Fig 2: Panx1 channels regulate thermogenesis in brown adipocytes. Temperature measurement using ratiometric fluorescence thermal probes in brown adipocyte. iBACs were pre-treated with Panx1 inhibitors (50 µM) and then treated with 100 of nM CL316243 for 2 h. Normalized fluorescence (top) as well as the fluorescence ratio (F575/F513) (bottom) are shown. Gray bars indicate area that was used for calculation of the ratio. Statistical significance was calculated using two-way ANOVA with Tukey's post-hoc test. * indicates p < 0.05. Error bars, s. e.m. Experiments were performed with a minimum of 3 replicates and repeated at least twice. CBX, carbenoxolone; Spiro, spironolactone.

Fig 3: Apoptosis or ß3-adrenergic stimulation activate Panx1 channels in brown adipocytes. A, Immortalized pre-adipocytes differentiated into mature adipocytes (iBACs) were exposed to 200 mJ/cm2 UV-irradiation and carbenoxolone-sensitive Panx1 currents were measured by whole-cell patch-clamp analysis. Representative current–voltage (I–V) graph (left), patch-clamp recording at +80 mV (middle), and average current densities (right) are shown. B, Panx1-dependent ATP release from iBACs subjected to UV-irradiation as in A, in the presence or absence of Panx1 inhibitors, or a pan-caspase inhibitor (50 µM). C, Schematic showing activated Panx1 channel that allows for the release of ATP and the entry of fluorescent dyes. D, ATP release in iBACs treated with 100 nM of CL216343 for 30 min in the presence or absence of Panx1 inhibitors (50 µM). E, ATP release from iBACs transfected with 50 nM control or Panx1 siRNA for 48 h and then treated with 100 nM of CL216343 for 30 min. F, Representative images and quantification of ß3-agonist induced Yo-Pro-1 dye uptake in iBACs, which were pre-treated with Panx1 inhibitors (50 µM of spironolactone or 1 mM of probenecid) for 60 min and then treated with 100 nM of CL316243 for 30 min. G, Flow cytometric analysis of To-Pro-3 uptake in 293T cells transfected with GFP-Panx1 and mCherry-ß3AR (left), or GFP control and mCherry-ß3AR (right). H, ATP release from 293T cells transfected with Panx1 and ß3AR as in G. Forty-eight hours after transfection, cells were treated with 200 nM of CL316243 for 30 min. Statistical significance was calculated using Two-way ANOVA with Tukey's post-hoc test. * indicates p < 0.05. Error bars, s. e.m. Experiments were performed with a minimum of 3 replicates and repeated at least twice. CBX, carbenoxolone; Trova, trovafloxacin; Spiro, spironolactone; Proben, probenecid.

Fig 4: Gß? subunits mediate ß3AR-induced, Panx1-dependent ATP release in brown adipocytes. A, ATP release from iBACs treated with 100 nM of CL316243, 50 µM of forskolin or 10 µM of 8-Bromo-cAMP for 30 min. B, ATP release from iBACs transfected with 50 nM of control siRNA or siRNAs for Gß sububits (GNB1-4) for 48 h and then treated with 100 nM of CL316243 for 30 min. C, Co-immunoprecipitation of Panx1-Gß?. 293T cells were transfected with plasmids encoding mouse GFP-Panx1 or mouse myc-ADRB3 alone or together. Gß subunits were immunoprecipitated and Panx1 in the immunocomplex analyzed by Western blotting. D, ß3-agonist induced Panx1-Gß? interaction in 293T cells. Forty-eight hours after transfection with mouse WT-Panx1 or ?Gß? mutant Panx1 and ADRB3, cells were treated with CL316243 for indicated time points. Gß subunits were immunoprecipitated, and Panx1 in the immunocomplex was analyzed by Western blotting. E, Flow cytometric analysis of To-Pro-3 uptake in 293T cells transfected with GFP-Panx1-wt or GFP-Panx1 mutant (Panx1-?ß?) and mCherry-ß3AR. F, ATP release from 293T cells transfected with Panx1-wt or Panx1 mutant (Panx1-?ß?) and ß3AR. Forty-eight hours after transfection, cells were treated with 200 nM of CL316243 for 30 min. UCP1 mRNA expression in iBACs transfected with Panx1-wt or Panx1 mutant (Panx1-?ß?) and ß3AR. Forty-eight hours after transfection, cells were treated with 100 nM of CL316243 for 6 h. Statistical significance was calculated using one-way ANOVA with Dunnett post-hoc test (A) two-way ANOVA with Tukeys post-hoc test (B, F-G), one-way ANOVA with Dunnett post-hoc test (D), or two-tailed unpaired Student's t-test (E). * indicates p < 0.05. Error bars, s. e.m.