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Results: 8

1.
Figure 8

Figure 8. Effect of increasing concentrations of nicotinic acid on forskolin stimulated cAMP levels.. From: Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation.

NHEK (squares), HaCaT (circles), and SCC-25 cells (triangles) were treated with different concentrations of nicotinic acid the effects on cAMP levels were determined. Representative data are shown with curve fitting lines using a two site-binding model. The R2 values for the curve fitting were NHEK (0.985), HaCaT (0.988) and SCC-25 (0.987). EC50 values for GPR109A and GPR109B, respectively, calculated by curve fitting of data from multiple experiments were: NHEK (6.9 nM and 25 µM); HaCaT (72 nM and 17 µM); SCC-25 (36 nM and 22 µM).

Yira Bermudez, et al. PLoS One. 2011;6(5):e20487.
2.
Figure 7

Figure 7. GPR109A/B couple through Gi in normal keratinocytes while SCC-25 cells are insensitive to pertussis toxin and nicotinic acid.. From: Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation.

NHEK (open columns) and SCC-25 (grey columns) cells were pretreated with or without 100 ng/mL pertussis toxin overnight and stimulated with 10 µM forskolin in the presence or absence of 100 µM nicotinic acid and intracellular cAMP levels were measured. Data represent the mean ± SEM from three independent experiments calculated as forskolin-induced cAMP relative to cellular protein. Students t-test was used to compare to cAMP produced without nicotinic acid to that produced with nicotinic acid, * p≤0.05.

Yira Bermudez, et al. PLoS One. 2011;6(5):e20487.
3.
Figure 5

Figure 5. Localization of GPR109A and GPR109B protein expression in cultured normal and malignant human skin cells.. From: Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation.

Panels A and B: Immunocytochemistry (ICC) analyses utilizing an antibody against GPR109A/B were performed on NHEK shown in red and merged with nuclear DAPI staining in blue. Panel A shows staining using the GPR109A/B antibody and Panel B shows staining in the presence of 1000 fold excess of peptides used to raise the antibody. Panel C: HaCaT, Panel D: Ras-transformed HaCaT, Panel E: A-431, Panel F: SCC-25. For all panels, magnification was 400× and size marker represents 10 microns.

Yira Bermudez, et al. PLoS One. 2011;6(5):e20487.
4.
Figure 4

Figure 4. Localization of GPR109A/B protein expression in human skin.. From: Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation.

Immunohistochemistry (IHC) analyses were performed on paraffin-embedded human skin sections utilizing antibody against GPR109A/B. Panels A and B utilized Streptavidin Quantum Dot 605 Conjugates for detection. Panels C and D used FITC Goat Anti-Rabbit IgG for detection. Panel A: Representative immunostaining sample shown at 200× magnification. Panel B: Representative IHC sample shown at 400× magnification. Panels C and D: Representative IHC samples shown at 400× magnification in the absence (Panel C) or presence (Panel D) of competition with peptide used to generate the antibody. Abbreviations: SC, stratum corneum; SG, stratum granulosum; SS, stratum spinousum; SB, stratum basale. Size marker represents 2 microns.

Yira Bermudez, et al. PLoS One. 2011;6(5):e20487.
5.
Figure 2

Figure 2. GPR109A and GPR109B are expressed in human tissues and cells including skin.. From: Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation.

Panel A: RT-PCR was performed on total RNA from adipose tissue, placenta, lung, and skin tissues and cells using primers specific for GPR109A (top row) and GPR109B (bottom row) as described in Materials and Methods. Panel B: qRT-PCR was performed on total RNA from six different normal human skin tissues using probes specific for GPR109A (dark grey column) and GPR109B (light grey column) receptors as described in Material and Methods. Students t-test was used to compare GPR109A to GPR109B, * p≤0.05. Panel C: qRT-PCR analyses were performed on total RNA from ten human squamous cell carcinoma tissues using probes specific for GPR109A (dark grey columns) and GPR109B (light grey columns) receptors. Results represent the mean ± SEM. Students t-test was used to compare to normal human skin, * p≤0.05.

Yira Bermudez, et al. PLoS One. 2011;6(5):e20487.
6.
Figure 6

Figure 6. GPR109A and GPR109B are functional in normal human keratinocytes but are defective in malignant cells.. From: Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation.

Panel A: NHEK, HaCaT, Ras-transformed HaCaT, A-431, SCC-25, and CF3 cells were treated in the presence of 10 µM forskolin (open columns) or 10 µM forskolin and 100 µM nicotinic acid (grey columns) for 1 h followed by measurement of intracellular cAMP levels. Data are from three independent experiments and show forskolin-induced cAMP production relative to cellular protein. Panel B: The percent inhibition by nicotinic acid is shown for each cell line. For both panels A and B, Students t-test was used to compare cAMP produced without nicotinic acid to that produced with nicotinic acid, * p≤0.05.

Yira Bermudez, et al. PLoS One. 2011;6(5):e20487.
7.
Figure 1

Figure 1. Nicotinic acid promotes epidermal differentiation in photodamaged human skin.. From: Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation.

Tissue arrays of skin biopsy samples from a clinical study of the effects of myristyl nicotinate (MN) in human subjects with photodamaged skin [5] were stained for the terminal differentiation markers caspase 14 and filaggrin. Panel A: An example of a biopsy sample at baseline and 12 weeks of MN treatment stained with H&E, and immunostaining for caspase 14 or filaggrin. Panel B: Quantification of staining for the placebo (n = 27) and MN treated (n = 31) groups for caspase 14 and filaggrin. Students t-test was used to compare placebo and MN treated groups and p values are shown.

Yira Bermudez, et al. PLoS One. 2011;6(5):e20487.
8.
Figure 3

Figure 3. GPR109A/B genes are transcribed and translated in human skin cells.. From: Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation.

Panel A: qRT-PCR was performed on total RNA from normal human epidermal keratinocytes (NHEK), immortalized human epidermal keratinocytes (HaCaT), immortalized Ras-transformed human epidermal keratinocytes (Ras-transformed HaCaT), human epidermoid carcinoma cells (A-431), squamous cell carcinoma cells (SCC-25), and human diploid fibroblasts (CF3), using probes specific for GPR109A (dark grey columns) and GPR109B (light grey columns) receptors. Students t-test was used to compare to NHEK, * p≤0.05. Panels B and C: Protein Expression of GPR109A and GPR109B in Human Skin Cells. Cell extracts from NHEK (lane 1), HaCaT (lane 2), Ras-transformed HaCaT (lane 3), A-431 (lane 4), and SCC-25 (lane 5) were subjected to SDS-PAGE and Western immunoblot analyses using an antibody against GPR109A/B pre-incubated in the absence (panel B) or presence (panel C) of 1000-fold excess peptide against which the antibody was generated relative to purified antibody. β-Actin was used as a loading control for Western immunoblot analyses. One representative blot is shown of three independent experiments. The relative densities were quantified using ImageJ. Graphical representation shows the average densitometric units of three independent experiments. Students t-test was used to compare to NHEK, * p≤0.05.

Yira Bermudez, et al. PLoS One. 2011;6(5):e20487.

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