then loaded onto the inserts at 1×105 cells/200 for each assay. Conditioned medium from μL adenoviral transduction assays was placed in the bottom chamber (500 After 1 hour of l). incubation at with 5% CO2, cells that migrated to the lower chamber were collected; the number of cells that migrated from the top to the bottom compartment was measured by counting. Respiration experiments on intact cells. THP-1 macrophages and SGBS adipocytes were obtained and cultured as described (3, Cells were introduced in the 2 mL chamber of an 4). Oroboros-2k™ respirometer (Oroboros® Instruments GmbH Corp, ) to perform high-resolution respirometry studies. respiration was assessed under conventional culture conditions at Routine 37 oC. To study respiration, oligomycin (inhibitor of ATP synthase) was added to the Leak chamber. To study (ETS) capacity, FCCP was titrated until reaching electron transfer system maximal respiration. Subsequently, complex I was inhibited by adding rotenone to the chamber and finally antimycin A was added to inhibit oxidative phosphorylation at the level of complex III. The oxygen consumption that remains is considered (ROX). residual oxygen consumption ROX values are subtracted from previous oxygen measurements to calculate oxygen flux at the different respiratory states. In this case, oxygen consumption values were normalized by total number of cells. Lactate and succinate release. Extracellular lactate and succinate measurements were performed on conditioned medium. THP-1 or SGBS cells were plated, infected with PTG or GFP, and supernatant media were collected after 24 hours. Supernatant from at least three independent experiments was assayed using a lactate assay kit (BioVision, Mountain View, CA) and succinate assay kit (BioVision, Mountain View, CA) following the manufacturer´s instructions. Animals. Adipose tissue gene expression analysis was performed on Wild-type (WT) and homozygous PTG KO male mice maintained on a C57BL/6J background (5). All mice were maintained in temperature- and humidity-controlled conditions with a 12-h light/dark cycle and
then loaded onto the inserts at 1×105 cells/200 μL for each assay. Conditioned medium from adenoviral transduction assays was placed in the bottom chamber (500 l). After 1 hour of incubation at with 5% CO2, cells that migrated to the lower chamber were collected; the number of cells that migrated from the top to the bottom compartment was measured by counting. Respiration experiments on intact cells. THP-1 macrophages and SGBS adipocytes were obtained and cultured as described (3, 4). Cells were introduced in the 2 mL chamber of an Oroboros-2k™ respirometer (Oroboros® Instruments GmbH Corp, ) to perform high-resolution respirometry studies. Routine respiration was assessed under conventional culture conditions at 37 oC. To study Leak respiration, oligomycin (inhibitor of ATP synthase) was added to the chamber. To study electron transfer system (ETS) capacity, FCCP was titrated until reaching maximal respiration. Subsequently, complex I was inhibited by adding rotenone to the chamber and finally antimycin A was added to inhibit oxidative phosphorylation at the level of complex III. The oxygen consumption that remains is considered residual oxygen consumption (ROX). ROX values are subtracted from previous oxygen measurements to calculate oxygen flux at the different respiratory states. In this case, oxygen consumption values were normalized by total number of cells. Lactate and succinate release. Extracellular lactate and succinate measurements were performed on conditioned medium. THP-1 or SGBS cells were plated, infected with PTG or GFP, and supernatant media were collected after 24 hours. Supernatant from at least three independent experiments was assayed using a lactate assay kit (BioVision, Mountain View, CA) and succinate assay kit (BioVision, Mountain View, CA) following the manufacturer´s instructions. Animals. Adipose tissue gene expression analysis was performed on Wild-type (WT) and homozygous PTG KO male mice maintained on a C57BL/6J background (5). All mice were maintained in temperature- and humidity-controlled conditions with a 12-h light/dark cycle and
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