Journal: The Journal of Cell Biology

Article Title: TGFβ 3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

doi: 10.1083/jcb.200306024

Figure Lengend Snippet: Effect of inhibitors of TGF β 3 , RhoA, and MEK pathways on palatal EMT. Hematoxylin and eosin-stained sections of palates fixed at different stages of palatogenesis (A–D) and after treatment with TGFβ 3 -blocking antibody (E and F), C3 (G–I) and U0126 (J–L). (A–D) Normal palatal shelves adhere to form the MEE seam by 12 h (A, arrow) Then, at 24 h the seam starts to break into small islands of epithelial cells (B). By 48 h, a few remnants of MEE are left (C). EMT is complete by 60 h (D, arrow). (E and F) Palates treated with anti-TGFβ 3 antibody (2 ng/μL) form a seam (E, arrow) by 12 h, but do not transform the MEE by 60 h (F, arrow). (G–I) Palates treated with C3, a RhoA inhibitor, form an MEE by 12 h (G, arrow) that transforms into mesenchyme by 24–48 h (H and I). The palates reach complete confluence by 60 h (not depicted). (J–L) Palates treated with U0126, a MEK1/2 inhibitor, form MEE seams that also undergo transformation (J, 24 h; K, 36 h) to complete EMT at 60 h (L, arrow). There were no effects of RhoA inhibitor (C3) or MEK inhibitor (U0126) on normal palatogenesis.

Article Snippet: The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINE™ PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/μl anti–mouse TGFβ 3 -blocking antibody (R&D Systems). (5) 2 ng/μl recombinant TGFβ 3 (R&D Systems). (6) AS β-catenin oligonucleotides 5′-GGAGTTTAACCACAACAGGCAGTCC-3′ and control sense 5′-CCTGACGGACAACACCAATTTGAGG-3′, as described by , used by , were tested in our laboratory. (7) 15 μM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem).

Techniques: Staining, Blocking Assay, Transformation Assay

Journal: The Journal of Cell Biology

Article Title: TGFβ 3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

doi: 10.1083/jcb.200306024

Figure Lengend Snippet: Quantification of disappearance of cells from epithelial seam during EMT. Using LCM on 8-μm-thick frozen sections from the middle one third of the palate, we estimated cell numbers in each section of the MEE seam from 12 to 60 h after the beginning of the culture. On the ordinate, we estimated seam transformation by the number of cells remaining in it. Stage 1, 0 cells (confluence); stage 2, 1–19 cells; stage 3, 20–39 cells; stage 4, 40–59 cells; stage 5, 60–76 cells; stage 6, untransformed seam (75 cells). Palates were treated from the beginning of culture with the following reagents: TGFβ 3 -blocking antibody, TGFβ 3 -blocking antibody plus LEF1, RhoA inhibitor C3, MEK1/2 inhibitor U0126, DN LEF1 virus, DN Smad4 adenovirus, and AS β-catenin. A (a) is the untreated control. Length of time is indicated by the decoration inside the graph bars. Untreated/control palates are confluent by 60 h (A, a), and palates treated with anti-TGFβ 3 antibody do not transform (A, b). Treatment with C3 (A, c), U0126 (A, d), and AS β-catenin (B, d) have no effect on MEE seam transformation. LEF1 does not rescue blockage by anti-TGFβ 3 antibody (B, a). Treatment with DN LEF1 (B, b) or DN SMAD4 (B, c) inhibits transformation.

Article Snippet: The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINE™ PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/μl anti–mouse TGFβ 3 -blocking antibody (R&D Systems). (5) 2 ng/μl recombinant TGFβ 3 (R&D Systems). (6) AS β-catenin oligonucleotides 5′-GGAGTTTAACCACAACAGGCAGTCC-3′ and control sense 5′-CCTGACGGACAACACCAATTTGAGG-3′, as described by , used by , were tested in our laboratory. (7) 15 μM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem).

Techniques: Transformation Assay, Blocking Assay, Virus

Journal: The Journal of Cell Biology

Article Title: TGFβ 3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

doi: 10.1083/jcb.200306024

Figure Lengend Snippet: LEF1 gene expression (real-time quantitative PCR). LEF1 mRNA expression was measured by highly sensitive real-time PCR in LCM-dissected MEE cells from frozen sections. (A) Palates were treated with exogenous LEF1 virus, anti-TGFβ 3 antibody, DN LEF1 virus, and exogenous recombinant TGFβ 3 . (B) Palates were treated with exogenous LEF1 virus plus anti-TGFβ 3 -blocking antibody, AS β-catenin oligodeoxynucleotide, DN Smad4 adenovirus, C3, a Rho inhibitor, and U0126, a MEK1/2 inhibitor. Length of time is indicated within each graph bar, as in . (A, a) In the untreated palates (Untr/Cntrl), LEF1 expression peaks at 36 h, at which time EMT also peaks. (A, b) In presence of exogenous LEF1, there is a large increase in LEF1 mRNA expression that peaks at 24 h instead of 36 h. (A, c) TGFβ 3 inhibition by blocking antibody causes complete inhibition of LEF1 mRNA expression and EMT (A, d) DN LEF1 causes palates to cease endogenous LEF1 mRNA expression and remain untransformed. (A, e) Exogenous TGFβ 3 causes LEF1 mRNA and protein to up-regulate and reach a peak at 24 h without significantly hastening the EMT process. (B, a) Exogenous LEF1 DNA increases LEF1 mRNA, but the seam remains untransformed if palates are treated with TGFβ 3 -blocking antibody. (B, b) AS β-catenin did not affect LEF1 mRNA expression as compared with the control (A, a). (B, c) DN Smad4 inhibits LEF1 mRNA expression and MEE remains untransformed. (B, d) RhoA inhibitor C3 had no effect on normal LEF1 mRNA expression or EMT, (B, c) and neither did MEK1/2 inhibitor U0126 (B, e).

Article Snippet: The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINE™ PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/μl anti–mouse TGFβ 3 -blocking antibody (R&D Systems). (5) 2 ng/μl recombinant TGFβ 3 (R&D Systems). (6) AS β-catenin oligonucleotides 5′-GGAGTTTAACCACAACAGGCAGTCC-3′ and control sense 5′-CCTGACGGACAACACCAATTTGAGG-3′, as described by , used by , were tested in our laboratory. (7) 15 μM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Virus, Recombinant, Blocking Assay, Inhibition

Journal: The Journal of Cell Biology

Article Title: TGFβ 3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

doi: 10.1083/jcb.200306024

Figure Lengend Snippet: Immunolocalization of Smad2, phospho-Smad2, and Smad4 in untreated and treated (TGF β 3 -blocking antibody and DN Smad4) palates. (A) Phospho-Smad2 is intensely present in the nuclei of normal palatal MEE (12 h), indicating it is translocated by Smad4 into the nucleus (arrow) during normal palatogenesis. (B) Phospho-Smad2 in MEE of palates treated (36 h) with DN Smad4 is located in the cytoplasm and no nuclear localization occurs (arrow), confirming that Smad4 is required for nuclear translocation of phospho-Smad2. (C) Unphosphorylated Smad2 is located in the cytoplasm of MEE cells not in the nucleus (arrow) of palates lacking Smad4 after 12 h of culture. (D) Smad2 is also located in cytoplasm of the MEE cells (36 h), but not in the nucleus (arrow) in the presence of blocking TGFβ 3 , confirming that TGFβ 3 is required for the phosphorylation of Smad2. (E) Smad2 in MEE of palates (36 h) treated with DN Smad4 is located in the cytoplasm of the cell, but not in the nucleus (arrow). At this late phase, the MEE is still untransformed. (F) DN Smad4 inhibits the transport of Smad4 itself to the nucleus. Thus, nuclear Smad2/4 is not available to activate LEF1, and these palates do not transform.

Article Snippet: The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINE™ PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/μl anti–mouse TGFβ 3 -blocking antibody (R&D Systems). (5) 2 ng/μl recombinant TGFβ 3 (R&D Systems). (6) AS β-catenin oligonucleotides 5′-GGAGTTTAACCACAACAGGCAGTCC-3′ and control sense 5′-CCTGACGGACAACACCAATTTGAGG-3′, as described by , used by , were tested in our laboratory. (7) 15 μM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem).

Techniques: Blocking Assay, Translocation Assay

Journal: International immunopharmacology

Article Title: Foxp2 inhibits Th9 cell differentiation and attenuates allergic airway inflammation in a mouse model of ovalbumin-induced asthma.

doi: 10.1016/j.intimp.2022.109060

Figure Lengend Snippet: Fig. 1. Anti-IL-9 antibody inhibits Th9 cells differentiation in asthmatic mice. Forty BALB/c mice were randomly divided into four groups: control group (Control), OVA-induced asthma model group (OVA), mouse IgG injection treatment group (IgG) and anti-IL-9 mAb injection treatment group (anti-IL-9). Four hours after the last OVA stimulation, the mice were anesthetized and killed to collect bronchoalveolar lavage fluid (BALF) and lung tissue. A-B. The percentage of Th9 cells in BALF was evaluated by flow cytometry in each group. C-D. The percentage of Th9 cells in lung tissue was evaluated by flow cytometry in each group. E-F. The levels of IL-9 in BALF and lung tissue were measured by ELISA in each group. G-H. The mRNA expression of BATF and IRF4 in lung tissue were detected by RT-PCR. I-K. The protein expression of BATF and IRF4 in lung tissue were detected by western blot. **P < 0.01,***p < 0.001 vs Control group, #P < 0.05, ##P < 0.01,###p < 0.001 vs IgG group.

Article Snippet: Subsequently, CD4+ T cells were cultured in Th9 induction medium (10 ng/mL IL-4, 3 ng/mL TGF-β, and 10 mg/mL anti-IFN-γ, Proteintech).

Techniques: Control, Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International immunopharmacology

Article Title: Foxp2 inhibits Th9 cell differentiation and attenuates allergic airway inflammation in a mouse model of ovalbumin-induced asthma.

doi: 10.1016/j.intimp.2022.109060

Figure Lengend Snippet: Fig. 3. Down regulation of Foxp2 expression in Th9 cells Spleen cells were isolated from normal mice and CD4+T cells were sorted by magnetic beads. IL-4, anti–IFN-ɣ and TGF-β were used to induce the differentiation of Th9 cells. After 4 days of induction, cells were collected. A.The levels of IL-9 in the supernatant of pre-induction (CD4+T) and post-induction (Th9) cells were were measured by ELISA. B-C. The mRNA expres sion of IL-9 and Foxp2 in pre-induction (CD4+T) and post-induction (Th9) cells were detected by RT-PCR in each group. D-E. The protein expression of IL-9 and Foxp2 in pre-induction (CD4+T) and post-induction (Th9) cells were analyzed by western blot in each group. **P < 0.01,***p < 0.001 vs CD4 + T cell group.

Article Snippet: Subsequently, CD4+ T cells were cultured in Th9 induction medium (10 ng/mL IL-4, 3 ng/mL TGF-β, and 10 mg/mL anti-IFN-γ, Proteintech).

Techniques: Expressing, Isolation, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International immunopharmacology

Article Title: Foxp2 inhibits Th9 cell differentiation and attenuates allergic airway inflammation in a mouse model of ovalbumin-induced asthma.

doi: 10.1016/j.intimp.2022.109060

Figure Lengend Snippet: Fig. 4. Overexpression of Foxp2 inhibits Th9 cell differentiation in vitro Th9 cells were infected withFoxp2 overexpressed lentivirus (LV-Foxp2) and control lentivirus (LV-NC), respectively, and no infected group as control. A-B. The percentage of Th9 cells was evaluated by Flow cytometry. C. The levels of IL-9 in cell supernatant was measured by ELISA. D. The mRNA expression of IL-9 in cells was detected by RT-PCR. E-F. The mRNA expression of BATF and IRF4 in cells were detected by RT- PCR. **P < 0.01, ***p < 0.001 vs LV-NC group.

Article Snippet: Subsequently, CD4+ T cells were cultured in Th9 induction medium (10 ng/mL IL-4, 3 ng/mL TGF-β, and 10 mg/mL anti-IFN-γ, Proteintech).

Techniques: Over Expression, Cell Differentiation, In Vitro, Infection, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International immunopharmacology

Article Title: Foxp2 inhibits Th9 cell differentiation and attenuates allergic airway inflammation in a mouse model of ovalbumin-induced asthma.

doi: 10.1016/j.intimp.2022.109060

Figure Lengend Snippet: Fig. 5. Overexpression of Foxp2 improves airway inflammation in asthmatic mice by inhibiting Th9 cell differentiation. Ten BALB/c mice were fed adaptively for one week. In the second week, 10 mice were divided into two groups (OVA + NC group and OVA + Foxp2 group) to establish asthma model. In OVA + NC group, OVA sensitized/challenged asthmatic mice were given control lentivirus by tracheal delivery at 3 days before aerosol challenge. In OVA + Foxp2 group, OVA sensitized/challenged asthmatic mice were given Foxp2 overexpression lentivirus by tracheal delivery at 3 days before aerosol challenge. Four hours after the last challenge, the mice were anesthetized and killed to get the lung tissue. A. HE staining was used to observe the pathological changes of lung tissue. B. PAS glycogen staining was used to determine the airway mucus secretion. C-F. The mRNA expression of IL-9, FOXP2, BATF and IRF4 in lung tissue were detected by RT-PCR. *P < 0.05,**P < 0.01,***p < 0.001 vs OVA + NC group.

Article Snippet: Subsequently, CD4+ T cells were cultured in Th9 induction medium (10 ng/mL IL-4, 3 ng/mL TGF-β, and 10 mg/mL anti-IFN-γ, Proteintech).

Techniques: Over Expression, Cell Differentiation, Control, Aerosol, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of pharmacological sciences

Article Title: Compensatory role of neuregulin-1 in diabetic cardiomyopathy.

doi: 10.1016/j.jphs.2023.08.009

Figure Lengend Snippet: Fig. 4. Klf10 was upregulated in the ventricles of STZ mice, A qRT-PCR analysis of Klf4 in the ventricle of control, STZ-4W or insulin-treated STZ-4W STZ-4W þ Ins) mice. Each data represents the mean ± SEM (n ¼ 6e9). ****p < 0.0001 by ANOVA post hoc Tukey's test. B. The correlation between the expression levels of Klf4 and Klf10 (r ¼ 0.3989, p ¼ 0.0733). C qRT-PCR analysis of Klf10. Each data represents the mean ± SEM (n ¼ 6e9). *p < 0.05 by ANOVA post hoc Tukey's test. D. The correlation between the expression levels of Nrg1 and Klf10 (r ¼ 0.7935, p ¼ 0.0001). E. Perinuclear localization of Klf 10 (green), CD31 (magenta) and DAPI (cyan) in the ventricles of control and STZ-4W mice. For RT-PCR, relative quantification (RQ) of the gene expression was calculated with the comparative Ct method. Hprt was used as an internal reference.

Article Snippet: Cryostat sections (15 mm) were incubated overnight at 4 C with the following primary antibodies: mouse antieneuregulin-1 monoclonal antibody (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), rat anti-CD31 monoclonal antibody (1:50; BD Pharmingen, Franklin Lakes, NJ, USA), or mouse anti-TIEG1 (KLF10) monoclonal antibody (1:50; Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression