Ed on cultures derived from at least 3 different subjects. We did

Ed on cultures derived from at least 3 different subjects. We did not notice any variations in the effects of vitamin D as a function of the BMI of the donor. In separate experiments, we also tested the effects of 1,25(OH)2D3 in the absence of a TZD in the differentiation cocktail.differentiated human adipocytes were incubated with 25(OH)D3 (1028 M) for 24 h in DMEM/F12 with no other additions. The quantity of 1,25(OH)2D3 in the incubation media was assayed with an enzyme immunoassay (Immunodiagnostic Systems Inc.). Data were expressed as picograms of 1,25(OH)2D3 produced per million cells.RNA Extraction and Measurement of Gene ExpressionTotal RNA was extracted using Trizol (Invitrogen) and quantity and quality were assessed spectrophotometrically. 1 mg total RNA was reverse transcribed using High-Capacity cDNA Reverse Title Loaded From File Transcription Kits (Applied Biosystems) and qPCR was Title Loaded From File performed with the Light Cycler 480 (Roche) with Taqman probes (Applied Biosystems). Cyclophilin A (PPIA) was used as a reference gene.3T3-L1 Cell Culture3T3-L1 fibroblasts were cultured in 10 FBS supplemented DMEM. 2d post-confluent (day 0) cells were differentiated in DMEM with 10 FBS, 500 mM IBMX, 100 nM bovine insulin, and 1 mM dexamethasone. Medium was replenished with DMEM+10 FBS with 100 nM insulin on d2, and with DMEM+10 FBS on d4. 1,25(OH)2D3 (10211, 10210, 1028 M), 25(OH)D3 (1029 M), or vehicle (ethanol) was added to the media during differentiation at times specified in the figure legends.Western BlottingCells were washed with ice-cold PBS and scraped in cell lysis buffer (Cell Signaling) supplemented with 5 SDS and protease inhibitors (Pierce). 5?0 mg total protein was resolved in 10 or 15 Tris-HCl gels (Biorad), transferred to PVDF membranes, and blocked in 5 milk in Tris buffered saline with 0.2 tween-20. Membranes were probed for FABP4 (a gift from Dr. Judith Storch at Rutgers University), VDR (D-6, Santa Cruz), adiponectin (BD Biosciences), CYP27B1 (C-12 and H-90, Santa Cruz), and loading controls [(a-tubulin (Santa Cruz) and total ERK (Cell Signaling)]. Chemiluminescence images were captured using an Imager (LAS 4000, Fuji) and quantified using software (Multi Guage, Fuji).Mouse Primary Preadipocyte Culture and DifferentiationStromal vascular cells from the inguinal adipose tissue of C57BL/6J mice were prepared as described for human preadipocytes. Cells were grown and differentiated as described for 3T3-L1 cells, except that the differentiation cocktail with Rosiglitazone (1 mM) was added only during the initial 2dinduction period. 1,25(OH)2D3 or vehicle control (ethanol) was added continuously until harvest on day 7. Animal studies were conducted in conformity with PHS policy and approved by IACUC of Boston University Medical Campus.Triglyceride (TG) and DNA QuantificationTotal TG and DNA quantity in cell lysates were measured using a triglyceride determination kit (Sigma) and Quant-iTTM PicoGreen dsDNA reagent (Invitrogen). Statistical analysis. Data are expressed as means6standard error mean (SEM). After log transformation, the differences between groups were determined by analysis of variance with repeated measures and 2-tailed Student t tests using GraphPad (GraphPad Software). Means were considered statistically different when p values were less than 0.05.Production of 1,25(OH)2D3 from 25(OH)DThe ability of preadipocytes and newly-differentiated adipocytes to produce 1,25(OH)2D3 from 25(OH)D3 was tested. Upon reaching confluence, preadipo.Ed on cultures derived from at least 3 different subjects. We did not notice any variations in the effects of vitamin D as a function of the BMI of the donor. In separate experiments, we also tested the effects of 1,25(OH)2D3 in the absence of a TZD in the differentiation cocktail.differentiated human adipocytes were incubated with 25(OH)D3 (1028 M) for 24 h in DMEM/F12 with no other additions. The quantity of 1,25(OH)2D3 in the incubation media was assayed with an enzyme immunoassay (Immunodiagnostic Systems Inc.). Data were expressed as picograms of 1,25(OH)2D3 produced per million cells.RNA Extraction and Measurement of Gene ExpressionTotal RNA was extracted using Trizol (Invitrogen) and quantity and quality were assessed spectrophotometrically. 1 mg total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) and qPCR was performed with the Light Cycler 480 (Roche) with Taqman probes (Applied Biosystems). Cyclophilin A (PPIA) was used as a reference gene.3T3-L1 Cell Culture3T3-L1 fibroblasts were cultured in 10 FBS supplemented DMEM. 2d post-confluent (day 0) cells were differentiated in DMEM with 10 FBS, 500 mM IBMX, 100 nM bovine insulin, and 1 mM dexamethasone. Medium was replenished with DMEM+10 FBS with 100 nM insulin on d2, and with DMEM+10 FBS on d4. 1,25(OH)2D3 (10211, 10210, 1028 M), 25(OH)D3 (1029 M), or vehicle (ethanol) was added to the media during differentiation at times specified in the figure legends.Western BlottingCells were washed with ice-cold PBS and scraped in cell lysis buffer (Cell Signaling) supplemented with 5 SDS and protease inhibitors (Pierce). 5?0 mg total protein was resolved in 10 or 15 Tris-HCl gels (Biorad), transferred to PVDF membranes, and blocked in 5 milk in Tris buffered saline with 0.2 tween-20. Membranes were probed for FABP4 (a gift from Dr. Judith Storch at Rutgers University), VDR (D-6, Santa Cruz), adiponectin (BD Biosciences), CYP27B1 (C-12 and H-90, Santa Cruz), and loading controls [(a-tubulin (Santa Cruz) and total ERK (Cell Signaling)]. Chemiluminescence images were captured using an Imager (LAS 4000, Fuji) and quantified using software (Multi Guage, Fuji).Mouse Primary Preadipocyte Culture and DifferentiationStromal vascular cells from the inguinal adipose tissue of C57BL/6J mice were prepared as described for human preadipocytes. Cells were grown and differentiated as described for 3T3-L1 cells, except that the differentiation cocktail with Rosiglitazone (1 mM) was added only during the initial 2dinduction period. 1,25(OH)2D3 or vehicle control (ethanol) was added continuously until harvest on day 7. Animal studies were conducted in conformity with PHS policy and approved by IACUC of Boston University Medical Campus.Triglyceride (TG) and DNA QuantificationTotal TG and DNA quantity in cell lysates were measured using a triglyceride determination kit (Sigma) and Quant-iTTM PicoGreen dsDNA reagent (Invitrogen). Statistical analysis. Data are expressed as means6standard error mean (SEM). After log transformation, the differences between groups were determined by analysis of variance with repeated measures and 2-tailed Student t tests using GraphPad (GraphPad Software). Means were considered statistically different when p values were less than 0.05.Production of 1,25(OH)2D3 from 25(OH)DThe ability of preadipocytes and newly-differentiated adipocytes to produce 1,25(OH)2D3 from 25(OH)D3 was tested. Upon reaching confluence, preadipo.