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1.
Fig. 3.

Fig. 3. From: MKP-1 Is Essential for Canonical Vitamin D-Induced Signaling through Nuclear Import and Regulates RANKL Expression and Function.

MKP-1 is needed for maximal 1,25-(OH)2D3 target mRNA expression and RANKL promoter activity in a UAMS32 Luc-reporter stromal cell line. A, UAMS-32 stromal cells were treated with 10−8 m 1,25-(OH)2D3 for 18 h followed by qPCR analysis. B, MKP-1 siRNA-transfected UAMS-32 stromal cells were stimulated with 10−8 m 1,25-(OH)2D3 for 18 h, followed by qPCR analysis. (n = 4; *, P < 0.05; **, P < 0.01). SCR, Scrambled.

Alfred C. Griffin, et al. Mol Endocrinol. 2012 Oct;26(10):1682-1693.
2.
Fig. 7.

Fig. 7. From: MKP-1 Is Essential for Canonical Vitamin D-Induced Signaling through Nuclear Import and Regulates RANKL Expression and Function.

MKP-1 is required for maximal 1,25-(OH)2D3-induced osteoclastogenesis. WT and MKP-1−/− BMSC were cocultured with RAW264.7 cells to test the osteoclast-inducing potential of 1,25-(OH)2D3-stimulated stromal cells. A, Quantification of osteoclasts per well and nuclei per osteoclast. Coculture osteoclast-resorbing activity was quantified on osteologic discs. B, Representative pit images and C) pits per well and total pit area per well. (n = 3; P < 0.05; **, P < 0.01; ***, P < 0.001). Ctrl, Control; OPG, osteoprotegerin.

Alfred C. Griffin, et al. Mol Endocrinol. 2012 Oct;26(10):1682-1693.
3.
Fig. 1.

Fig. 1. From: MKP-1 Is Essential for Canonical Vitamin D-Induced Signaling through Nuclear Import and Regulates RANKL Expression and Function.

MKP-1 is required in BMSC for 1,25-(OH)2D3 (D3) induction of RANKL. A, RT-qPCR analysis of RANKL mRNA expression in response to 18 h treatment with inflammatory stimuli and 10−8 m 1,25-(OH)2D3. B, ELISA of soluble RANKL expression from cultured media containing indicated cytokines, LPS, or 10−8 m 1,25-(OH)2D3 for 48 h (n = 4; ***, P < 0.001). Aa, Aggregatibacter actinomycetemcomitans; Pg, Porphyromonas gingivalis.

Alfred C. Griffin, et al. Mol Endocrinol. 2012 Oct;26(10):1682-1693.
4.
Fig. 4.

Fig. 4. From: MKP-1 Is Essential for Canonical Vitamin D-Induced Signaling through Nuclear Import and Regulates RANKL Expression and Function.

EMSA analysis shows 1,25-(OH)2D3-stimulated MKP-1−/− BMSC have a decreased VDRE binding capacity. A, The α-32P-labeled mOP VDRE was incubated with 1,25-(OH)2D3-treated nuclear extracts from WT and MKP-1−/− BMSC and electrophoresed through a nondenaturing gel. R, RXRα supershifts; V, VDR antibody supershifts; *, the putative mOP-RXRα/VDR heterodimer complex. Results are representative of three independent experiments. B, ChIP analysis of the RANKL VDRE enhancer region located −76 kb from the RANKL TSS (mRLD5) and control non-VDRE region located −50 kb from the RANKL TSS (IS4). Results are representative of three independent experiments. Ctrl, Control.

Alfred C. Griffin, et al. Mol Endocrinol. 2012 Oct;26(10):1682-1693.
5.
Fig. 6.

Fig. 6. From: MKP-1 Is Essential for Canonical Vitamin D-Induced Signaling through Nuclear Import and Regulates RANKL Expression and Function.

MKP-1 is required for 1,25-(OH)2D3-induced nuclear translocation of the VDR-RXRα heterodimer. WT and MKP-1−/− BMSC were treated with vehicle control or 10−7 m 1,25-(OH)2D3 for 1 h and were subjected to PLA with antibodies against VDR (mouse) and RXRα (rabbit); confocal images were captured for quantification. A, Representative confocal images of PLA signals in WT and MKP-1−/− BMSC. B, Quantification of total and nuclear PLA signals in the Z-range of the DAPI stained nuclei for the indicated treatments. White scale bar, 10 μm (n = 3; *, P < 0.05). Ctrl, Control.

Alfred C. Griffin, et al. Mol Endocrinol. 2012 Oct;26(10):1682-1693.
6.
Fig. 5.

Fig. 5. From: MKP-1 Is Essential for Canonical Vitamin D-Induced Signaling through Nuclear Import and Regulates RANKL Expression and Function.

MKP-1 is required for 1,25-(OH)2D3-induced RXRα nuclear import. A, Representative Western blot of cytoplasmic/nuclear expression of VDR and RXRα from BMSC treated with 10−7 m 1,25-(OH)2D3 for 1 h. B, Densitometric quantification of RXRα and VDR protein levels normalized to GAPDH or lamin A/C. C, Densitometric comparison of VDR and RXRα nuclear import in primary WT and MKP-1−/− BMSC and total relative VDR and RXRα present in nuclear and cytoplasmic fractions. D, Immunofluorescence cytochemistry of WT and MKP-1−/− BMSC for RXRα and VDR protein localization. White scale bar, 10 μm. (n = 3; *, P < 0.05). Ctrl, Control; Veh, vehicle.

Alfred C. Griffin, et al. Mol Endocrinol. 2012 Oct;26(10):1682-1693.
7.
Fig. 2.

Fig. 2. From: MKP-1 Is Essential for Canonical Vitamin D-Induced Signaling through Nuclear Import and Regulates RANKL Expression and Function.

MKP-1 is necessary for maximal genomic expression of 1,25-(OH)2D3 target genes RANKL, VDR, and CYP24a1. A, WT and MKP-1−/− BMSC were stimulated with 10−8 m 1,25-(OH)2D3 for 0, 2, 4, 8, 12, 18, and 24 h. RT-qPCR results above were normalized to GAPDH expression and shown as fold change relative to WT control (n = 3). B,) Western blot analysis for VDR after 24 h 10−8 m 1,25-(OH)2D3 stimulation indicates that MKP-1 is required for 1,25-(OH)2D3 induction of VDR protein expression. C, Densitometric analysis of VDR expression normalized to GAPDH (n = 3; *, P < 0.05, **, P < 0.01).

Alfred C. Griffin, et al. Mol Endocrinol. 2012 Oct;26(10):1682-1693.

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