Figure 2
Figure 2. Activin A inhibits FRβ expression and mediates the inhibitory effect of GM-CSF or M1 macrophage–conditioned medium. (A) FOLR2 mRNA expression levels in macrophages differentiated for 3 days (left panel) or 7 days (right panel) in the presence of M-CSF, GM-CSF, or M-CSF + activin A (ActA; 10 ng/mL) determined by qRT-PCR. (B) FOLR2 mRNA expression levels determined by qRT-PCR on monocytes (Mo.) or macrophages cultured for 7 days in the presence of the indicated cytokine combinations. For panels A and B, results are expressed as mRNA levels relative to GAPDH RNA and referred to the expression level observed in the presence of M-CSF (A) or in monocytes (B). The means and SD of triplicate determinations are shown; *P < .005; **P < .05. (C) Transcriptional activity of the pFOLR2-200Luc reporter construct in Mv1Lu cells incubated in the absence (−) or presence of IL-6 or activin A (ActA; 25 μg/mL). 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; *P < .005. (D) FOLR2 mRNA expression levels determined by qRT-PCR on monocytes exposed for 72 hours to increasing concentrations of GM-CSF–free M1 macrophage–conditioned medium (GM-CSF–free M1 SN %) and in the presence of 100 ng/mL of a blocking anti–activin A antibody (α-ActA) or an isotype-matched antibody (IgG). Results are expressed as relative mRNA levels (relative to GAPDH RNA levels). The means and SD of triplicate determinations are shown; *P < .005. (E) FOLR2 mRNA expression levels determined by qRT-PCR on M1 macrophages or M2 macrophages generated in the presence of GM-CSF–free M1 macrophage–conditioned medium (50%) and with the daily addition of a blocking anti–activin A antibody (α-ActA) or an isotype-matched antibody (IgG). Results are expressed as relative mRNA levels (relative to GAPDH RNA levels). The means and SD of triplicate determinations are shown; **P < .05. (F) FOLR2 mRNA expression levels determined by qRT-PCR on M1 macrophages generated in the presence of a blocking anti–activin A antibody (α-ActA) or an isotype-matched antibody (IgG). Results are expressed as relative mRNA levels (relative to GAPDH RNA levels). The means and SD of triplicate determinations are shown; **P < .05.

Activin A inhibits FRβ expression and mediates the inhibitory effect of GM-CSF or M1 macrophage–conditioned medium. (A) FOLR2 mRNA expression levels in macrophages differentiated for 3 days (left panel) or 7 days (right panel) in the presence of M-CSF, GM-CSF, or M-CSF + activin A (ActA; 10 ng/mL) determined by qRT-PCR. (B) FOLR2 mRNA expression levels determined by qRT-PCR on monocytes (Mo.) or macrophages cultured for 7 days in the presence of the indicated cytokine combinations. For panels A and B, results are expressed as mRNA levels relative to GAPDH RNA and referred to the expression level observed in the presence of M-CSF (A) or in monocytes (B). The means and SD of triplicate determinations are shown; *P < .005; **P < .05. (C) Transcriptional activity of the pFOLR2-200Luc reporter construct in Mv1Lu cells incubated in the absence (−) or presence of IL-6 or activin A (ActA; 25 μg/mL). 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; *P < .005. (D) FOLR2 mRNA expression levels determined by qRT-PCR on monocytes exposed for 72 hours to increasing concentrations of GM-CSF–free M1 macrophage–conditioned medium (GM-CSF–free M1 SN %) and in the presence of 100 ng/mL of a blocking anti–activin A antibody (α-ActA) or an isotype-matched antibody (IgG). Results are expressed as relative mRNA levels (relative to GAPDH RNA levels). The means and SD of triplicate determinations are shown; *P < .005. (E) FOLR2 mRNA expression levels determined by qRT-PCR on M1 macrophages or M2 macrophages generated in the presence of GM-CSF–free M1 macrophage–conditioned medium (50%) and with the daily addition of a blocking anti–activin A antibody (α-ActA) or an isotype-matched antibody (IgG). Results are expressed as relative mRNA levels (relative to GAPDH RNA levels). The means and SD of triplicate determinations are shown; **P < .05. (F) FOLR2 mRNA expression levels determined by qRT-PCR on M1 macrophages generated in the presence of a blocking anti–activin A antibody (α-ActA) or an isotype-matched antibody (IgG). Results are expressed as relative mRNA levels (relative to GAPDH RNA levels). The means and SD of triplicate determinations are shown; **P < .05.

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