Journal: Scientific Reports

Article Title: MAD2B acts as a negative regulatory partner of TCF4 on proliferation in human dermal papilla cells

doi: 10.1038/s41598-017-10350-w

Figure Lengend Snippet: Effect of MAD2B on TCF4-induced DPC proliferation. DPCs were transfected with the vectors, as indicated, for 1‒7 d. The cell viability was determined through CCK-8 assays. The results shown are representative of three independent experiments. * P < 0.05, ** P < 0.01 vs. control; n = 3. CCK-8, cell counting kit-8; TCF4, T cell factor 4; DPCs, dermal papilla cells.

Article Snippet: Cells were lysed with RIPA buffer (Beyotime) and the lysates were incubated with immunoglobulin G (IgG; negative control) or a monoclonal antibody against MAD2B or TCF4 (R&D Systems) using a Co-IP kit (Thermo Fisher Scientific).

Techniques: Transfection, CCK-8 Assay, Control, Cell Counting

Journal: Scientific Reports

Article Title: MAD2B acts as a negative regulatory partner of TCF4 on proliferation in human dermal papilla cells

doi: 10.1038/s41598-017-10350-w

Figure Lengend Snippet: Effect of MAD2B on TCF4-mediated mRNA and protein expression and secretion of cytokines in DPCs. DPCs were transfected with the vectors, as indicated, for 24 h. Q-PCR, Western blot and ELISA were performed to examine the mRNA expression ( A ), protein expression ( B ), and secretion ( C ) of HGF, IGF-1, and VEGF, respectively. The results shown are representative of three independent experiments. * P < 0.05, ** P < 0.01 vs. control; n = 3. TCF4, T cell factor 4; DPCs, dermal papilla cells.

Article Snippet: Cells were lysed with RIPA buffer (Beyotime) and the lysates were incubated with immunoglobulin G (IgG; negative control) or a monoclonal antibody against MAD2B or TCF4 (R&D Systems) using a Co-IP kit (Thermo Fisher Scientific).

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: MAD2B acts as a negative regulatory partner of TCF4 on proliferation in human dermal papilla cells

doi: 10.1038/s41598-017-10350-w

Figure Lengend Snippet: MAD2B physically interacts with TCF4. DPCs were transduced with the vectors, as indicated, for 24 h. Cell lysates were collected and immunoprecipitated with IgG (negative control), TCF4 antibody ( A and C ), or MAD2B antibody ( B ). The immunoprecipitates were blotted with MAD2B ( A ), TCF4 ( B ), and MAD2B/β-catenin ( C ). The results shown are representative of three independent experiments. IgG, Immunoglobulin G; TCF4, T cell factor 4; DPCs, dermal papilla cells.

Article Snippet: Cells were lysed with RIPA buffer (Beyotime) and the lysates were incubated with immunoglobulin G (IgG; negative control) or a monoclonal antibody against MAD2B or TCF4 (R&D Systems) using a Co-IP kit (Thermo Fisher Scientific).

Techniques: Transduction, Immunoprecipitation, Negative Control

Journal: Scientific Reports

Article Title: MAD2B acts as a negative regulatory partner of TCF4 on proliferation in human dermal papilla cells

doi: 10.1038/s41598-017-10350-w

Figure Lengend Snippet: Effect of MAD2B on TCF4-mediated transactivation. ( A ) MAD2B suppresses TCF4-mediated transactivation through inhibiting TCF4 binding to DNA. DPCs were transfected with the vectors, as indicated. TOP-FLASH or FOP-FLASH was co-transfected into DPCs. Relative luciferase activity was determined 48 h after transfection and is shown as the TOP/FOP ratio. pRL-TK was also co-transfected as an internal control. ( B ) MAD2B knockdown increased TCF4-mediated transactivation. The results shown are representative of three independent experiments. ** P < 0.01 vs. control; n = 3. DPCs, dermal papilla cells; TCF4, T cell factor 4.

Article Snippet: Cells were lysed with RIPA buffer (Beyotime) and the lysates were incubated with immunoglobulin G (IgG; negative control) or a monoclonal antibody against MAD2B or TCF4 (R&D Systems) using a Co-IP kit (Thermo Fisher Scientific).

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Control, Knockdown

Journal: Nature Communications

Article Title: Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen

doi: 10.1038/s41467-022-30134-9

Figure Lengend Snippet: a DAXX degradation after infection. 293T-ACE2 cells were infected with SARS-CoV-2 at MOI 0.1. After 24 h, cells were harvested and levels of DAXX, Lamin B, HSP90, Actin, GAPDH, Tubulin, TRIM22, RIG-I and of the viral protein spike were analyzed by Western Blot. A Western Blot representative of 3 independent experiments is shown. b GRL0617 and MG132 treatments restore DAXX expression. 293T-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.1. When indicated, cells were pretreated 2 h before infection with GRL0617 (at the indicated concentrations), or with MG132 (10 µM), a proteasome inhibitor, or Masitinib (10 µM) a 3CL inhibitor. After 24 h, cells were harvested and levels of DAXX, GAPDH and of the viral protein spike were analyzed by Western Blot. A Western Blot representative of 3 independent experiments is shown. c GRL0617 treatment restores DAXX localization. 293T-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.1. 24 h post-infection, cells were labelled with Hoescht and with antibodies against dsRNA (detecting viral RNA, in green) and HA (detecting DAXX, in red). When indicated, cells were treated with 50 µM of GRL0617 at the time of infection. Scale bars correspond to 10 µm. Images are representative of 3–6 different fields from 2 independent experiments. d – f : Nsp3 induces DAXX degradation. d 293T-ACE2 cells were transfected with 1 μg of the indicated viral proteins. After 24 h, the levels of DAXX and GAPDH were analyzed by Western Blot. e 293T-ACE2 cells were transfected with the indicated amounts of Nsp3. After 24 h, the levels of DAXX and GAPDH were analyzed by Western Blot. f 293T-ACE2 cells were transfected with 1 μg of Nsp3 or of pcDNA. 6 h post transfection, cells were also, when indicated, treated with 50 µM of GRL0617. Of, 24 h after transfection, the levels of DAXX and GAPDH were analyzed by Western Blot. Western Blots representative from 2 independent experiments are shown. The quantification of band intensity for Fig. 6d–f is shown in Fig. . Source data are provided as a Source Data file.

Article Snippet: The plasmids encoding mCherry-tagged viral proteins were a gift from Bruno Antonny and ordered through Addgene: Nsp3 -mCherry (#165131); Nsp4-mCherry (#165132); Nsp6-mCherry (#165133); Nsp7-mCherry (#165134); Nsp10-mCherry (#165135); Nsp13-mCherry (#165136); Nsp14-mCherry (#165137).

Techniques: Infection, Western Blot, Expressing, Transfection

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: MIN6 cells were plated at 5 × 10 5 cells per well in 6-well plates and exposed to a TCF7L2-specific short hairpin RNA (shTCF7L2) or a scrambled shRNA (shScr) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A ) Western blots showing the TCF7L2 protein content after E 2 or P 4 treatment. ( B ) Viable cells. ( C,D ) Basal and stimulated insulin/proinsulin secretions (normalized to viable cell numbers). ( E,F ) Stimulatory indexes. ( G ) Proinsulin-to-insulin ratio. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 shTCF7L2 vs. shScr.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: shRNA, Cell Culture, Western Blot, Control

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: MIN6 cells were plated at 5 × 10 5 cells per well in 6-well plates and exposed to high glucose concentration (33.3 mM) and transfected with TCF7L2-IRES2-EGFP (OE-TCF7L2) or a control vector (CV) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A ) Western blots showing the TCF7L2 protein content after E 2 or P 4 treatment. ( B ) Viable cells. ( C , D ) Basal and stimulated insulin/proinsulin secretions (normalized to viable cell numbers). ( E,F ) Stimulatory indexes. ( G ) Proinsulin-to-insulin ratio. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 OE-TCF7L2 vs. CV.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: Concentration Assay, Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot

Journal: Scientific Reports

Article Title: TCF7L2 involvement in estradiol- and progesterone-modulated islet and hepatic glucose homeostasis

doi: 10.1038/srep24859

Figure Lengend Snippet: HepG2 cells (2.5 × 10 5 cells per well) were seeded in 6-well plates and exposed to a TCF7L2-specific short hairpin RNA (shTCF7L2) or a scrambled shRNA (shScr) for 72 h, or transfected with TCF7L2-IRES2-EGFP (OE-TCF7L2) or a control vector (CV) for 72 h, then cultured for 24 h in the presence of 100 nM E 2 or 1 μM P 4 . ( A,B ) 2-NBDG uptake. ( C,D ) Glucose production. ( E,F ) Western blot. * P < 0.05 vs. sex hormone treatment control; # P < 0.05 shTCF7L2 vs. shScr or OE-TCF7L2 vs. CV.

Article Snippet: The antibodies previously used for Western blots were the anti-human TCF7L2 antibody (1:800; 13838-1-AP, Proteintech, USA), anti-human PEPCK antibody (1:1000; 14892-1-AP; Proteintech, USA), anti-human GLUT2 antibody (1:800; 20436-1-AP; Proteintech, USA), anti-human IRS2 antibody (1:1000; 20702-1-AP; Proteintech, USA), anti-human pAKT antibody (1:1000; 60072-1-Ig; Proteintech, USA), anti-human AKT antibody (1:1000; 10176-2-AP; Proteintech, USA), anti-human pGSK antibody (1:1000; 14850-1-AP; Proteintech, USA), anti-human GSK antibody (1:1000; 22104-1-AP; Proteintech, USA), anti-human pERK1/2 antibody (1:1000; 3441-100; BioVision, USA), anti-human ERK1/2 antibody (1:1000; 16443-1-AP; Proteintech, USA), and anti-human GAPDH antibody (1:1000; 10494-1-AP; Proteintech, USA).

Techniques: shRNA, Transfection, Control, Plasmid Preparation, Cell Culture, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Effect of Trinucleotide Repeat Expansion on the Expression of TCF4 mRNA in Fuchs' Endothelial Corneal Dystrophy

doi: 10.1167/iovs.18-25760

Figure Lengend Snippet: Schematic image of TCF4 and the genomic regions recognized by hydrolysis probes. Hs00971338 recognized the N-terminal side of active domain 1 (AD1), Hs00162613 recognized the N-terminal side of active domain 2 (AD2), and Hs00972432 recognized the N-terminal side of basic helix-loop-helix (bHLH) of TCF4.

Article Snippet: The expression level of TCF4 determined by Hs00971338 was significantly higher in patients with FECD with or without CTG TNR expansion >50 than in non-FECD subjects (control: 7.2 ± 4.8; FECD without expansion: 22.3 ± 29.9; and FECD without expansion: 47.4 ± 46.3; P < 0.01).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Effect of Trinucleotide Repeat Expansion on the Expression of TCF4 mRNA in Fuchs' Endothelial Corneal Dystrophy

doi: 10.1167/iovs.18-25760

Figure Lengend Snippet: Expression of TCF4 mRNA in corneal endothelium of patients with FECD. (A) Total RNA was extracted from the corneal endothelium of the 203 patients with FECD and 35 non-FECD subjects, and cDNA was synthesized. The expression level of TCF4 determined by Hs00971338 was significantly higher in FECD with or without a CTG expansion of a trinucleotide repeat (TNR) expansion larger than 50 when compared with non-FECD subjects. In addition, TCF4 level was significantly higher in FECD with expansion than in FECD without expansion. * P < 0.01. (B) Expression level of TCF4 determined by Hs00162613 was significantly higher in FECD with or without a CTG TNR expansion larger than 50 than in non-FECD subjects. *P < 0.01. (C) Expression level of TCF4 determined by Hs00972432 was higher in FECD with or without CTG-TNR expansion larger than 50 when compared to non-FECD subjects, but TCF4 level showed a statistically significant difference only in FECD with expansion when compared to the control subjects. The TCF4 level determined by Hs00972432 was significantly higher in FECD with expansion than in FECD without expansion. *P < 0.01. The statistical significance was determined with the Steel-Dwass test.

Article Snippet: The expression level of TCF4 determined by Hs00971338 was significantly higher in patients with FECD with or without CTG TNR expansion >50 than in non-FECD subjects (control: 7.2 ± 4.8; FECD without expansion: 22.3 ± 29.9; and FECD without expansion: 47.4 ± 46.3; P < 0.01).

Techniques: Expressing, Synthesized, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Effect of Trinucleotide Repeat Expansion on the Expression of TCF4 mRNA in Fuchs' Endothelial Corneal Dystrophy

doi: 10.1167/iovs.18-25760

Figure Lengend Snippet: Correlation between CTG-TNR length and expression of TCF4 mRNA. (A) CTG TNR length was evaluated by analyzing the genomic DNA from peripheral blood and expression levels were plotted for TCF4 mRNA in the corneal endothelium of patients with FECD. Spearman's correlation coefficient by rank test revealed a weak positive correlation between CTG-TNR length and expression level of TCF4 determined by Hs00971338 (ρ = 0.24, P < 0.01). (B) CTG-TNR length did not show a significant correlation with the expression level of TCF4 determined by Hs00162613 (ρ = 0.01, P = 0.884). (C) CTG-TNR length showed a weak positive correlation with the expression level of TCF4 determined by Hs00972432 (ρ = 0.22, P < 0.01). Correlation was determined by a rank test using Spearman's correlation coefficient.

Article Snippet: The expression level of TCF4 determined by Hs00971338 was significantly higher in patients with FECD with or without CTG TNR expansion >50 than in non-FECD subjects (control: 7.2 ± 4.8; FECD without expansion: 22.3 ± 29.9; and FECD without expansion: 47.4 ± 46.3; P < 0.01).

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Effect of Trinucleotide Repeat Expansion on the Expression of TCF4 mRNA in Fuchs' Endothelial Corneal Dystrophy

doi: 10.1167/iovs.18-25760

Figure Lengend Snippet: Correlation between the genotype of TCF4 SNP, rs613872, and TCF4 expression level. Genotyping of rs613872 in TCF4 was performed by PCR. No statistically significant correlation was revealed by the Steel-Dwass test between the genotype of TCF4 SNP rs613872 and the expression level of TCF4 determined by three probes: (A) Hs00971338, (B) Hs001612613, and (C) Hs00972432.

Article Snippet: The expression level of TCF4 determined by Hs00971338 was significantly higher in patients with FECD with or without CTG TNR expansion >50 than in non-FECD subjects (control: 7.2 ± 4.8; FECD without expansion: 22.3 ± 29.9; and FECD without expansion: 47.4 ± 46.3; P < 0.01).

Techniques: Expressing

Journal: The EMBO Journal

Article Title: TCF / LEF dependent and independent transcriptional regulation of Wnt/β‐catenin target genes

doi: 10.15252/embj.201798873

Figure Lengend Snippet: The presence of the desired genomic deletions at TCF/LEF or CTNNB1 loci was confirmed by PCR, using primers flanking the regions near the gRNAs (upper panels). Uncropped version of the same Western blot panels shown in Fig 1B indicates that TCF7L, LEF1, TCF7L1, TCF7L2 and β‐catenin proteins were undetectable in the new cell lines generated in this study. Note that the PCR panel showing TCF7L1 has been obtained by adjoining the “ladder” lane close to the sample lanes. The artificial merging of the lanes is marked by a black vertical line.Source data are available online for this figure.

Article Snippet: The following plasmids were obtained from Addgene: pSpCas9(BB)‐2A‐Puro (PX459), pcDNA3flagFKHRL1 (FOXO3) (#10709), pcDNA‐HA‐TCF1 (TCF7) (#40620), pcDNA‐myc‐TCF4 (TCF7L2) (#16512), pc‐DNA3hE47 (TCF7L1) (#16059).

Techniques: Western Blot, Generated