F3917

Fully differentiated HBECs on Snapwell filters were mounted in Ussing chambers (Easymount chamber; Physiologic Instruments) connected to a VCC MC6 voltage clamp unit (Physiologic Instruments, San Diego, CA, USA). Solutions were maintained at 37 °C by heated water jackets and bubbled with air. For BK activity, basolateral membranes were permeabilized for 30 min with 20 µM amphotericin B, 10 µM nigericin and 10 µM valinomycin because whole cell short circuit current recordings cannot distinguish K⁺ efflux (it measures net current, a combination of K⁺ and Cl⁻ efflux)^{10 (link)}. Furthermore, cells were exposed to a basolateral (140 mM) to apical (5 mM) K⁺ gradient, in the presence of apically applied 10 µM amiloride (Sigma-Aldrich #A7410, St. Louis, MO, USA) and 10 µM ATP (Sigma-aldrich #A1852). CFTR activity was measured with apical 5 mM Cl⁻ in the presence of apically applied 10 µM amiloride, 10 µM forskolin (Sigma-Aldrich #F3917) followed by 10 µM CFTR_inh172 (Sigma-Aldrich #C2992)^{8 (link)}.

All stock solutions were prepared at 1000× concentration. 100 mM caffeine (108160100, ACROS Organics) was prepared as an aqueous solution and was stored at 4 °C for up to 4 weeks. The solution was warmed to 37 °C before experiments to dissolve precipitates. For dose–response experiments, working solutions were prepared by serial dilution in DMEM with 10% (v/v) FBS (F7524, Sigma-Aldrich) and 1% (v/v) streptomycin/penicillin (L0022, Biowest), prewarmed to 37 °C. 10 µg/mL recombinant human TNFα (300-01A, Peprotech),
Aqueous solutions of 10 µg/mL recombinant human FGF-basic (154 a.a.) (100-18B, Peprotech) and 10 µg/mL IL-6 (200−06, PeproTech) were prepared and stored at −20 °C prior to use.
A solution of 10 mM forskolin (F3917, Sigma-Aldrich) in ethanol was prepared and stored at −20 °C prior to use.
An aqueous solution of 4 M KCl (P9541, Sigma-Aldrich) was prepared and stored at 4 °C.
A solution of 10 µM rapamycin (AG-CN2-0025-C100, AdipoGen) in DMSO was prepared and stored in aliquots at −80 °C.

For the establishment of primary cell cultures, single fibers of 3 weeks old male WT mice were isolated as described in the Supplemental Experimental Procedures. Primary myoblasts were differentiated at around 60–70% confluency using differentiation medium (DMEM Glutamax, 4% HS, 1% P/S, 1% CEE) for 3 days. The next day, cells were serum starved with low glucose medium (LG, D6046, Sigma) for 16 h before treatment of the different compounds for 6 h. Compounds used were forskolin (100 μM, F3917, Sigma), 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP, 100 μM, C3912, Sigma), 3-isobutyl-1-methylxanthine (IBMX, 1 mM, I5879, Sigma). All compounds were diluted in dimethyl sulfoxid (DMSO, 1%, D2650, Sigma).

To induce preadipocyte differentiation, white and brown adipocyte precursors at day 2 post confluence were shifted in induction medium composed of DMEM plus 10% FCS supplemented with 0.85 µM insulin (19278, Sigma), 0.2 nM triiodothyronine (T3) (T-2877, Sigma), 1 µM dexamethasone (265005, CalBiochem), 0.5 mM isobutylmethylxanthine (I-5879, Sigma) and 125 nM indomethacin.
For white adipogenic differentiation, after 3 days of induction, the medium was replaced by white differentiation medium (WAT medium): DMEM plus 10% FCS supplemented with 0.85 µM insulin (19278, Sigma), 0.2 nM triiodothyronine (T3) (T-2877, Sigma), 100 nM pioglitazone (E6910, Sigma), and 125 nM indomethacin. WAT medium was replaced every other day and cells were allowed to differentiate for 6 additional days.
For brown adipogenic differentiation, after 3 days of induction, the medium was replaced by brown differentiation medium (BAT medium): DMEM plus 10% FCS, 0.85 µM insulin, 20 nM T3, and 100 nM pioglitazone. Cells were maintained in BAT medium for 17 days and the medium was replaced every other day. Twelve hours before the end of the differentiation protocol, cells were treated with 10 μM forskolin (F-3917, Sigma) and 1 μM pioglitazone directly added to the differentiation medium (total days 20).

Mature adipocyte cell layers were washed twice in plain pre-warmed DMEM and cultured in plain DMEM with inhibitors prior to stimulation with the following isoproterenol (Sigma-Aldrich I6504) concentrations: 0.1 μM immortalized day 8 and day 10 brown adipocytes; 1 and 0.1 μM d8 brown adipocytes. After 6 h of treatment, the cells were washed once with cold PBS and RNA was harvested using TRizol or QIAzol lysis reagent as described below. For immunoblotting, the cultures were harvested after indicated treatment times with isoproterenol after washing with cold PBS on ice and adding 50 μl RIPA buffer per well of a 6 well plate or 100 μl RIPA buffer to a 10 cm dish. For stimulation with forskolin (Sigma-Aldrich F3917) 1μM dissolved in DMSO was used.