IPP and VPP inhibit TNFα mediated activation of the pro-inflammatory NF-κB pathway downstream of IκB degradation.

<p>3T3-F442A cells were incubated for 48 hr in presence of insulin (10 μg/mL) to induce differentiation. Cells were then washed and further incubated for 30 min with pro-inflammatory cytokine TNFα (10 ng/mL) with/without addition of IPP (50 μM) or VPP (50 μM). Afterwards, the cells were lysed and western blotting of the lysates was performed to determine (A) IκBα degradation (using antibodies against IκBα and the loading control, α-tubulin) and (B) p65 phosphorylation (using antibodies against phosphorylated and total p65). A set of representative images was shown. Data were presented as mean±SEM of 4 independent experiments. All data were normalized to the values from untreated (i.e. undifferentiated) cells. * indicates p<0.05 compared to the TNFα treated cells. NS means: not significant (compared to TNFα treated group).</p

IPP and VPP induce lipid accumulation in 3T3-F442A cells.

<p>3T3-F442A cells were incubated in presence of insulin (10 μg/mL), IPP (50 μM) or VPP (50 μM) for 72 hr. (A) For one study, the cells were fixed, stained with the neutral lipid-specific dye LipidTox and visualized under fluorescence microscopy. A set of representative images are shown. (B) For another set of experiments, the cells were lysed and their lipid contents were estimated by a biochemical assay. Data were presented as mean±SEM of 3–4 independent experiments. * and ** indicate p<0.05 and p<0.01 respectively compared to the untreated control (Untr).</p

IPP and VPP promote expression of the adipocyte differentiation markers PPARγ and adiponectin in 3T3-F442A cells.

<p>3T3-F442A cells were incubated in presence of insulin (10 μg/mL), IPP (50 μM) or VPP (50 μM) for 72 hr. (A) The cells were lysed and western blotting of the lysates was performed with antibodies against PPARγ and α-tubulin (loading control). A set of representative images (including cropped images obtained from the same membrane) was shown. (B) The cell-free culture supernatants were collected and analyzed by ELISA to determine levels of adiponectin. Data were presented as mean±SEM of 5 independent experiments. * and *** indicate p<0.05 and p<0.001 compared to the untreated control (Untr) respectively.</p

IPP and VPP prevent TNFα mediated loss of adiponectin release from insulin-differentiated 3T3-F442A cells.

<p>3T3-F442A cells were incubated for 48 hr in presence of insulin (10 μg/mL) to induce differentiation. Cells were then washed and further incubated for 24 hr with the pro-inflammatory cytokine TNFα (10 ng/mL) with/without addition of IPP (50 μM) or VPP (50 μM). At the end of this incubation period, the cell-free supernatants were collected and analyzed for their adiponectin content by ELISA. Data were presented as mean±SEM of 4 independent experiments. * indicates p<0.05 compared to the insulin alone (Alone). NS means: not significant (compared to Alone).</p

IPP and VPP increase the protein levels of adipocyte differentiation regulators c-Jun and C/EBPα.

<p>3T3-F442A cells were incubated in presence of insulin (10 μg/mL), IPP (50 μM) or VPP (50 μM) for 72 hr. The cells were lysed and western blotting of the lysates was performed with antibodies against c-Jun (A), C/EBPα (B) and α-tubulin (loading control). A set of representative images was shown. Data were presented as mean±SEM of 4–6 independent experiments. * and ** indicate p<0.05 and p<0.01 respectively, compared to the untreated control (Untr). # and ## indicate p<0.05 and p<0.01 respectively, compared to the insulin treated cells.</p

Human endothelial cells express GPR30 protein in the cell nucleus.

<p>(A) Confluent monolayers of HUVECs from 3 different cords were lysed and the protein lysates were immunoblotted for GPR30. α-tubulin was used as loading control. (B) HUVEC monolayers at 30–40% confluence were treated with 40 nM siRNA (control or GPR30_4) for 48 hours prior to lysis followed by immunoblotting of the cell lysates for GPR30. α-tubulin was used as loading control. Data shown are mean ± SEM of 4 independent experiments. ** and ## indicate p<0.01 compared to untreated and control siRNA-treated cells, respectively. (C) Confluent HUVECs grown on glass coverslips were fixed, permeabilized and immunostained with anti-GPR30 antibody. Nuclei were stained with Hoechst33342 dye. The merged image shows GPR30 (red) and nuclei (blue) in pseudocolor. Representative images from 3 independent experiments are shown. Bar, 20 µm. (D) Confluent HUVECs were lysed and fractionated into cytosolic (<i>C</i>) and nuclear (<i>N</i>) fractions prior to western blotting for eNOS, GPR30, p65, α-tubulin and c-Jun. A representative set of images (obtained from different membranes) from 3 independent experiments is shown.</p

4-hydroxy tamoxifen (4-HT) exerts anti-inflammatory effects similar to G-1.

<p>(A and B) Confluent HUVEC monolayers were pre-treated with either G-1 (1 µM) or 4-HT (10 µM) for 45 minutes prior to 4 hour stimulation with TNF (5 ng/ml). Cells were lysed and the lysates were immunoblotted with antibodies against ICAM-1 (A), VCAM-1 (B) and α-tubulin. Data are shown as mean ± SEM of 4 independent experiments. (C and D) Confluent HUVECs were pre-treated with G-15 (2 µM) followed by treatment with 4-HT (10 µM) for 45 minutes prior to 4 hour stimulation with TNF (5 ng/ml). Cells were lysed and the lysates were immunoblotted with antibodies against ICAM-1 (C), VCAM-1 (D) and α-tubulin. Data are shown as mean ± SEM of 5 independent experiments. ** and *** indicate p<0.01 and p<0.001 respectively, compared to the untreated control. #, ## and ### indicate p<0.05, p<0.01 and p<0.001 respectively, compared to TNF alone.</p

G-1 does not alter TNF-mediated endothelial activation of NF-κB.

<p>(A) Confluent HUVEC monolayers were pre-treated with/without G-1 (1 µM) for 45 minutes prior to 15 minute stimulation with TNF (5 ng/ml). Cells were immediately fixed, permeabilized and then immunostained with anti-p65 antibody. Representative images from 3 independent experiments are shown. Magnification (X100). (B) Confluent HUVECs were pre-treated with/without G-1 (1 µM) for 45 minutes prior to stimulation with TNF (5 ng/ml) for the indicated time periods. Cells were lysed and the lysates were immunoblotted with antibodies against IκBα and the loading control α-tubulin. Data are summarized as mean ± SEM of 3 independent experiments. *** indicates p<0.001.</p

GPR30 activation attenuates TNF-mediated endothelial expression of ICAM-1 and VCAM-1.

<p>(A and B) Confluent HUVEC monolayers were pre-treated with the selective GPR30 agonist G-1 (0.1–3 µM) for 45 minutes prior to 4 hour stimulation with TNF (5 ng/ml). Cells were lysed and the lysates were immunoblotted with antibodies against ICAM-1 (A), VCAM-1 (B) and α-tubulin. DMSO (1∶5000) was used as a solvent control for G-1. (C and D) Confluent HUVECs were pre-treated for 30 minutes with/without the specific GPR30 inhibitor G-15 (2 µM) followed by treatment with G-1 (1 µM) for 45 minutes prior to 4 hour stimulation with TNF (5 ng/ml). Cells were lysed and the lysates were immunoblotted with antibodies against ICAM-1 (C), VCAM-1 (D) and α-tubulin. Data shown are mean ± SEM of 6–8 independent experiments. *, ** and *** indicate p<0.05, p<0.01 and p<0.001 respectively, compared to the untreated control.</p

17-β-estradiol (E2) alone has no effect on TNF-mediated endothelial ICAM-1 and VCAM-1 expression.

<p>Confluent HUVEC monolayers were pre-treated with different concentrations (10–1000 nM) of E2 for 45 minutes prior to 4 hour stimulation with TNF (5 ng/ml). Cells were lysed and the lysates were immunoblotted with antibodies against ICAM-1 (A), VCAM-1 (B) and α-tubulin. Data are shown as mean ± SEM of 4 independent experiments.</p