Journal: Arthritis Research & Therapy

Article Title: Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

doi: 10.1186/ar2986

Figure Lengend Snippet: Vascular fibrosis in transgenic mice is associated with increased TGF-β expression and signaling . H&E, Masson trichrome, and picrosirius red (viewed under polarized light) staining of wild-type and transgenic thoracic aorta sections (a-c) . Smooth muscle layer architecture, elastic fibers, and smooth muscle layer are normal in the transgenic animals. However, adventitial collagen content is increased, and fibers are thicker in the transgenic animals on Masson trichrome and sirius red staining. LAP(TGF-β1) and free TGF-β1 expression is increased particularly in the adventitia (arrows) but also in smooth muscle cells (d-f) . Immunostaining for pSmad2/3 confirms increased nuclear translocation in the transgenic compared with the wild-type. Original magnification, ×20; representative images for panels (a-f) from transgenic (n = 6) and wild-type (n = 6) littermate sex-matched controls. (g) Serial measurements of adventitial and smooth muscle thickness show transgenic adventitial thickening and smooth muscle layer attenuation; summary data are expressed as mean ± SEM. * P < 0.05, from transgenic (n = 6) and wild-type (n = 6) littermate sex-matched controls. (h) Summary data from measurement of non-crosslinked collagen concentration compared with collagen standards (Sircol assay) show significantly higher collagen in transgenic animals compared with wild-type. Data are expressed as mean ± SEM.* P < 0.05, from transgenic (n = 8) and wild-type (n = 8) littermate sex-matched controls.

Article Snippet: Primary antibodies were as follows: CD34 (Abcam, Cambridge, UK); TGF-β1 and pSmad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA); LAP(TGFβ1) (R&D Biosystems, Minneapolis, MN); and α-SMA (Sigma-Aldrich, St. Louis, MO).

Techniques: Transgenic Assay, Expressing, Staining, Immunostaining, Translocation Assay, Concentration Assay

Journal: Arthritis Research & Therapy

Article Title: Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

doi: 10.1186/ar2986

Figure Lengend Snippet: Cultured vascular smooth muscle cells from transgenic mice have a TGF-β- activated phenotype . (a) Reporter gene assay for β-galactosidase shows equal chemiluminescence in vSMCs from wild-type and transgenic mice. Transgenic fibroblasts from the same animals were used as a positive control. Data are expressed as mean ± SEM; * P < 0.05 from WT (n = 3) and TG (n = 3) animals. (b) β-Galactosidase immunostaining results comparing vSMCs and fibroblasts from wild-type and transgenic animals show negative staining in all vSMCs and positive staining only in transgenic fibroblasts. Images shown are representative of three independent experiments. (c) vSMCs from transgenic mice show increased expression of smoothelin gene by qPCR. Recombinant TGF-β1 induced smoothelin mRNA in wild-type vSMCs, but the response in cells from transgenic mice was attenuated. Immunostaining confirms constitutive overexpression of smoothelin in vSMCs from transgenic mice. (d) Data for transgelin, a second gene important for vSMC cytoskeletal function, show the same trends. Data are expressed as mean ± SEM; * P < 0.05, ** P < 0.001; and are representative of three independent experiments examining four littermates for each condition. Original magnification, ×40.

Article Snippet: Primary antibodies were as follows: CD34 (Abcam, Cambridge, UK); TGF-β1 and pSmad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA); LAP(TGFβ1) (R&D Biosystems, Minneapolis, MN); and α-SMA (Sigma-Aldrich, St. Louis, MO).

Techniques: Cell Culture, Transgenic Assay, Reporter Gene Assay, Positive Control, Immunostaining, Negative Staining, Staining, Expressing, Recombinant, Over Expression

Journal: Arthritis Research & Therapy

Article Title: Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

doi: 10.1186/ar2986

Figure Lengend Snippet: Vascular smooth muscle cells from Tβ RIIΔk-fib mice show enhanced remodeling of floating type I collagen gel lattices . vSMCs from transgenic animals promoted more contraction of free-floating collagen lattices, resulting in gels of reduced diameter and weight when compared with wild-type. Induction by exogenous TGF-β1 resulted in further contraction by wild-type cells, but cells from transgenic mice were refractory to further induction. Data are expressed as mean ± SEM;* P < 0.05, ** P < 0.001, and are representative of three independent experiments examining three littermates for each condition.

Article Snippet: Primary antibodies were as follows: CD34 (Abcam, Cambridge, UK); TGF-β1 and pSmad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA); LAP(TGFβ1) (R&D Biosystems, Minneapolis, MN); and α-SMA (Sigma-Aldrich, St. Louis, MO).

Techniques: Transgenic Assay

Journal: Arthritis Research & Therapy

Article Title: Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

doi: 10.1186/ar2986

Figure Lengend Snippet: Perturbed endothelin receptor expression and function in vascular smooth muscle cells from transgenic animals . (a, b) vSMCs from transgenic mice have reduced expression of ETRA mRNA and protein when compared with wild-type cells. Exogenous administration of TGF-β or ET-1 to cells from both wild-type and transgenic animals further suppressed ETRA expression. Data are representative of three independent experiments examining four littermates for each condition and are expressed as mean ± SEM,* P < 0.05, ** P < 0.001. (c) Vasoconstrictor response of aortic rings to ET-1 was attenuated in transgenic mice. Bosentan attenuates the response in both wild-type and transgenic mice. Representative data from dose response curves of WT (n = 6) and TG (n = 6) aortic rings before (-) and after (+) response to 10 -8 mol/L ET-1 concentrations, before (-) and after (+) bosentan 2 μmol/L pretreatment. Data are expressed as mean ± SEM; * P < 0.05.

Article Snippet: Primary antibodies were as follows: CD34 (Abcam, Cambridge, UK); TGF-β1 and pSmad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA); LAP(TGFβ1) (R&D Biosystems, Minneapolis, MN); and α-SMA (Sigma-Aldrich, St. Louis, MO).

Techniques: Expressing, Transgenic Assay

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Experimental design to dissect TGFβ-regulated genes in CCA cells and LINC00313 expression in HuCCT1 and Huh28 in response to TGFβ1, as identified by gene expression profiling. ( B ) LINC00313 expression in response to TGFβ1 stimulation for 16 h, in normal human cholangiocytes (NHC) and CCA cell lines. ( C ) LINC00313 expression in response to the indicated doses of TGFβ1 in HuCCT1 and Huh28 cell lines. ( D ) LINC00313 expression in response to TGFβ1 for the indicated time periods in HuCCT1 and Huh28 cell lines. ( E ) MALAT1 , RNU48 , GAPDH and LINC00313 RNA levels in nuclear and cytoplasmic fractions of HuCCT1 and Huh28 cell lines. Data information: Data in graphs are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test), n.s. not significant. .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Expressing, Gene Expression

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) LINC00313 expression in HuCCT1 and Huh28 cells treated with LY2157299 or DMSO, and stimulated with TGFβ1 or BSA/HCl for 16 h. ( B ) LINC00313 and SERPINE1 expression in HuCCT1 and Huh28 treated with SIS3 or DMSO and stimulated with TGFβ1 or BSA/HCl for 16 h. ( C ) LINC00313 expression in HuCCT1 transiently transfected with siRNA targeting SMAD2 , SMAD3 , or SMAD4 , alone or in combination, with or without TGFβ1. ( D ) LINC00313 expression in Huh28 transiently transfected with siRNA targeting SMAD2 , SMAD3 , or SMAD4 , alone or in combination, with or without TGFβ1. ( E ) Snapshot of the UCSC genome browser showing predicted SMAD-binding regions in LINC00313 gene locus, using the JASPAR CORE 2022 collection. Active histone mark (H3K27ac) is also shown around LINC00313 TSS. Data information: Data are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test), n.s.: not significant. In panel ( A ) the experiment in HuCCT1 cells was performed twice ( n = 2 biological replicates) and data are presented as single data points. .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Expressing, Transfection, Binding Assay

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) LINC00313 levels in HuCCT1 or Huh28 treated with MEK, p38, JNK or PI3K inhibitors, with or without TGFβ1 stimulation for 16 h ( n = 3 biological replicates). ( B ) Immunoblotting for detection of phosphorylated p44/42 (Thr202/Tyr204), phosphorylated p-SAPK/JNK (Thr183/Tyr185), phosphorylated AKT (Ser473), phosphorylated p38 (Thr180/Tyr182), phosphorylated MAPKAPK-2 (Thr334), total p44/42, SAPK/JNK and AKT protein levels and β-tubulin (used as a loading control) in HuCCT1 cells treated with MEK, p38, JNK or PI3K inhibitors, with or without TGFβ1 stimulation for 16 h. DMSO was used as a vehicle treatment. ( C ) Immunoblotting for detection of the p38 isoforms p38α, p38β, p38γ and p38δ in HuCCT1 cells transiently silenced for MAPK11 or MAPK12 or MAPK13 or MAPK14 and treated or not with TGFβ1 for 16 h. β-tubulin was used as a loading control. ( D ) Real-time qPCR analysis of LINC00313 , MAPK11 , MAPK12 , MAPK13 and MAPK14 expression in HuCCT1 and Huh28 cell lines transiently transfected with siRNAs targeting MAPK11 or MAPK12 or MAPK13 or MAPK14 and treated or not with TGFβ1 for 16 h. Data information: In Panel ( A ) data are presented as mean ± SD ( n = 3 biological replicates). In panel ( D ) data are presented as single data points ( n = 2 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, n.s.: not significant (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Western Blot, Control, Expressing, Transfection

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Experimental approach to identify LINC00313 transcriptional targets. ( B ) Number of differentially expressed genes in pcLINC00313 versus pcDNA3 HuCCT1 cells (shrunken log2FC > 1, adjusted p -value < 0.1). Volcano plot shows differentially expressed genes (green colour: upregulated genes, red colour: downregulated genes). ( C ) KEGG pathway analysis of up or downregulated genes upon LINC00313 over-expression. ( D ) Example of GSEA of differentially expressed genes in response to LINC00313 over-expression. Shown is the enrichment of a Hippo signalling signature in the gene expression of cells overexpressing LINC00313. ( E ) WNT5A , AXIN2 and SULF2 mRNA levels in HuCCT1 expressing LINC00313 (pcLINC) or empty vector (pcDNA3). ( F ) WNT5A , AXIN2 and SULF2 expression in HuCCT1 transfected with siLINC00313 and stimulated with TGFβ1 or BSA/HCl for 16 h. Data information: In panel ( B ) statistically significant differential expressed genes were identified using DEseq2. In panel ( C ) adjusted p -value was calculated using the Benjamini-Hockberg method for correction for multiple hypotheses testing. In panel ( D ) GSEA results are displayed as a normalized enrichment score (NES) and presented as an enrichment plot. The Kolmogorov-Smirnov test was used for statistical analysis of GSEA. In ( E , F ) data are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Over Expression, Gene Expression, Expressing, Plasmid Preparation, Transfection

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Efficient ligation of LINC00313 insert to pcDNA3.1 plasmid vector was verified by double digestion with restriction enzymes, followed by agarose gel electrophoresis. Molecular size (in kbp) marker ladder is shown in the first lane. ( B ) Real-time qPCR analysis of LINC00313 expression in HuCCT1 cells stably over-expressing pcDNA3.1 or pcLINC00313 expression vectors stimulated or not with TGFβ1 for 16 h. ( C ) Subcellular fractionation followed by RT-qPCR analysis of LINC00313 RNA levels in nuclear and cytoplasmic fractions of HuCCT1 cells transiently over-expressing an empty vector (pcDNA3.1) or LINC00313 and HuCCT1 clones, stably over-expressing pcDNA3.1 or LINC00313. ( D ) smiFISH for LINC00313 RNA in HuCCT1 cells stably over-expressing LINC00313 and stimulated with TGFβ for 16 h. DAPI was used to stain nuclei. Scale bars: 15 μm. ( E ) Heatmap presenting the top 40 differentially regulated genes in HuCCT1 pcLINC00313 versus pcDNA3-expressing cells (shrunken log2FC > 1, adjusted p -value < 0.1). Biological triplicates (1-3) were used per condition. ( F ) GSEA analysis of differentially expressed genes in pcLINC00313 versus pcDNA3 HuCCT1 cells. ( G ) Real-time qPCR analysis of LINC00313 , TCF7 , WNT5A , AXIN2 , and SULF2 expression in a monoclonal HuCCT1 cell line stably expressing pcDNA3.1 and in two monoclonal HuCCT1 cell lines stably expressing pcLINC00313, in the presence or not of TGFβ1 for 16 h. Data information: Data in panels ( B ), ( C ) and ( G ) are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). In panel ( F ) the Kolmogorov-Smirnov test was used for statistical analysis of GSEA. .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Ligation, Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Expressing, Stable Transfection, Fractionation, Quantitative RT-PCR, Clone Assay, Staining

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Venn diagram showing merged regions with overlapping and unique ATAC-seq peaks between pcDNA3 and pcLINC00313 expressing HuCCT1 cells. ( B ) Bar plot illustrating the top differential regions. ( C ) Venn diagram highlighting the 44 genes, whose chromatin accessibility and expression are altered. ( D ) GO analysis for biological process of the 44 genes highlighted in panel ( C ) using Enrichr. ( E ) Snapshot of the UCSC genome browser with ATAC-seq peaks at human TCF7 gene locus, in pcDNA3 versus pcLINC00313 over-expressing HuCCT1 cells. ( F ) TCF7 mRNA and protein levels in HuCCT1 cells over-expressing LINC00313 (pcLINC) or pcDNA3 control. ( G ) TCF7 RNA and protein levels in HuCCT1 cells, transfected with siLINC00313 and stimulated with TGFβ1 for 16 h. Data information: In panel ( D ) p -value was calculated by Enrichr using Fisher’s exact test or hypergeometric test. In ( F , G ) qPCR data are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Expressing, Control, Transfection

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) Schematic representation of the TCF/LEF-luciferase reporter assay. Activators (Wnt ligands, CHIR) and inhibitors (XAV939) of TCF/LEF-dependent responses are also shown. ( B ) TCF/LEF-luciferase reporter assay in HuCCT1 cells transiently transfected with TOP-Flash or FOP-Flash luciferase expression vectors and treated with CHIR99021 or DMSO for 24 h. Cells were co-transfected with a Renilla luciferase expression vector for normalization of the firefly luciferase activity. ( C ) TCF/LEF-luciferase reporter assay in HuCCT1 cells stably expressing the pGreenFire 2.0 TCF/LEF reporter construct and treated with CHIR99021 or DMSO for 24 h. TCF/LEF-luciferase reporter assay in HuCCT1 cells stably expressing the pGreenFire 2.0 TCF/LEF reporter construct and treated with the indicated concentrations of TGFβ1 or BSA/HCl (Ctr) for 16 h. ( D ) Subcellular fractionation followed by immunoblotting for β-catenin, lamin B1 (nuclear marker), β-tubulin and 14-3-3 (cytoplasmic markers) using nuclear and cytoplasmic fractions of HuCCT1 cells stably over-expressing an empty vector (pcDNA3.1) or LINC00313 and treated with CHIR99021 or DMSO for 24 h. ( E ) Immunofluorescence to detect β-catenin subcellular localization in control or LINC00313 over-expressing HuCCT1 cells, treated or not with CHIR99021 or DMSO for 24 h. Scale bars: 150 μm. ( F ) Cell viability assays in control or LINC00313 over-expressing HuCCT1 cells treated with CHIR99021 or DMSO for 24 h. ( G ) Colony formation assay in control or LINC00313 over-expressing HuCCT1 cells, treated with CHIR99021 or XAV939 or DMSO for 24 h. Quantification of the number of colonies for each condition is also shown. ( H ) Real-time qPCR analysis of AXIN2 , TCF7 and LINC00313 expression in control or LINC00313 overexpressing HuCCT1 cells, treated with CHIR99021, XAV939, or DMSO for 24 h. Data information: In panel ( B ) data are presented as mean ± SD ( n = 3 biological replicates). In panel ( F ) data are presented as mean ± SD ( n = 6 biological replicates). ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). In panels ( G ) and ( H ) data are presented as mean ± SD ( n = 3 biological replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Luciferase, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Activity Assay, Stable Transfection, Construct, Fractionation, Western Blot, Marker, Immunofluorescence, Control, Colony Assay

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) In vitro transcribed F-luc and LINC00313 RNAs. ( B ) Overview of interacting proteins identified by mass spectrometry. ( C ) Venn diagram representing the strategy to narrow down LINC0313 -binding proteins. ( D ) Protein network analysis depicting physical and functional interactions between the 51 nuclear interactors, using STRING. ( E ) GO analysis for molecular function of 32 nuclear interactors predicted by CatRAPID. ( F ) Heatmap depicting the probability of interaction between LINC00313 and ACTL6A. ( G ) Immunofluorescence of ACTL6A in control or LINC00313 over-expressing HuCCT1 cells, treated or not with TGFβ1. Scale bars: 150 μm. ( H ) RNA pull-down assays in HuCCT1 cells, stimulated with TGFβ1 or not, using in vitro synthesized F-luc mRNA or LINC00313 lncRNA, followed by immunoblotting for ACTL6A, HuR and β-actin. ( I ) RNA pull-down assay in HEK293T cells, over-expressing an empty vector or HA-tagged ACTL6A using in vitro synthesized LINC00313 lncRNA. An arrow depicts the specific HA-ACTL6A protein band, while an asterisk marks unspecific protein bands. ( J ) RIP assay for endogenous ACTL6A followed by qPCR for LINC00313 in HuCCT1 cells treated with TGFβ or BSA/HCl (vehicle control) for 16 h ( n = 3). An immunoblotting to verify the efficiency of ACTL6A immunoprecipitation is also shown. The arrows depict the specific endogenous ACTL6A protein bands. Data information: In panel J RIP-qPCR data are presented as mean ± SD ( n = 3 technical replicates). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: In Vitro, Mass Spectrometry, Binding Assay, Functional Assay, Immunofluorescence, Control, Expressing, Synthesized, Western Blot, Pull Down Assay, Plasmid Preparation, Immunoprecipitation

Journal: EMBO Reports

Article Title: TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma

doi: 10.1038/s44319-024-00075-z

Figure Lengend Snippet: ( A ) TCF7 , LINC00313 and ACTL6A mRNA levels in HuCCT1 transfected with siACTL6A or siNC and in the presence or not of TGFβ. ( B ) Immunoblotting for TCF7, ACTL6A and β-actin, upon silencing ACTL6A, using the indicated siRNA concentrations. ( C ) TCF/LEF luciferase reporter assay in siACTL6A or siNC HuCCT1 cells treated with CHIR99021 or DMSO for 24 h. ( D – F ) qPCR analysis of ACTL6A ( D ), AXIN2 ( E ) and TCF7 ( F ) mRNAs in the same conditions as these of panel ( C ). ( G – I ) qPCR analysis of SULF2 ( G ), TCF7 ( H ) and ACTL6A ( I ) mRNA levels in pcDNA3 or LINC00313 over-expressing HuCCT1 transiently transfected with siACTL6A or siNC with or without TGFβ. ( J ) Immunoblotting for TCF7, ACTL6A and β-actin in the same conditions as these of panels G-I. ( K ) qPCR analysis of TCF7 , ACTL6A and LINC00313 RNA levels in HuCCT1 cells transiently transfected with siRNAs against LINC00313 or ACTL6A individually or simultaneously with both siRNAs and treated or not with TGFβ for 16 h. ( L ) RIP assay for endogenous Brg1 followed by qPCR for LINC00313 in HuCCT1 cells treated with TGFβ or BSA/HCl (vehicle control) for 16 h. ( M ) Proposed model for the molecular mechanism of LINC00313 . Data information: In panels ( A ), (D–I) and (K) qPCR data are presented as mean ± SD ( n = 3 biological replicates). In panel ( C ), luciferase assay data are presented as mean ± SD ( n = 6 biological replicates). In panel ( L ), RIP-qPCR data are presented as mean ± SD ( n = 3 technical replicates). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test). .

Article Snippet: Serum-starved cells were stimulated with the indicated concentrations of human recombinant TGFβ1 (R&D Systems) and for the indicated time periods as shown in the figures and figure legends.

Techniques: Transfection, Western Blot, Luciferase, Reporter Assay, Expressing, Control

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: (A) Supplementation of serum-free KPC3 cultures with LDL (100 μg/ml), but not with 50 μM ethanol-solubilized cholesterol, reverses pSMAD2 induction by compactin. Shown are results of 3 independent repeats and a representative panel below. (B) Compactin-induced activation of SREBP1 is reversed by addition of LDL quantified as ratio of nuclear and full length (FL) protein. (C) Secreted TGFβ1 suppression by SREBP inhibitor. Fatostatin (20 μM) was added to KPC3 cells grown in the indicated media for 48 hours. Averaged results of 3 independent ELISA assays are shown. (D) Representative Western blot of pSMAD2 of cellular lysates corresponding to (C). (E) Human TGFB1 promoter-dependent luciferase reporter activity following co-transfection into HEK293T cells with plasmids expressing nuclear fragments of SREBP1 (aa 1–480), SREBP2 (aa 1–473), constitutively active MEK1 (S218D/S222D), or dominant negative MEK1 mutant (S218A/S222A). Empty vectors (EV) were used as negative controls. (F) TGFB1-luciferase reporter activity in human PDAC cells MiaPaCa2 co-transfected with nuclear SREBP1 or SREBP2. Fatostatin at 10 μM was used to block the endogenous SREBP activation. (G) Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) determination of genomic Tgfb1 DNA binding (amplicon +390 bp to +564 bp) by the endogenous SREBP1 in KPC3 cells conditioned for 48 hours in FBS, or LDS+ 1 μM compactin. Amplicon −3204 bp to −3032 bp distant to TSS served as negative control, whereas a canonical SREBP1 binding site in Ldlr promoter (−38 bp to +60 bp) served as a positive control for SREBP1 activity. (H) Increased association of open chromatin (H3K4me3) and reduced association of repressed chromatin marks (H3K27me3) with the proximal Tgfb1 promoter of cholesterol-depleted KPC3 cells as determined by ChIP-qPCR. The map of genomic Tgfb1 locus is drawn to scale. Data were pooled from two independent experiments. In all figures, data are represented as mean±SEM, p-values determined by independent two-sample Student t-test: *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S6.

Article Snippet: The amount of TGFβ secreted by mouse pancreatic tumor cells was quantified using the Mouse TGFβ1 DuoSet ELISA (#DY1679–05, R&D systems), complemented with Sample Activation Kit 1 (#DY010, R&D systems) and DuoSet ELISA Ancillary Reagent Kit 1 (#DY007, R&D systems) according to the manufacturer’s instructions.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Activity Assay, Cotransfection, Expressing, Dominant Negative Mutation, Mutagenesis, Transfection, Blocking Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Amplification, Negative Control, Positive Control

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: (A) Enumeration of pancreatic epithelial lesion by grade per section in KPC and KPCN mice aged 5–6 months; p=0.035 for PDAC (Fisher’s exact test); p=0.02 for PanIN2/3; p=0.0006 for ADM, Wilcoxon rank-sum test; error bars, SEM. (B) Kaplan-Meier representation of PDAC-free survival of KPC (n=34) and KPCN (n=37) mice. p<0.0001, logrank test. (C, D) Activated TGFβ pathway signaling in NSDHL-deficient pancreatic lesions with homozygous (C) and heterozygous (D) Trp53 knockout as assessed by phosphorylated SMAD2 immunohistochemistry. Top, 5–6 months old KPC and KPCN mice; bottom, 5 week old KPPC and KPPCN pancreatic lesions. Right panel, quantification of pSMAD2-positive nuclei in acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasm (PanIN) lesions. (E) Cleaved caspase 3 in pancreatic ADM and PanIN lesions. In C-E, staining intensity was quantified separately in ADM and PanIN lesions; p-values are calculated by Wilcoxon test; ns, not significant; data are represented as boxplots: median (black bar), box (25% to 75% confidence interval), whiskers (full range of measurements). See also Figure S4.

Article Snippet: The amount of TGFβ secreted by mouse pancreatic tumor cells was quantified using the Mouse TGFβ1 DuoSet ELISA (#DY1679–05, R&D systems), complemented with Sample Activation Kit 1 (#DY010, R&D systems) and DuoSet ELISA Ancillary Reagent Kit 1 (#DY007, R&D systems) according to the manufacturer’s instructions.

Techniques: Knock-Out, Immunohistochemistry, Staining

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: (A) NSDHL inactivation in well-differentiated KPC3 PDAC cells by CRISPRi is confirmed by absence of NSDHL band on Western blot of total cellular lysates; KPC3 parental cells and GFP-targeted gRNA used as controls. (B) Secreted TGFB1 as determined by ELISA using 48 hour supernatants. Shown, averaged results from 3 independent repeats; error bars, SEM. (C) Histological grading of glandular (grades 1–2) versus solid (grades 3–4) tumor areas in tumors generated via orthotopic implantation of KPC3wt or KPC3 NsdhlCRISPRi cells; p=0.007, two-way Student t-test. Symbols represent individual tumors; black bars, mean±SEM. (D) Cholesterol level in KPPC (n=10) and KPPCN (n=10) clones grown for 48 hours in FBS or LDS media; (1) p=0.004; (2) p=0.0001, Wilcoxon test. Boxplots represent median (black bar) and full range of measurements. (E) Cholesterol levels in PDAC cells conditioned for 48 hours as indicated; L+C, 5%LDS with 1 μM compactin. (F) Representative Western blot of phosphorylated pSMAD2 (Ser465/467) and pSmad3 (Ser423/425) in KPC3 cells cultured for 48 hours in fetal bovine serum (FBS), lipid depleted serum (LDS), or LDS with 1 μM of compactin (L+C) followed by incubation in serum-free DMEM for 4 hours. Indicated samples were treated with TGFβ1 at 10 ng/ml for 30 minutes, and/or SB431542 at 25 μM for 1 hour. (G) Summary results of levels of phosphorylated pSMAD2(Ser465/467) and pSmad3 (Ser423/425) in cholesterol depleted PDAC cells. Results from 3 independent experiments normalized to α-tubulin are shown. (H) Phosphorylated pSMAD2(Ser465/467) in human Capan-2 carcinoma cells conditioned for 48 hours in FBS, LDS with or without 1 μM compactin. Summary results from 3 independent experiments normalized to α-tubulin are shown. (J) Increased nuclear SREBP1 and SREBP2 in Capan-2 cells as in H. (I) Surface versus internalized pools of TGFBR1 and TGFBR2 in KPC3 cells conditioned for 48 hours in 5% FBS or in 5% LDS with 1 μM compactin. Biotinylated (surface) and non-biotinylated (internalized) proteins were affinity separated using streptavidin-agarose beads. (K) Levels of Tgfb1, Tgfb2 and Tgfb3 mRNA as assessed by qRT-PCR, in cells grown in indicated media for 48 hrs. (L) ELISA measurement of secreted TGFβ1 in supernatants of KPC3 cells conditioned in indicated media for 48 hrs; (M) Expression of Zeb2, Tgfb1 and Wnt10b mRNA as assessed by qRT-PCR in KPC3 and KPC634 PDAC cells cultured for 48 hours in media supplemented with FBS, LDS or LDS+compactin (1 μM). In graphs B, E, G-J and K-M, statistical p-values by two-way Student t-test are indicated as: *, <0.05, **, <0.01, ***, <0.001. See also Figure S5.

Article Snippet: The amount of TGFβ secreted by mouse pancreatic tumor cells was quantified using the Mouse TGFβ1 DuoSet ELISA (#DY1679–05, R&D systems), complemented with Sample Activation Kit 1 (#DY010, R&D systems) and DuoSet ELISA Ancillary Reagent Kit 1 (#DY007, R&D systems) according to the manufacturer’s instructions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Clone Assay, Cell Culture, Incubation, Quantitative RT-PCR, Expressing

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The amount of TGFβ secreted by mouse pancreatic tumor cells was quantified using the Mouse TGFβ1 DuoSet ELISA (#DY1679–05, R&D systems), complemented with Sample Activation Kit 1 (#DY010, R&D systems) and DuoSet ELISA Ancillary Reagent Kit 1 (#DY007, R&D systems) according to the manufacturer’s instructions.

Techniques: Polymer, Plasmid Preparation, Virus, Clone Assay, Mutagenesis, Modification, Cloning, Recombinant, Antibody Labeling, Transfection, Marker, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Activation Assay, Viability Assay, Gentle, Reporter Assay, Purification, Multiplex Assay, RNA Sequencing, Sequencing, Software, Imaging

Journal: G3: Genes|Genomes|Genetics

Article Title: Chi-miR-370-3p regulates hair follicle morphogenesis of Inner Mongolian cashmere goats

doi: 10.1093/g3journal/jkab091

Figure Lengend Snippet: Verification of chi-miR-370-3p sequencing results and confirmation that it directly targets TGF-βR2 and FGFR2 . (A) Validation of chi-miR-370-3p sequencing results. (B) qRT-PCR verification of the targeting of TGF-βR2 and FGFR2 by chi-miR-370-3p. (C) Plasmid map of the dual luciferase reporter system and location of the target fragment inserted into the vector. (D) Verification of the interaction between chi-miR-370-3p and TGF-βR2-3′-UTR by dual luciferase reporter gene assay. (E) Verification of the interaction between chi-miR-370-3p and FGFR2-3′-UTR by dual luciferase reporter gene assay.

Article Snippet: Antibodies were as follows: rabbit polyclonal antibody against FGFR2 (AF0159; Affinity, China), rabbit polyclonal antibody against TGF-βR2 (AF5449; Affinity), mouse monoclonal antibody against β-actin, horseradish peroxidase (HRP)-conjugated goat anti-rabbit second antibody (BA1054; BOSTER, China), and HRP-conjugated goat anti-mouse second antibody (BA1051; BOSTER).

Techniques: Sequencing, Biomarker Discovery, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Reporter Gene Assay

Journal: G3: Genes|Genomes|Genetics

Article Title: Chi-miR-370-3p regulates hair follicle morphogenesis of Inner Mongolian cashmere goats

doi: 10.1093/g3journal/jkab091

Figure Lengend Snippet: Verification of the regulatory effect of chi-mir-370-3p on TGF-βR2 and FGFR2 at epithelial cell and dermal fibroblast levels. (A) Construction of chi-miR-370-3p (lo) and chi-miR-370-3p (hi) dermal fibroblast and epithelial cell lines. (B) Relative expression of chi-miR-370-3p in various cell lines. (C) Relative expression of TGF-βR2 and FGFR2 in various cell lines. (D) Expression of β-actin, TGF-βR2, and FGFR2 proteins in each cell line. (E) Relative abundance of TGF-βR2 and FGFR2 proteins in different epithelial cell lines. (F) Relative abundance of TGF-βR2 and FGFR2 proteins in different dermal fibroblast cell lines.

Article Snippet: Antibodies were as follows: rabbit polyclonal antibody against FGFR2 (AF0159; Affinity, China), rabbit polyclonal antibody against TGF-βR2 (AF5449; Affinity), mouse monoclonal antibody against β-actin, horseradish peroxidase (HRP)-conjugated goat anti-rabbit second antibody (BA1054; BOSTER, China), and HRP-conjugated goat anti-mouse second antibody (BA1051; BOSTER).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Interrogation of human microglial phagocytosis by CRISPR genome editing

doi: 10.3389/fimmu.2023.1169725

Figure Lengend Snippet: Optimization of HMC3 genome editing by Cas9 ribonucleoproteins (RNP) electroporation. (A) Cas9 RNP and DNA repair templates were electroporated into HMC3 cells by a Lonza 4D nucleofector system. The editing efficiency was determined genotypically by DNA sequencing and ICE analysis, and phenotypically using immunofluorescent staining and flow cytometry. Off-target editing was analyzed by next generation sequencing (NGS). Gene-edited cells were isolated by fluorescence-activated cell sorting (FACS), and assayed for in vitro phagocytosis of fluorescently labeled amyloid beta peptide (fAβ 1-42 ). (B) Screening of electroporation pulse codes for the optimal DNA delivery using plasmid pmaxGFP encoding the turboGFP gene. (C) Screening of pulse codes for Cas9 RNP delivery using CD40 -targeting Cas9. (D) Shift in CD40 intensity after Cas9 RNP electroporation by the indicated pulse codes. The complete list of pulse codes is in Table S3 .

Article Snippet: Plasmid pmaxGFP (encoding the turboGFP reporter gene, Lonza) was added at 0.4 μg to screen for the DNA electroporation condition.

Techniques: Electroporation, DNA Sequencing, Staining, Flow Cytometry, Next-Generation Sequencing, Isolation, Fluorescence, FACS, In Vitro, Labeling, Plasmid Preparation

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: (A) Supplementation of serum-free KPC3 cultures with LDL (100 μg/ml), but not with 50 μM ethanol-solubilized cholesterol, reverses pSMAD2 induction by compactin. Shown are results of 3 independent repeats and a representative panel below. (B) Compactin-induced activation of SREBP1 is reversed by addition of LDL quantified as ratio of nuclear and full length (FL) protein. (C) Secreted TGFβ1 suppression by SREBP inhibitor. Fatostatin (20 μM) was added to KPC3 cells grown in the indicated media for 48 hours. Averaged results of 3 independent ELISA assays are shown. (D) Representative Western blot of pSMAD2 of cellular lysates corresponding to (C). (E) Human TGFB1 promoter-dependent luciferase reporter activity following co-transfection into HEK293T cells with plasmids expressing nuclear fragments of SREBP1 (aa 1–480), SREBP2 (aa 1–473), constitutively active MEK1 (S218D/S222D), or dominant negative MEK1 mutant (S218A/S222A). Empty vectors (EV) were used as negative controls. (F) TGFB1-luciferase reporter activity in human PDAC cells MiaPaCa2 co-transfected with nuclear SREBP1 or SREBP2. Fatostatin at 10 μM was used to block the endogenous SREBP activation. (G) Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) determination of genomic Tgfb1 DNA binding (amplicon +390 bp to +564 bp) by the endogenous SREBP1 in KPC3 cells conditioned for 48 hours in FBS, or LDS+ 1 μM compactin. Amplicon −3204 bp to −3032 bp distant to TSS served as negative control, whereas a canonical SREBP1 binding site in Ldlr promoter (−38 bp to +60 bp) served as a positive control for SREBP1 activity. (H) Increased association of open chromatin (H3K4me3) and reduced association of repressed chromatin marks (H3K27me3) with the proximal Tgfb1 promoter of cholesterol-depleted KPC3 cells as determined by ChIP-qPCR. The map of genomic Tgfb1 locus is drawn to scale. Data were pooled from two independent experiments. In all figures, data are represented as mean±SEM, p-values determined by independent two-sample Student t-test: *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S6.

Article Snippet: Human TGFβ1 in serum samples from patients was measured by human DuoSet ELISA kit (#DY240, R&D systems).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Activity Assay, Cotransfection, Expressing, Dominant Negative Mutation, Mutagenesis, Transfection, Blocking Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Amplification, Negative Control, Positive Control

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: (A) NSDHL inactivation in well-differentiated KPC3 PDAC cells by CRISPRi is confirmed by absence of NSDHL band on Western blot of total cellular lysates; KPC3 parental cells and GFP-targeted gRNA used as controls. (B) Secreted TGFB1 as determined by ELISA using 48 hour supernatants. Shown, averaged results from 3 independent repeats; error bars, SEM. (C) Histological grading of glandular (grades 1–2) versus solid (grades 3–4) tumor areas in tumors generated via orthotopic implantation of KPC3wt or KPC3 NsdhlCRISPRi cells; p=0.007, two-way Student t-test. Symbols represent individual tumors; black bars, mean±SEM. (D) Cholesterol level in KPPC (n=10) and KPPCN (n=10) clones grown for 48 hours in FBS or LDS media; (1) p=0.004; (2) p=0.0001, Wilcoxon test. Boxplots represent median (black bar) and full range of measurements. (E) Cholesterol levels in PDAC cells conditioned for 48 hours as indicated; L+C, 5%LDS with 1 μM compactin. (F) Representative Western blot of phosphorylated pSMAD2 (Ser465/467) and pSmad3 (Ser423/425) in KPC3 cells cultured for 48 hours in fetal bovine serum (FBS), lipid depleted serum (LDS), or LDS with 1 μM of compactin (L+C) followed by incubation in serum-free DMEM for 4 hours. Indicated samples were treated with TGFβ1 at 10 ng/ml for 30 minutes, and/or SB431542 at 25 μM for 1 hour. (G) Summary results of levels of phosphorylated pSMAD2(Ser465/467) and pSmad3 (Ser423/425) in cholesterol depleted PDAC cells. Results from 3 independent experiments normalized to α-tubulin are shown. (H) Phosphorylated pSMAD2(Ser465/467) in human Capan-2 carcinoma cells conditioned for 48 hours in FBS, LDS with or without 1 μM compactin. Summary results from 3 independent experiments normalized to α-tubulin are shown. (J) Increased nuclear SREBP1 and SREBP2 in Capan-2 cells as in H. (I) Surface versus internalized pools of TGFBR1 and TGFBR2 in KPC3 cells conditioned for 48 hours in 5% FBS or in 5% LDS with 1 μM compactin. Biotinylated (surface) and non-biotinylated (internalized) proteins were affinity separated using streptavidin-agarose beads. (K) Levels of Tgfb1, Tgfb2 and Tgfb3 mRNA as assessed by qRT-PCR, in cells grown in indicated media for 48 hrs. (L) ELISA measurement of secreted TGFβ1 in supernatants of KPC3 cells conditioned in indicated media for 48 hrs; (M) Expression of Zeb2, Tgfb1 and Wnt10b mRNA as assessed by qRT-PCR in KPC3 and KPC634 PDAC cells cultured for 48 hours in media supplemented with FBS, LDS or LDS+compactin (1 μM). In graphs B, E, G-J and K-M, statistical p-values by two-way Student t-test are indicated as: *, <0.05, **, <0.01, ***, <0.001. See also Figure S5.

Article Snippet: Human TGFβ1 in serum samples from patients was measured by human DuoSet ELISA kit (#DY240, R&D systems).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Clone Assay, Cell Culture, Incubation, Quantitative RT-PCR, Expressing

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human TGFβ1 in serum samples from patients was measured by human DuoSet ELISA kit (#DY240, R&D systems).

Techniques: Polymer, Plasmid Preparation, Virus, Clone Assay, Mutagenesis, Modification, Cloning, Recombinant, Antibody Labeling, Transfection, Marker, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Activation Assay, Viability Assay, Gentle, Reporter Assay, Purification, Multiplex Assay, RNA Sequencing, Sequencing, Software, Imaging

Journal: Cell Communication and Signaling : CCS

Article Title: Secreted RCN3 acts as an early epithelial-fibroblast mediator via TGFβR1-Smad signaling in post-ALI pulmonary fibrosis

doi: 10.1186/s12964-026-02690-w

Figure Lengend Snippet: RCN3 activates fibroblasts via canonical TGF‑β signaling and engages TGFβR1. A RNA-Sequencing analysis of total RNA from CP-H011 treated with vehicle or 0.1 µg/mL rhRCN3 ( n = 3). A total of 543 differentially-expressed genes, (DEGs) with the foldchange (FC) > 1.5 and FDR < 0.05 were identified. Hierarchical clustering presented the gene expression profiles segregated based on RCN3 treatment. B GO enrichment of these DEGs, prioritized by gene ratio and BH-corrected FDR ( p < 0.05); top processes are shown. C The KEGG pathway analysis of the differentially expressed genes. D Immunoblot indicates that the phosphorylation of Smad3 was upregulated, while the phosphorylation of AKT, Erk1/2 and JNK remained unchanged. The ratios of phosphorylated/total proteins are presented in scatter plots as values relative to control ( n = 4, ** p < 0.01 versus vehicle). E The SBE (SMAD binding element) reporter assay validates that rhRCN3 activates the TGFβ signaling pathway ( n = 5, *** p < 0.001). F Immunoprecipitation assay shows that his-RCN3 interacts with TGFβR1 but not TGFβR2. The His-immunoprecipitates and total lysate (input) were analyzed by immunoblot using anti-TGFβR1 and anti-TGFβR2 antibodies. G Immunoblot of Smad3 in the fibroblast cells treated with 0.1 µg/mL rhRCN3 in response to TGFβ receptor inhibitors, LY364947 and LY2109761. H Knockdown of TGFβR2 did not inhibit the phosphorylation of Smad3 induced by rhRCN3 (0.1 µg/mL). I Knockdown of TGFβR1 inhibited the phosphorylation of Smad3 induced by rhRCN3 (0.1 µg/mL). J Biolayer interferometry (BLI) studies using rhRCN3 and TGFβR1 proteins indicate the interaction between RCN3 and TGFβR1 exhibiting a clear concentration gradient with an affinity (KD) of 26.91nM. BLI binding representative of at least 4 independent traces. K The best potential direct interaction models of RCN3 with the extracellular domain of the TGFβR1 predicted by the protein-protein docking tool ClusPro server. The predicted structures of RCN3 obtained from the UniProt by AlphaFold model (AF- Q15910 and AF- Q96D15 ) and the solution structure of the extracellular domain of the TGFβR1 (PDB: 2L5S) were submitted to the ClusPro web-based server to analyze their interaction ( http://cluspro.bu.edu/ ). The model shows that residues 60–70 and 80–93 of RCN3 fold and form a docking surface that inserts into the cavity of extracellular domain of TGFβR1. Abbreviations: DEG, differentially expressed gene; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; SBE, SMAD‑binding element; IP, immunoprecipitation; BLI, biolayer interferometry; FC, fold change; FDR, false discovery rate

Article Snippet: Reagents and drugs used were human/mouse recombinant RCN3 (Abcam, ab276552/ab277008), human TGF-β (Sigma, GF346), LPS (Sigma, L6529), SIS3 (MCE, HY-13013), anti-RCN3 antibody (Abcepta, AP5788c), TGFβ receptor inhibitors- LY364947 (Abcam, ab141890) /LY2109761 (MCE, HY-12075), TGFβR2 siRNA (Santa Cruz, sc-36657), control siRNA-A (Santa Cruz, sc-37007), MiQumn G25 centrifugal desalting column (Chromsep, 110607), anti-His antibody (CST, 12698), anti-TGFβR1 antibody (Abcam, ab235578), anti-TGFβR2 antibody (Abcam, ab259360), anti-Smad3 (phospho S423 + S425) antibody (Abcam, ab52903), anti-Smad3 antibody (Abcam, ab208182), anti-Histone H3 antibody (CST, 9715).

Techniques: RNA Sequencing, Gene Expression, Western Blot, Phospho-proteomics, Control, Binding Assay, Reporter Assay, Immunoprecipitation, Knockdown, Concentration Assay

Journal: bioRxiv

Article Title: Oct1 recruits the histone lysine demethylase Utx to canalize lineage specification

doi: 10.1101/2020.12.01.406488

Figure Lengend Snippet: (A) HOMER motif analysis of Oct1 peaks that are both shared with Oct4 in ESCs, and maintained after D6 of MD differentiation. (B) Smad3 immunoblot using cell lysates immunoprecipitated with Oct1 antibodies, or rabbit IgG controls. D6 MD-differentiated cells were used. 20% input is shown (lane 1). (C) Smad3 gene expression is shown in violin plots for parental ESCs at D6 of MD differentiation. (D) Schema for Oct1/Smad reporter assay. A segment of a mouse Myog enhancer element containing multiple octamer and Smad motifs was cloned with the core CMV promoter upstream of secreted nLuc and co-transfected into Oct1-deficient MEFs together with a construct encoding constitutive secreted mCherry as a normalization control. Added TGFb1 and co-transfected mouse Oct1 supply Oct1 and Smad3 activity. (E) Transfected WT (left panel) or Oct/Smad mutant (right panel) Myog enhancer constructs were supplied with Oct1, recombinant purified TGFb1 treatment, or both. For each construct, secreted luciferase activity was assessed relative to secreted mCherry expressed from a co-transfected plasmid. An average of three experimental replicates is shown. Error bars denote ±SEM. For the situation in which both Oct1 and TGFb1 are both supplied, fold changes relative to a double-mutant construct are also shown. (F) Signal tracks ( Mm10 v. D191020) showing Oct1ChIP-seq enrichment at the Myog locus. Shown above are RNA-seq tracks in differentiated and undifferentiated parental and cKO cells. Annotated enhancer elements are shown below.

Article Snippet: Where indicated, transfected cells were provided with 5 ng recombinant mouse TGFb1 protein (R&D Systems). mCherry fluorescence was determined first by exciting at 570 nm and measuring emission at 610 nm with a 100 msec time delay using an Envision Xcite Multilabel Plate Reader.

Techniques: Western Blot, Immunoprecipitation, Gene Expression, Reporter Assay, Clone Assay, Transfection, Construct, Control, Activity Assay, Mutagenesis, Recombinant, Purification, Luciferase, Plasmid Preparation, RNA Sequencing

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet: miR-194-3p, TAB2 and iPSC gene expression in GSC vs. ML counterparts, miR-194-3p modulates NF-κB activation in GSC vs. ML matched pairs, TAB2 expression correlates with P and M subtype (A) The expression levels of miR-194-3p in matched pair ML and GSC cell lines. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (B) Relative expression levels of TAB2 and iPSC markers in matched GSC and ML pairs. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (C) Top: Quantification of TAB2 protein levels in panel of ML and GSC cell lines. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. Middle: TAB2 protein levels in OSU13, OSU20 and OSU53 matched pair ML and GSC cell lines. Bottom: TAB2 protein levels in commercially available GBM cell lines; Corresponding mRNA expression are represented above images. (D) Western blot analysis of NF-κB activation in matched pair ML and GSC cell lines. (E) NF-κB activation following the transfection of 100 nM miR-194-3p mimic and inhibitor in U87 ML and OSU68 ML cells. (F) The heatmap represents the relative expression of TAB2, mesenchymal and proneural markers in the GSC cell lines. Data is row-normalized data. (G) Expression levels of proneural and mesenchymal markers in miR-194-3p isogenic cells. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. ns not significant; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Gene Expression, Activation Assay, Expressing, Two Tailed Test, Western Blot, Transfection

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet: Luciferase reporter assay confirming inhibition of TAB2 by miR-194-3p, expression level of TAB2, NF-κB, and iPSC markers in U87-TAB2 isogenic cells (A) Schematic of the TAB2 3′ UTR miRNA reporter system. Sequences representing the TAB2 3′ UTR and a mismatched control were cloned into the reporter system downstream of the firefly luciferase protein (PGK). The Renilla luciferase protein was driven off a second promoter (SV40) and used as a control for transfection efficiency. (B) The miRNA reporter vectors were transfected in U87 cells for 24 h before treating with 100 nM miR-194-3p mimic or inhibitor. Activity is represented by the amount of firefly luciferase normalized to the amount of Renilla luciferase. Data are shown as mean ± SEM. Statistical significance was tested using two-way ANOVA (DF = 16). (C) U87-EV and U87-194-3p-OE were transfected using the same conditions as B. Data are shown as mean ± SEM. Statistical significance was tested using two-way ANOVA (DF = 12). (D) Relative mRNA expression of TAB2 in U87 cells transduced with shTAB2. (E) Relative TAB2, NF-κB, and iPSC marker expression levels in TAB2 isogenic cell lines. ns not significant; ∗∗∗∗p < 0.0001.

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Luciferase, Reporter Assay, Inhibition, Expressing, Control, Clone Assay, Transfection, Activity Assay, Transduction, Marker

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet: TAB2 silencing transforms GSC/P phenotype by downregulating NF-κB and iPSC genes promoting adherent/M phenotype (A) Western blot analysis of the indicated proteins in U87-shTAB2 and U87-miR-194-3p isogenic cells. (B) TAB2 expression levels in OSU53-shTAB2 cell lines. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (C) Microscopic image showing the morphological features of isogenic shTAB2 cells. (D and E) Relative NF-κB activity in TAB2 and miR-194-3p isogenic cells ± competitive inhibitor measured using the NF-κB p65 Transcription Factor Assay Kit. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (F) Expression levels of P/M and iPSC marker in isogenic OSU53-shTAB2 cells. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test. (G) Western blot analysis of TAB2, NF-κB, and iPSC markers in OSU53-shTAB2 and OSU53-miR-194-3p isogenic cells. ns not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Western Blot, Expressing, Two Tailed Test, Activity Assay, Transcription Factor Assay, Marker

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet: TAB2 silencing enhances radiation sensitivity and prolongs survival of nude mice bearing intracranial tumors and graphical summary (A) Protein expression levels of TAB2 and NF-κB activation in T98G and OSU2 cell lines transduced with shTAB2. (B–D) Clonogenic assay comparing the radiosensitivity of NT and shTAB2 cell lines. Cells were fixed and stained 14 days following radiation. Data are shown as mean ± SEM. Statistical significance was tested using two-tailed unpaired t-test comparing the 6 Gy RER values of shTAB2 to NT in each cell line. The p value for each cell line was less than 0.01. (E) Kaplan-Meier curves demonstrating the survival of mice following intracranial implantation of U87-, OSU2-, and OSU53-shTAB2 isogenic cell lines. The p value was calculated using the two-sided log rank test. (F) H&E-stained coronal sections of tumors from nude mice bearing OSU53-shTAB2 isogenic cells. (G) Schematic summary representing the role of miR-194-3p in regulating PMT by inhibiting TAB2 and NF-κB activity. ns not significant; ∗∗p < 0.01.

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Expressing, Activation Assay, Transduction, Clonogenic Assay, Staining, Two Tailed Test, Activity Assay

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet:

Article Snippet: Taqman gene expression assay probes were used to measure the mRNA levels of 194-1 (Thermo Fisher: Cat# Hs04231530_s1), 194-2 (Thermo Fisher: Cat# Hs04331541_s1), ALDH1A3 (Thermo Fisher: Cat# Hs00167476_m1), CD44 (Thermo Fisher: Cat# Hs05662929_s1), LYN (Thermo Fisher: Cat# Hs01015818_g1), WT1 (Thermo Fisher: Cat# Hs01103751_m1), CD133 (Thermo Fisher: Cat# Hs01009259_m1), Nestin (Thermo Fisher: Cat# Hs04187831_g1), OLIG1 (Thermo Fisher: Cat# Hs00907227_s1), SOX2 (Thermo Fisher: Cat# Hs04234836_s1), klf4 (Thermo Fisher: Cat# Hs00358836_m1), OCT4 (Thermo Fisher: Cat# Hs00999632_g1), FUT4 (Thermo Fisher: Cat# Hs01106466_s1), NCAM1 (Thermo Fisher: Cat# Hs00941830_m1), Nanog (Thermo Fisher: Cat# Hs02387400_g1), TAB2 (Thermo Fisher: Cat# Hs00248373_m1), CMYC (Thermo Fisher: Cat# Hs00153408_m1), DOCK5 (Thermo Fisher: Cat# Hs07287975_m1), FAS (Thermo Fisher: Cat# Hs00236330_m1), HFE (Thermo Fisher: Cat# Hs00373474_m1), LGALS3 (Thermo Fisher: Cat# Hs07288627_m1), MAL (Thermo Fisher: Cat# Hs00707014_s1), RHOF (Thermo Fisher: Cat# Hs00368032_m1), SOWAHA (Thermo Fisher: Cat# Hs00703106_s1), C21orf91 (Thermo Fisher: Cat# Hs05040994_s1), TRIM23 (Thermo Fisher: Cat# Hs01106626_m1).

Techniques: Recombinant, Protease Inhibitor, Transfection, Isolation, cDNA Synthesis, MTT Assay, Extraction, Transcription Factor Assay, Expressing, Plasmid Preparation, Reporter Assay, Staining, Negative Control, shRNA, Software