Journal: Scientific reports

Article Title: Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice.

doi: 10.1038/s41598-019-43730-5

Figure Lengend Snippet: Figure 3. Fibrogenic gene and EMT-related gene expression levels were induced in the BSA-injected diabetic mouse kidney; TENE treatment suppressed these gene expression levels. (a) Microarray analysis of the kidney samples. Heat map analysis of the gene expression. BSA injection (particularly in the diabetic mice) induced genes, such as DPP-4, TGF-β/smad3 signaling, CAV1, integrin β1 and the EMT program; TENE treatment suppressed these alterations (n = 2 mice per group, and the average value is shown in the figure). Red indicates high and green indicates low relative expression levels. (b–p) qPCR analysis of the expression of the indicated genes in the kidney of mice in each group (n = 7 mice per group). Gene expression was normalized to the control mice value. *P < 0.05, **P < 0.01. Data are presented as mean ± s.e.m. (q) Representative western blot analysis. As a densitometric analysis, each protein level was normalized with actin. n = 7 per group were analyzed.

Article Snippet: Smad3 inhibitor SIS3 (CAS 1009104-85-1) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Gene Expression, Injection, Microarray, Expressing, Control, Western Blot

Journal: Scientific reports

Article Title: Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice.

doi: 10.1038/s41598-019-43730-5

Figure Lengend Snippet: Figure 6. TENE treatment suppressed the crosstalk among DPP-4, integrin β1 and CAV1 via inhibition of TGF-β/smad3 signaling pathway in vitro. Duolink in situ analysis of (a-c) DPP-4/integrin β1, (d–f) DPP-4/ CAV1 and (g–i) integrin β1/CAV1 in HK-2 cells with or without TGF-β1 (10 ng/ml) was performed by confocal microscopy (×1260). Scale bar: 50 μm in each panel. (j) Representative western blot analysis. As a densitometric analysis, each protein level was normalized with actin. n = 6 per group were analyzed. (k–n) Duolink in situ analysis of integrin β1/CAV1 in DPP-4 overexpressed HK-2 cells with or without TENE and SIS3. (o) Immunoprecipitation analysis revealed TGF-β treatment increased crosstalk among DPP-4, integrin β1 (ITGβ1) and CAV1. (p) Immunoprecipitation assay revealed TGF-β neutralization suppressed crosstalk among DPP-4, integrin β1 and CAV1 induced by DPP-4 overexpression.

Article Snippet: Smad3 inhibitor SIS3 (CAS 1009104-85-1) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Inhibition, In Vitro, In Situ, Confocal Microscopy, Western Blot, Immunoprecipitation, Neutralization, Over Expression

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 4. The identification of YAP-pSmad2/3-SOX9 axis during MSCs chondrogenesis. (A) RNA-seq analyses in human SMSCs after static and CTS treatment [(A1) volcano plot of differentially expressed genes (DEGs) in CTS group versus static group; (A2) KEGG pathway enrichment, red boxes represent Hippo and TGFβ signaling]. (B) Volcano plot of differentially expressed genes in regenerated tissue of PCL scaffold + synovium transplant group versus native porcine menisci [(B1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (B2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (B3) PCL scaffold + synovium transplant group at 2 versus 4 months]. (C) Heatmap of differential gene cluster in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (C2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (C3) PCL scaffold + synovium transplant group at 2 versus 4 months]. (D) KEGG pathway enrichment in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(D1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (D2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (D3) PCL scaffold + synovium transplant group at 2 versus 4 months, the red boxes represent Hippo and TGFβ signaling]. (E) Protein and mRNA levels in human SMSCs after static and CTS treatment [(E1) Western blot analysis; (E2) semiquantitative analysis of Western blot, n = 3, unpaired t test; (E3) mRNA levels, n = 8, unpaired t test]. (F) Cell immunofluorescence of human SMSCs after static and CTS treatment [(F1) cell immunofluorescence; (F2) six regions are randomly selected for semiquantitative analysis; the intensity of immunofluorescence is calculated by ImageJ software, n = 6, unpaired t test] (continued with Fig. 5). **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: RNA Sequencing, Western Blot, Immunofluorescence, Software

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 5. The identification of YAP-pSmad2/3-SOX9 axis during MSCs chondrogenesis. (A) Protein and mRNA levels in human SMSCs after being treated with dihy- drexidine and SIS3 [(A1) the Western blot analysis; (A2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (A3) the alcian blue staining; (A4) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (A5) cell immunofluorescence; (A6) the semiquantitative analysis of cell immunofluorescence, Dihy represents dihydrexidine, n = 6, one-way ANOVA; (A7) the mRNA levels, n = 8, one-way ANOVA]. (B) Immunofluorescent colocalization of YAP and Smad2/3 in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(B1) immunofluorescent colocalization of YAP and Smad2/3; (B2) immu- nofluorescence overlap coefficient, n = 9]. (C) Immunofluorescent colocalization of pSmad2/3 and SOX9 in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) immunofluorescent colocalization of pSmad2/3 and SOX9; (C2) immunofluorescence overlap coefficient, n = 9]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 6. Piezo1 mediates mechanotransduction on YAP-pSmad2/3-SOX9 axis through concerted activation of calcineurin and NFATc1 during MSCs chondro- genesis. (A) Protein and mRNA levels of Piezo1 in human SMSCs after static and CTS treatment [(A1) the Western blot analysis; (A2) the semiquantitative analysis of Western blot, n = 3, unpaired t test; (A3) the cell immunofluorescence; (A4) the semiquantitative analysis of cell immunofluorescence, n = 6, unpaired t test; (A5) the mRNA levels, n = 8, unpaired t test]. (B) Immunofluorescent assessment of Piezo1 expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. (C) Protein and mRNA levels in human SMSCs after being treated with YODA1 or GsMTx-4 [(C1) the Western blot analysis; (C2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (C3) the mRNA levels, n = 8, unpaired t test; (C4) the alcian blue staining; (C5) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (C6) the cell immunofluorescence; (C7) the semiquantitative analysis of cell immunofluorescence, n = 6, one-way ANOVA]. (D) Assessment of intracellular calcium content in human SMSCs [(D1) intracellular calcium content after static and CTS treatment; (D2) the semiquantitative analysis of calcium content, n = 5, unpaired t test; (D3) intracellular calcium content after being treated with YODA1 or GsMTx-4; (D4) the semiquantitative analysis of calcium content, n = 5, one-way ANOVA]. (E) Protein and mRNA levels in human SMSCs after CsA treatment [(E1) Western blot analysis, the red arrow represents dephosphorylated NFATc1, the blue arrows represent phosphorylated NFATc1; (E2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (E3) the alcian blue staining; (E4) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (E5) the mRNA levels, n = 8, unpaired t test; (E6) the cell immunofluorescence; (E7) the semiquantitative analysis of cell immunofluorescence, n = 6, one-way ANOVA]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing, Staining

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 7. The effect of matrix stiffness on YAP-pSmad2/3-SOX9 axis. (A) Protein and mRNA levels in human SMSCs after being treated with soft or stiff matrix [(A1) the mechanical properties of silicon rubber and polystyrene culture plate, n = 3; (A2) Western blot analysis, the red arrow indicates dephosphorylated NFATc1, the blue arrows indicate phosphorylated NFATc1; (A3) the semiquantitative analysis of Western blot, n = 3, unpaired t test; (A4) the alcian blue staining; (A5) the semiquantitative analysis of alcian blue staining, n = 3, unpaired t test; (A6) the mRNA levels, n = 8, unpaired t test]. (B) Immunofluorescent assessment of FAK in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(B1) the immunofluorescence of FAK; (B2) the semiquantitative analysis of FAK, n = 6, one-way ANOVA]. (C) Immunofluorescent assessment of pFAK in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) the immunofluorescence of pFAK; (C2) the semiquantitative analysis of pFAK, n = 6, one-way ANOVA]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 8. The biomechanical stimulus and matrix stiffness regulate the expression of collagen cross-link enzymes (LOX and LH) through Piezo1. (A) Protein and mRNA levels of LOX and LH in human SMSCs after static or CTS treatment [(A1) the Western blot analysis; (A2) the semiquantitative analysis of LOX, n = 3, unpaired t test; (A3) the semiquantitative analysis of LH2, n = 3, unpaired t test; (A4) the cell immunofluorescence of LOX; (A5) the semiquantitative analysis of LOX immunofluorescence, n = 6, unpaired t test; (A6) the cell immunofluorescence of LH2; (A7) the semiquantitative analysis of LH2 immunofluorescence, n = 6, unpaired t test; (A8) the mRNA levels, n = 8, unpaired t test]. (B) Protein and mRNA levels of LOX and LH in human SMSCs after being treated with YODA1 or GsMTx-4 [(B1) the Western blot analysis; (B2) the semiquantitative analysis of LOX, n = 3, one-way ANOVA; (B3) the semiquantitative analysis of LH2, n = 3, one-way ANOVA; (B4) the cell immunofluorescence of LOX; (B5) the semiquantitative analysis of LOX immunofluorescence, n = 6, one-way ANOVA; (B6) the cell immunofluorescence of LH2; (B7) the semiquantitative analysis of LH2 immunofluorescence, n = 6, one-way ANOVA; (B8) the mRNA levels, n = 8, one-way ANOVA]. (C) Protein and mRNA levels of LOX and LH in human SMSCs after being treated with soft or stiff matrix [(C1) the Western blot analysis; (C2) the semiquantitative analysis of LOX, n = 3, unpaired t test; (C3) the semiquantitative analysis of LH2, n = 3, unpaired t test; (C4) the mRNA levels, n = 8, unpaired t test]. (D) Immunofluorescent assessment of LOX expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. (E) Immunofluorescent assessment of LH2 expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: BMC Cancer

Article Title: Machine learning-based bulk RNA analysis reveals a prognostic signature of 13 cell death patterns and potential therapeutic target of SMAD3 in acute myeloid leukemia

doi: 10.1186/s12885-025-13658-3

Figure Lengend Snippet: Flow diagram of this study. PCD, Programmed cell death; Dox, Doxrubicin; Ara-C, Cytarabine; SIS3, SAMD3 inhibitor; P-SMAD3, phosphorylated SMAD3

Article Snippet: After treatment with SIS3 (0, 10 µM) (T3636, 521984-48-5, TargetMol, China), doxorubicin (70 nM) (T1456, 23214-92-8, Target Mol, China), or cytarabine (Ara-c, 1 µM) (T1272, 147-94-4, Target Mol, China) at 37 °C for 72 h, the cells were incubated with 10 µL of CCK-8 reagent (WK464, Dojindo, JAP) for 2 h at 37 °C.

Techniques:

Journal: BMC Cancer

Article Title: Machine learning-based bulk RNA analysis reveals a prognostic signature of 13 cell death patterns and potential therapeutic target of SMAD3 in acute myeloid leukemia

doi: 10.1186/s12885-025-13658-3

Figure Lengend Snippet: The level of phosphorylated-SMAD3/SMAD3 and drug sensitivity-related programmed cell death in AML cell strains. ( A ) The corresponding genotypes of different AML cell lines. ( B - C ) The expression levels of SMAD3 and its phosphorylated form (P-SMAD3) in different AML cell lines were detected by western blotting. ( D - F ) Survival analysis of AML cell lines treated with the SMAD3 inhibitor SIS3, traditional chemotherapeutic drugs (doxorubicin plus Ara-C), or their combination. The optical density (OD) value at 450 nm was via a microplate reader, and the OD value was proportional to the cell viability. (G-H) PCD-related molecular markers in drug-treated (SIS3, DOX + Ara-c, DOX + Ara-c + SIS3) MV4-11 and THP-1 cell lines were detected via western blotting

Article Snippet: After treatment with SIS3 (0, 10 µM) (T3636, 521984-48-5, TargetMol, China), doxorubicin (70 nM) (T1456, 23214-92-8, Target Mol, China), or cytarabine (Ara-c, 1 µM) (T1272, 147-94-4, Target Mol, China) at 37 °C for 72 h, the cells were incubated with 10 µL of CCK-8 reagent (WK464, Dojindo, JAP) for 2 h at 37 °C.

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: AKT2-mediated nuclear deformation leads to genome instability during epithelial-mesenchymal transition.

doi: 10.1016/j.isci.2023.106992

Figure Lengend Snippet: Figure 4. Smad3 contributes to AKT2 phosphorylation and TGFb-induced nuclear deformation (A) A549 cells were infected with lentiviruses encoding shRNAs specific to luciferase (sh-Luciferase), Smad2 (sh-Smad2), or Smad3 (sh-Smad3). The cells were treated with (+) or without () TGFb for 24 h and lysed. An equal amount of whole cell lysates was analyzed by immunoblotting with anti-Smad2/3 or anti-actin antibody. (B) The cells as described in (A) were treated with or without TGFb for 24 h and stained for lamin A (white) and DNA (blue). Scale bars, 10 mm. The percentage of the cells with a crumpled or lobulated nucleus was measured (n R 600). (C) A549 cells were transiently transfected with the plasmid encoding FLAG-Smad3 or HA-Smad2 for 24 h, and then stained for FLAG (green), HA (green), and DNA (white). Scale bars, 20 mm. The percentage of the transfection-positive cells with a deformed nucleus was measured (n R 269). (D) A549 cells expressing shRNAs to luciferase or Smad3 were treated with or without TGFb for 1 h. An equal amount of whole cell lysates was analyzed by immunoblotting with anti-AKT2 pS474 or anti-AKT2 antibody. The phosphorylation of AKT2 at Ser474 was quantified and expressed as –fold relative to the level of the control. (E) A549 cells were pre-treated with the Smad3 inhibitor SIS3 for 4 h and then co-treated with TGFb for 1 h. An equal amount of whole cell lysates was analyzed by immunoblotting with the antibodies as indicated. The phosphorylation of AKT2 at Ser474 was quantified and expressed as –fold relative to the level of the control. Data information: In B and C, values (means G SD) were from three independent experiments. ***p < 0.001. See also Figure S3.

Article Snippet: SP600125 Merck Millipore Cat#420128 SB23580 Calbiochem Cat#559389 Y27632 EMD Chemicals Cat#688000 Cytochalasin D Sigma-Aldrich Cat#C8273 ML-7 Sigma-Aldrich Cat#12764 Blebbistatin Sigma-Aldrich Cat#B0560 methylstat Sigma-Aldrich Cat#SML0343 SIS3 Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Infection, Luciferase, Western Blot, Staining, Transfection, Plasmid Preparation, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Activin A activation of Smad3 mitigates innate inflammation in mouse models of psoriasis and sepsis

doi: 10.1172/JCI187063

Figure Lengend Snippet: ( A ) RT-qPCR analysis in macrophages stimulated by LPS. ( n = 4–6.) ( B ) Activin A levels (measured by ELISA) in the supernatant of macrophages stimulated by LPS. ( C ) Abundance of the indicated proteins in macrophages stimulated by LPS for 6 hours. ( D ) RT-qPCR analysis of INHBA expression in human macrophages stimulated by LPS for 6 hours. ( n = 4.) ( E ) Abundance of the indicated proteins in human macrophages pretreated with follistatin or an anti–activin A or anti–TGF-β blocking antibody for 1 hour followed by LPS stimulation for 6 hours. ( F – G ) Activin A levels in serum of WT mice injected with LPS ( F ) or subjected to CLP surgery ( G ) and harvested after 6 hours. ( n = 6–10.) ( H – I ) Flow cytometry analysis of pSmad2/3 levels in macrophages from WT or Acvr1b -Lyz2cre mice injected i.p. with LPS ( H ) or subjected to CLP surgery ( I ) and harvested after 6 hours. ( J ) RT-qPCR analysis of the indicated genes in macrophages stimulated or not by LPS for 24 hours. ( n = 4–6.) ( K ) RT-qPCR analysis of macrophages stimulated or not by LPS for 24 hours. ( n = 4.) ( L ) RT-qPCR analysis of IL6 expression in human macrophages pretreated with follistatin or an anti–activin A blocking antibody for 1 hour followed by LPS stimulation for 24 hours. ( n = 4.) ( M ) RT-qPCR analysis of IL6 expression in human macrophages pretreated with a Smad3 inhibitor (SMAD3i) for 1 hour followed by LPS stimulation for 24 hours. ( n = 4.) ( N ) RT-qPCR analysis in macrophages stimulated by LPS for 24 hours in the presence of activin A. ( n = 6.) Representative or pooled from at least 2 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001 by Student’s t test ( A , B , D , F , G , and N ) or 1-way ANOVA ( H – M ).

Article Snippet: Macrophages were pretreated for 1 hour before these treatments with follistatin (0.5 μg/mL; BioLegend), cycloheximide (5 μM; Cayman Chemical), anti–TGF-β antibody (50 μg/mL; Bio X Cell), anti–activin A antibody (2 μg/mL; R&D Systems), Smad3 inhibitor (SIS3, 2 μM; Cayman Chemical), TAK1 inhibitor (5 nM; Sigma-Aldrich), MEK inhibitor (10 μM; Cayman Chemical), ERK inhibitor (1 μM; Cayman Chemical), STAT5 inhibitor (100 μM; Cayman Chemical), CD73 inhibitor (10 μM; Cayman Chemical), and CREB inhibitor (1 μM; Cayman Chemical).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Blocking Assay, Injection, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Activin A activation of Smad3 mitigates innate inflammation in mouse models of psoriasis and sepsis

doi: 10.1172/JCI187063

Figure Lengend Snippet: ( A ) Heatmap representing significantly downregulated genes in macrophages from Smad3 -KO mice (compared with WT macrophages) stimulated with LPS for 24 hours. ( B and C ) MitoTracker staining in macrophages stimulated with LPS for 24 hours and isolated from Smad3 -KO mice ( B ) or Acvr1b -Lyz2cre mice ( C ). ( D and E ) ATP production (intracellular) in macrophages stimulated with LPS for 24 hours and isolated from Smad3 -KO mice ( D ) ( n = 6) or Acvr1b -Lyz2cre mice ( E ). ( F and G ) RT-qPCR analysis of Arg1 and Tgfbi expression in macrophages stimulated with LPS for 24 hours in combination (or not) with 20 μM of ATP and isolated from Smad3 -KO mice ( F ) or Acvr1b -Lyz2cre mice ( G ). ( H ) RT-qPCR analysis of Nt5e (encoding CD73) expression in macrophages stimulated with LPS for 24 hours and isolated from Smad3 -KO or Acvr1b -Lyz2cre mice. ( I and J ) RT-qPCR analysis of Tgfbi and Arg1 expression in macrophages stimulated with LPS for 24 hours in combination (or not) with ATP and a CD73 inhibitor or a CREB inhibitor and isolated from Smad3 -KO mice ( I ) or Acvr1b -Lyz2cre mice ( J ). ( F – J , n = 4.) Pooled or representative of at least 2 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001 by Student’s t test ( B – G ) or 1-way ANOVA ( H – J ).

Article Snippet: Macrophages were pretreated for 1 hour before these treatments with follistatin (0.5 μg/mL; BioLegend), cycloheximide (5 μM; Cayman Chemical), anti–TGF-β antibody (50 μg/mL; Bio X Cell), anti–activin A antibody (2 μg/mL; R&D Systems), Smad3 inhibitor (SIS3, 2 μM; Cayman Chemical), TAK1 inhibitor (5 nM; Sigma-Aldrich), MEK inhibitor (10 μM; Cayman Chemical), ERK inhibitor (1 μM; Cayman Chemical), STAT5 inhibitor (100 μM; Cayman Chemical), CD73 inhibitor (10 μM; Cayman Chemical), and CREB inhibitor (1 μM; Cayman Chemical).

Techniques: Staining, Isolation, Quantitative RT-PCR, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Activin A activation of Smad3 mitigates innate inflammation in mouse models of psoriasis and sepsis

doi: 10.1172/JCI187063

Figure Lengend Snippet: ( A ) RT-qPCR analysis of Inhba expression in macrophages pretreated for 1 hour with the indicated inhibitors and stimulated with imiquimod (IMQ) for 2 hours. ( n = 4–6.) ( B ) Abundance of the indicated proteins in macrophages isolated from WT or Acvr1b -Lyz2cre mice (KO) and stimulated with IMQ for 6 hours. ( C ) RT-qPCR analysis of INHBA expression in human monocytes stimulated with IMQ for 2 hours. ( n = 4.) ( D ) Abundance of the indicated proteins in human monocytes pretreated with follistatin or an anti–activin A or anti–TGF-β blocking antibody for 1 hour followed by IMQ stimulation for 6 hours. ( E ) RT-qPCR analysis of the indicated genes in macrophages from WT or Acvr1b -Lyz2cre mice stimulated or not with IMQ for 24 hours. ( n = 4.) ( F ) RT-qPCR analysis of the indicated genes in macrophages from WT or Smad3 -KO mice stimulated or not with IMQ for 24 hours. ( n = 4.) ( G ) RT-qPCR analysis of Inhba expression in skin of WT mice treated with an IMQ topical application for 6 hours. ( n = 10.) ( H ) Abundance of the indicated proteins in skin of WT mice treated with an IMQ topical application for 6 hours. Each band represents a mouse. Macrophages from WT or Smad3 -KO macrophages were transferred intradermally in skin of CD45.1 WT mice followed by IMQ topical application for 6 consecutive days. Mice were then harvested and analyzed. ( I ) Skin thickness. ( n = 15.) ( J ) TCRγδ cytokine production in skin. ( n = 15.) WT or Acvr1b -Lyz2cre mice were treated with IMQ topical application for 6 consecutive days, then harvested and analyzed. ( K ) Skin thickness. ( L ) Macrophage frequency in skin. ( M ) Production of cytokines by macrophages. ( N ) TCRγδ cytokine production in skin. ( K – N , n = 8–9.) Representative or pooled from at least 2 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001 by Student’s t test ( C and G – N ) or 1-way ANOVA ( A , E , and F ).

Article Snippet: Macrophages were pretreated for 1 hour before these treatments with follistatin (0.5 μg/mL; BioLegend), cycloheximide (5 μM; Cayman Chemical), anti–TGF-β antibody (50 μg/mL; Bio X Cell), anti–activin A antibody (2 μg/mL; R&D Systems), Smad3 inhibitor (SIS3, 2 μM; Cayman Chemical), TAK1 inhibitor (5 nM; Sigma-Aldrich), MEK inhibitor (10 μM; Cayman Chemical), ERK inhibitor (1 μM; Cayman Chemical), STAT5 inhibitor (100 μM; Cayman Chemical), CD73 inhibitor (10 μM; Cayman Chemical), and CREB inhibitor (1 μM; Cayman Chemical).

Techniques: Quantitative RT-PCR, Expressing, Isolation, Blocking Assay