cloned with a T2A self-cleaving peptide linker to human KLF4 (plasmid #19777) into a retroviral construct pRetroX-TRE3G-puro construct.

Techniques: Derivative Assay, Immunofluorescence, Binding Assay, Mutagenesis, RNA Sequencing, Expressing, Gene Expression

Journal: Science Advances

Article Title: p63 establishes epithelial enhancers at critical craniofacial development genes

doi: 10.1126/sciadv.aaw0946

Figure Lengend Snippet: ( A ) Genetic association database results showing that cleft lip/cleft palate is the most enriched disease category among the 2213 genes up-regulated (fold change > 1.5; FDR < 0.05) by p63 and KLF4. ( B ) Forty identified risk loci for cleft lip/palate indicated by black lines at the respective chromosomes with dark blue circles at topologically associated domains (TADs) containing at least one gene up-regulated by both p63 and KLF4 (respective genes in dashed triangles). ( C ) List showing association values of top 10 genes calculated by gene-based analysis with MAGMA as implemented in FUMA in the following categories: red, genes known to cause CL/P with asterisk marking genes outside of the known 40 risk loci; green, CL/P candidate genes located at 40 risk loci with significant ( P < 0.05) association to CL/P; blue, new candidate genes associated with CL/P outside of known risk loci. ( D and E ) Regional association plots of ZNF296 and CPNE9 , showing nsCL/P-associated SNPs with nominal significant gene-based P value (<0.05) within the genes and up to 200 kb away from TSSs.

Article Snippet: We then cloned ∆NP63α-Flag (plasmid #26979, Addgene) and ∆NP63α-Flag cloned with a T2A self-cleaving peptide linker to human KLF4 (plasmid #19777) into a retroviral construct pRetroX-TRE3G-puro construct.

Techniques:

Journal: Science Advances

Article Title: p63 establishes epithelial enhancers at critical craniofacial development genes

doi: 10.1126/sciadv.aaw0946

Figure Lengend Snippet: ( A ) GREGOR analysis showing that p63/KLF4 shared peaks (green) and newly established H3K27ac (orange) flanking these peaks are significantly ( P < 0.05) enriched for SNPs associated with nsCL/P. ( B ) Colocalization of shared p63/KLF4 peaks at inactive (inact.)/preestablished (preestbl.)/new enhancers, with nominal significant nsCL/P-associated GWAS SNPs. The graph shows that only p63/KLF4 peaks flanked by de novo H3K27ac peaks are enriched for nsCL/P-associated SNPs. ( C ) Colocalization of H3K27ac peaks with nominal significant nsCL/P-associated GWAS SNPs. The graph shows that only new enhancers in converted cells flanking p63/KLF4 peaks are significantly enriched for nsCL/P-associated SNPs. ( D and E ) Overlay of SNPs and UCSC genome browser tracks highlighting (in orange) that p63/KLF4 peaks strongly colocalize with highly associated nsCL/P SNPs near MAFB and the known 8q24 locus; binding of both proteins at promoters is highlighted in gray.

Article Snippet: We then cloned ∆NP63α-Flag (plasmid #26979, Addgene) and ∆NP63α-Flag cloned with a T2A self-cleaving peptide linker to human KLF4 (plasmid #19777) into a retroviral construct pRetroX-TRE3G-puro construct.

Techniques: Binding Assay

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 2 Wnt/β-catenin signalling promotes HSC contraction. LX2 human HSCs were treated with Wnt3a (50 ngml−1), XAV (10 μM), or IWR (5 μM) for 24 h or transfected with β-catenin siRNA with or without Wnt3a (50 ngml−1) for 48 h. (a) Collagen gel contraction; n = 5. (b) Cytoskeleton immunofluorescence (400× magnification, scale bars: 10 μm); n = 3. (c) Western blotting of phospho-MLC2; n = 5. (d) Immunofluorescence of phospho-MLC2 (400× magnification, scale bars: 10 μm); n = 3. In (a) and (c), *P < .05, significantly different from vehicle control or control siRNA, #P < .05, significantly different from Wnt3a + control siRNA

Article Snippet: Cells were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% FBS (Wisent Biotechnology Co., Ltd., Nanjing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 C. β-Catenin siRNA (sc-29209), Gli1 siRNA (sc37911), Lrp6 siRNA (sc-37233), LARG siRNA (sc-41800), Sufu siRNA (sc-36572), TCF3 siRNA (sc-36618), TCF4 siRNA (sc-43525), and control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Immunofluorescence, Western Blot, Control

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 3 Wnt stimulates Smo-independent Gli1 nuclear translocation followed by LARG-mediated RhoA activation, leading to HSC contraction. LX2 human HSCs were treated with Wnt3a (50 ngml−1) and/or SIS3 (Smad3 inhibitor, 5 μM), SCH772984 (ERK inhibitor, 10 μM), MK2206 (AKT inhibitor, 5 μM), GANT-58 (Gli1 inhibitor, 5 μM), or PDTC (NF-κB inhibitor, 5 μM) for 24 h or transfected with β-catenin siRNA or Gli1 siRNA for 48 h. (a) Western blotting of phospho-MLC2; n = 5. (b) Collagen gel contraction; n = 5. (c) Cytoskeleton immunofluorescence (400× magnification, scale bars: 10 μm); n = 3. (d) Western blotting of active and total RhoA; n = 5. (e) Western blotting of LARG in whole cell lysates; n = 5. (f) Western blotting of active LARG in membrane lysates; n = 5. *P < .05, significantly different from vehicle control or control siRNA, #P < .05, significantly different from Wnt3a or Wnt3a + control siRNA, N.S., no significance

Article Snippet: Cells were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% FBS (Wisent Biotechnology Co., Ltd., Nanjing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 C. β-Catenin siRNA (sc-29209), Gli1 siRNA (sc37911), Lrp6 siRNA (sc-37233), LARG siRNA (sc-41800), Sufu siRNA (sc-36572), TCF3 siRNA (sc-36618), TCF4 siRNA (sc-43525), and control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology.

Techniques: Translocation Assay, Activation Assay, Transfection, Western Blot, Immunofluorescence, Membrane, Control

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 4 Sufu negatively mediates Wnt interaction with Gli1 and induction of HSC contraction. LX2 human HSCs were treated with Wnt3a (50 ngml−1), XAV (10 μM), IWR (5 μM), or C59 (5 μM) for 24 h or transfected with β-catenin siRNA, Sufu siRNA, or Sufu overexpression plasmids with or without Wnt3a (50 ngml−1) for 48 h. (a) Western blotting of nuclear Gli1. *P < .05, significantly different from vehicle control or control siRNA, #P < .05, significantly different from Wnt3a + control siRNA; n = 5. (b) Immunofluorescence of Gli1 nuclear translocation (400× magnification, scale bars: 5 μm). Pearson's correlation coefficients were calculated to confirm Gli1 co-localization with cell nuclei. *P < .05, significantly different from vehicle control or control siRNA, #P < .05, significantly different from Wnt3a + control siRNA; n = 3. (c, d) Western blotting of nuclear Gli1. *P < .05, significantly different from control siRNA, #P < .05, significantly different from Sufu siRNA; n = 5. (e) Collagen gel contraction. *P < .05, significantly different from vector, #P < .05, significantly different from Wnt3a + vector; n = 5. (f) Cytoskeleton immunofluorescence (400× magnification, scale bars: 10 μm); n = 3

Article Snippet: Cells were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% FBS (Wisent Biotechnology Co., Ltd., Nanjing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 C. β-Catenin siRNA (sc-29209), Gli1 siRNA (sc37911), Lrp6 siRNA (sc-37233), LARG siRNA (sc-41800), Sufu siRNA (sc-36572), TCF3 siRNA (sc-36618), TCF4 siRNA (sc-43525), and control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Over Expression, Western Blot, Control, Immunofluorescence, Translocation Assay, Plasmid Preparation

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 5 Wnt/β-catenin signalling represses Sufu transcription via TCF4 in HSCs. LX2 human HSCs were treated with Wnt3a (50 ngml−1), XAV (10 μM), IWR (5 μM), or C59 (5 μM) for 24 h or transfected with β-catenin siRNA, TCF3 siRNA, or TCF4 siRNA for 48 h. (a, e) Luciferase activities of Sufu promoter; n = 5. (b) Real-time PCR for Sufu; n = 5. (c, d) Western blotting for Sufu; n = 5.*P < .05, significantly different from vehicle control or control siRNA, #P < .05, significantly different from Wnt3a + control siRNA. (f) Co-immunoprecipitation for β-catenin/TCF4 physical interaction; n = 3

Article Snippet: Cells were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% FBS (Wisent Biotechnology Co., Ltd., Nanjing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 C. β-Catenin siRNA (sc-29209), Gli1 siRNA (sc37911), Lrp6 siRNA (sc-37233), LARG siRNA (sc-41800), Sufu siRNA (sc-36572), TCF3 siRNA (sc-36618), TCF4 siRNA (sc-43525), and control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Luciferase, Real-time Polymerase Chain Reaction, Western Blot, Control, Immunoprecipitation

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 6 Wnt/β-catenin repression of Sufu transcription requires β-catenin/TCF4 interaction and TCF4 binding to Sufu promoter in HSCs. (a) Molecular simulation of β-catenin/TCF3 or β-catenin/TCF4 physical interactions. (b–d) LX2 human HSCs were treated with Wnt3a (50 ngml−1) and/or transfected with β-catenin WT plasmids or β-catenin site-directed mutant plasmids at Lys312 or Lys435 for 48 h. (b) Luciferase activities of Sufu promoter. *P < .05, significantly different from control, #P < .05, significantly different from Wnt3a + WT plasmids; n = 5. (c) Real-time PCR of Sufu. *P < .05, significantly different from control, #P < .05, significantly different fromWnt3a + WT plasmids; n = 5. (d) Collagen gel contraction. *P < .05, significantly different from control, #P < .05, significantly different from Wnt3a + WT plasmids; n = 5. (e) Chromatin immunoprecipitation-quantitative PCR of TCF4 binding to Sufu promoter using two pairs of primers of Sufu gene. *P < .05, significantly different from control; n = 5. (f) LX2 human HSCs were transfected with WT or mutant luciferase reporter plasmids of Sufu followed by transfection with TCF4 overexpression plasmids for 48 h. Luciferase activities of Sufu promoter. *P < .05, significantly different as indicated; n = 5

Article Snippet: Cells were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% FBS (Wisent Biotechnology Co., Ltd., Nanjing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 C. β-Catenin siRNA (sc-29209), Gli1 siRNA (sc37911), Lrp6 siRNA (sc-37233), LARG siRNA (sc-41800), Sufu siRNA (sc-36572), TCF3 siRNA (sc-36618), TCF4 siRNA (sc-43525), and control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology.

Techniques: Binding Assay, Transfection, Mutagenesis, Luciferase, Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Over Expression

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 7 Short-term liver-targeted deficiency of β-catenin inhibits cirrhotic portal hypertension via decreasing HSC contraction in mice. Mice with CCl4-induced cirrhosis were administrated with AAV8-β-catenin shRNA via caudal vein once for 1 or 4 weeks. (a) Measurements of portal pressure; n = 5. (b) Collagen gel contraction in mouse primary HSCs; n = 5. (c) Real-time PCR of Col 1α1 in mouse primary HSCs; n = 5. (d) Real-time PCR of Sufu in mouse primary HSCs; n = 5. (e) Western blotting of Sufu and nuclear Gli1 in mouse primary HSCs; n = 5. *P < .05, significantly different from olive oil + AAV8-control shRNA, #P < .05, significantly different from CCl4 + AAV8-control shRNA

Article Snippet: Cells were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% FBS (Wisent Biotechnology Co., Ltd., Nanjing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 C. β-Catenin siRNA (sc-29209), Gli1 siRNA (sc37911), Lrp6 siRNA (sc-37233), LARG siRNA (sc-41800), Sufu siRNA (sc-36572), TCF3 siRNA (sc-36618), TCF4 siRNA (sc-43525), and control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology.

Techniques: shRNA, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 8 Acute pharmacological blockade of β-catenin reduces portal hypertension via decreasing HSC contraction, and long-term treatment ameliorates liver fibrosis in mice. Mice with CCl4-induced cirrhosis were orally treated with XAV (20 mgkg−1) or IWR (10 mgkg−1) daily for 1 or 4 weeks. (a, b) Measurements of portal pressure; n = 5. (c) Collagen gel contraction in mouse primary HSCs; n = 5. (d) Real-time PCR of Col 1α1 in mouse primary HSCs; n = 5. (e) Real-time PCR of Sufu in mouse primary HSCs; n = 5. (f) Western blotting of Sufu and nuclear Gli1 in mouse primary HSCs; n = 5. *P < .05, significantly different from control, #P < .05, significantly different from CCl4

Article Snippet: Cells were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% FBS (Wisent Biotechnology Co., Ltd., Nanjing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 C. β-Catenin siRNA (sc-29209), Gli1 siRNA (sc37911), Lrp6 siRNA (sc-37233), LARG siRNA (sc-41800), Sufu siRNA (sc-36572), TCF3 siRNA (sc-36618), TCF4 siRNA (sc-43525), and control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 9 Scheme of the molecular mechanisms underlying HSC contraction. Activation of Wnt/β-catenin signalling represses transcription of Sufu in a TCF4-dependent manner. This stimulates Gli1 nuclear translocation, leading to LARG-associated RhoA activation and results in contraction of HSCs. β-Catenin inhibitors reduce HSC contraction by disrupting this cascade and thereby exhibit therapeutic effects in models of cirrhotic portal hypertension

Article Snippet: Cells were cultured in DMEM (Invitrogen, Grand Island, NY, USA) with 10% FBS (Wisent Biotechnology Co., Ltd., Nanjing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 C. β-Catenin siRNA (sc-29209), Gli1 siRNA (sc37911), Lrp6 siRNA (sc-37233), LARG siRNA (sc-41800), Sufu siRNA (sc-36572), TCF3 siRNA (sc-36618), TCF4 siRNA (sc-43525), and control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Translocation Assay