Effect of BPA on adipocyte gene expression.

<p>3T3-L1 cells have been differentiated in mature adipocytes in presence of BPA 1 nM. Next, mRNA levels of PPARγ (a), GLUT4 (b) and GLUT1 (c) during adipogenic differentiation were determined by real-time RT-PCR analysis. Bars represent the mean ± SD of four independent experiments and show the mRNA levels in these cells relative to those in 3T3-L1 cells in absence of BPA at day 2 of differentiation. Data were analyzed with Statview software (Abacus concepts) by one-factor analysis of variance. <i>p</i> values of less than 0.05 were considered statistically significant. Asterisks indicate statistically significant differences (* p<0.05). Error bars indicate mean ± S.D.</p

Effect of BPA on leptin mRNA levels.

<p>3T3-L1 adipocytes were incubated with 1 nM BPA for 24h and leptin mRNA levels were determined by real-time RT-PCR analysis. Bars represent the mean ± SD of three independent experiments and show the leptin mRNA levels relative to those in 3T3-L1 cells in absence of BPA. Data were analyzed with Statview software (Abacus concepts) by one-factor analysis of variance. <i>p</i> values of less than 0.05 were considered statistically significant. Asterisks indicate statistically significant differences (*<i>p</i>< 0.05). Error bars indicate mean ± S.D.</p

Effect of JAK2/STAT3 and JNK inhibition on BPA-impaired inflammatory and insulin pathways.

<p>Human adipocytes were incubated with 1 nM BPA for 24h and exposed to 20 µM SP600125 for 1 h. a) Cell lysates (50 µg protein/sample) were blotted with phospho-JNK and Phospho-Tyr₇₀₅ STAT3 antibodies and then reblotted with anti- JNK and STAT3 antibodies. c) Cells were treated with 100 nM insulin for 10 min and then solubilized. Cell lysates (50 µg protein/sample) were blotted with phospho- IR, phospho- Ser₄₇₃Akt/PKB and phospho-Thr₂₀₂/ERK and then reblotted with anti-IR, Akt/PKB and ERK antibodies. To ensure the equal protein transfer, membranes were blotted with actin antibodies. The filters were revealed by ECL and autoradiography. The autoradiographs shown are representative of four independent experiments. b-d) Filters obtained in <i>a</i> and <i>c</i> have been analyzed by laser densitometry as described under Materials and Methods. Data were analyzed with Statview software (Abacus concepts) by one-factor analysis of variance. <i>p</i> values of less than 0.05 were considered statistically significant. Asterisks indicate statistically significant differences (* <i>p</i><0.05). Error bars indicate mean± S.D.</p

Effect of BPA on insulin transduction pathways.

<p>Human (a) and 3T3-L1 adipocytes (c) were incubated with 1 nM BPA for 24 and 48h as indicated and exposed to 100 nM insulin for 10 min and then solubilized as described in Materials and Methods. Cell lysates (50 µg protein/sample) were blotted with phospho- Tyr₁₁₄₆ Insulin Receptor β (pIR), phospho- Ser₄₇₃Akt/PKB and phospho-Thr₂₀₂/Tyr₂₀₄Extracellular signal-Regulated Kinases (pERK) antibodies and then reblotted with anti-IR, Akt/PKB and ERK antibodies. To ensure the equal protein transfer, membranes were blotted with actin antibodies. The filters were revealed by ECL and autoradiography. The autoradiographs shown are representative of four independent experiments. b-d) Filters obtained in <i>a</i> and <i>c</i> have been analyzed by laser densitometry as described under Materials and Methods. Data were analyzed with Statview software (Abacus concepts) by one-factor analysis of variance. <i>p</i> values of less than 0.05 were considered statistically significant. Asterisks indicate statistically significant differences (*<i>p</i>< 0.05). Error bars indicate mean ± S.D.</p

Primer sequences used in Real-time RT-PCR analysis.

<p>Primer sequences used in Real-time RT-PCR analysis.</p

BPA interference on adipocyte-released cytokines.

<p>Supernatants from 3T3-L1 and human adipocytes treated with or without 1 nM BPA for 24h were collected and tested by using the Bioplex multiplex cytokine assay kit as described in Materials and Methods. Data were analyzed with Statview software (Abacus concepts) by one-factor analysis of variance. <i>p</i> values of less than 0.05 were considered statistically significant. Asterisks indicate statistically significant differences (* p<0.05; ** p<0.01).</p

Effect of BPA on adipocyte glucose utilization.

<p>Human adipocytes (a) and 3T3-L1 adipocytes (b) were incubated in serum free-media with 1 nM or 100 nM BPA and 100 nM insulin for 24 and 48h as indicated. Next, supernatants were collected and glucose consumption was determined as described in Materials and Methods. Bars represent the mean ± SD of three independent experiments. Data were analyzed with Statview software (Abacus concepts) by one-factor analysis of variance. <i>p</i> values of less than 0.05 were considered statistically significant. Asterisks indicate statistically significant differences (* p<0.05). Error bars indicate mean ± S.D.</p

Effect of JAK2/STAT3 and JNK inhibition on BPA-impaired glucose utilization.

<p>Human adipocytes were incubated in serum free-media with 1 nM BPA, 20 µM SP600125 with or without 100 nM insulin for 24h as indicated. Next, supernatants were collected and glucose consumption was determined as described in Materials and Methods.Bars represent the mean ± SD of three independent experiments. Data were analyzed with Statview software (Abacus concepts) by one-factor analysis of variance. <i>p</i> values of less than 0.05 were considered statistically significant. Asterisks indicate statistically significant differences (* p<0.05). Error bars indicate mean± S.D.</p

Effect of BPA on NFkB activation.

<p>3T3-L1 adipocytes were incubated with 1 nM BPA for 24h. Next, subcellular fractionation(nuclear and cytoplasmic) was performed and proteins were extracted and subjected to SDS–PAGE and immunoblotted with anti-NFKB antibody. Actin and Laminin A/C were used as loading control for the cytosolic and nuclear fractions, respectively. Blots were revealed by ECL and autoradiography. The autoradiographs shown are representative of four independent experiments.</p

BPA effect on cellular neutral lipid droplet accumulation and glucose utilization in 3T3-L1 mature adipocytes.

<p>The microphotographs were obtained with an optical microscope in two original magnifications (10X and 20X), following ORO staining <b>(A)</b> in adipocytes upon BPA incubation compared to control cells. Lipid quantification test <b>(B)</b> was expressed as optical density (OD). Insulin stimulated glucose utilization test, expressed as fold over basal, was shown in differentiated 3T3-L1 cells incubated with 1 nM of BPA <b>(C)</b>. Bars represent the mean ± SD of four independent experiments. Asterisks indicate statistically significant differences (*p<0.05 and ***p<0.001) between adipocytes cultured upon BPA compared to controls.</p