Figure 6
Figure 6. Smad2 is constitutively phosphorylated in M1 macrophages, in which activin A release activates Smad-dependent reporter genes. (A) Detection of phosphorylated Smad2 and total Smad2/3 in lysates of untreated or LPS-treated M1 and M2 macrophages determined by Western blot analysis. β-actin expression levels were determined in parallel as a loading control. The bands corresponding to Smad2 and Smad3 are indicated by arrowheads (n.s., indicates nonspecific band). (B) Detection of activated Smad2 in lysates of untreated or activin A–treated M2 macrophages determined by Western blot analysis. GAPDH expression levels were determined in parallel as a loading control. (C) Detection of activated Smad2 by Western blot analysis on lysates of M2 macrophages subjected to a 30-minute treatment with DMSO (−), SB431542 (SB), an anti–activin A antibody (α-ActA), or an isotype-matched antibody (IgG), and then treated with activin A for 1 hour. Total Smad2/3 and GAPDH expression levels were determined in parallel as loading controls. (D) Transcriptional activity of the p3TP-Lux reporter construct in Mv1Lu cells either unstimulated or exposed to 10 ng/mL of TGFβ1, 25 ng/mL of activin A, or conditioned medium from M1 (M1 SN) or M2 (M2 SN) macrophages. Where indicated, cells were preincubated for 30 minutes with 10μM SB431542 before treatment, maintained in culture medium with 0.1 μg/mL blocking antibody against activin A (anti-ActA), or cotransfected with expression vectors coding for dominant-negative mutants of either Smad2 (Smad2, d.n.) or Smad3 (Smad3, d.n.). For normalization purposes, cells were cotransfected with the Rous Sarcoma Virus promoter–β-gal expression plasmid, and results are presented as RLU (relative light units), which indicate the units of luciferase activity per unit of β-gal activity for each assay condition. The means and SD of triplicate determinations are shown. Six replicas of each experiment were performed and means and SD are shown; *P < .005; **P < .05. (E) Expression of the indicated genes in M1 macrophages generated in the presence of SB431542 (10μM), and compared with their expression in M1 macrophages generated without the inhibitor (Vehicle, DMSO) determined by qRT-PCR using microfluidic cards. The experiment was performed in triplicate on macrophages from 2 independent donors and data are presented on a log scale; *P < .005; **P < .05. The left panel shows the expression of M2 (M-CSF)–specific markers, and these results as expressed relative to the level of expression of each gene in the presence of DMSO (Fold induction SB431542/Vehicle). The right panel shows the expression of M1 (GM-CSF)–specific markers, and these results as expressed relative to the level of expression of each gene in the presence of the SB431542 inhibitor (Fold repression, Vehicle/SB431542).

Smad2 is constitutively phosphorylated in M1 macrophages, in which activin A release activates Smad-dependent reporter genes. (A) Detection of phosphorylated Smad2 and total Smad2/3 in lysates of untreated or LPS-treated M1 and M2 macrophages determined by Western blot analysis. β-actin expression levels were determined in parallel as a loading control. The bands corresponding to Smad2 and Smad3 are indicated by arrowheads (n.s., indicates nonspecific band). (B) Detection of activated Smad2 in lysates of untreated or activin A–treated M2 macrophages determined by Western blot analysis. GAPDH expression levels were determined in parallel as a loading control. (C) Detection of activated Smad2 by Western blot analysis on lysates of M2 macrophages subjected to a 30-minute treatment with DMSO (−), SB431542 (SB), an anti–activin A antibody (α-ActA), or an isotype-matched antibody (IgG), and then treated with activin A for 1 hour. Total Smad2/3 and GAPDH expression levels were determined in parallel as loading controls. (D) Transcriptional activity of the p3TP-Lux reporter construct in Mv1Lu cells either unstimulated or exposed to 10 ng/mL of TGFβ1, 25 ng/mL of activin A, or conditioned medium from M1 (M1 SN) or M2 (M2 SN) macrophages. Where indicated, cells were preincubated for 30 minutes with 10μM SB431542 before treatment, maintained in culture medium with 0.1 μg/mL blocking antibody against activin A (anti-ActA), or cotransfected with expression vectors coding for dominant-negative mutants of either Smad2 (Smad2, d.n.) or Smad3 (Smad3, d.n.). For normalization purposes, cells were cotransfected with the Rous Sarcoma Virus promoter–β-gal expression plasmid, and results are presented as RLU (relative light units), which indicate the units of luciferase activity per unit of β-gal activity for each assay condition. The means and SD of triplicate determinations are shown. Six replicas of each experiment were performed and means and SD are shown; *P < .005; **P < .05. (E) Expression of the indicated genes in M1 macrophages generated in the presence of SB431542 (10μM), and compared with their expression in M1 macrophages generated without the inhibitor (Vehicle, DMSO) determined by qRT-PCR using microfluidic cards. The experiment was performed in triplicate on macrophages from 2 independent donors and data are presented on a log scale; *P < .005; **P < .05. The left panel shows the expression of M2 (M-CSF)–specific markers, and these results as expressed relative to the level of expression of each gene in the presence of DMSO (Fold induction SB431542/Vehicle). The right panel shows the expression of M1 (GM-CSF)–specific markers, and these results as expressed relative to the level of expression of each gene in the presence of the SB431542 inhibitor (Fold repression, Vehicle/SB431542).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal