β counter

The 2-[1–3H] deoxyglucose (2-DG) uptake assay was performed as previously described [22] (link) with minor modifications. Cells differentiated in 24-well plates were incubated for 2 h in serum-free medium and then stimulated in triplicate for 1 h at 37°C with insulin (0, 100 nM and 2 µM). The test was initiated by incubating for 15 minutes at 37°C in a solution of D-glucose (50 µM) and 2-DG (1.5 µCi/ml) (GE Healthcare, Waukesha, Wisconsin, USA). The test was terminated by two rapid washes in ice-cold PBS. Cells were solubilized with 0.1% Triton X-100/PBS and the radioactivity was measured using a β-counter (PerkinElmer, Waltham, MA, USA). An aliquot of each cell lysate was removed for total protein quantification with a Micro BCA Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The insulin-induced glucose uptake was normalized to total protein content and reported as percent increase with respect to the basal value.

G9a activity was measured using the HMT assay reagent kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. For the in vitro methyltransferase assay, the recombinant Runt domain was incubated with the in vitro translated G9a enzyme using 1 μCi [³H]-adenosyl-L-methionine (SAM, PerkinElmer, Waltham, MA) as the methyl donor. ³H-methylated protein was transferred to a membrane, soaked in scintillation cocktail and radioactivity was measured using a β-counter (PerkinElmer).

For glucose uptake, bone marrow–derived macrophages were incubated for 10 min at 37 °C with 1 µCi/ml 2 deoxy-D-(1-³H) glucose (Amersham) and 10 µM deoxyglucose (Sigma) in uptake buffer (140 mM NaCl, 20 mM HEPES, 5 mM KCl, 2.5 mM MgSO₄*7H₂O, 1 mM CaCl₂*2H₂O, pH 7.4). Cells were washed 3 times with ice cold PBS and lysed with 500 µl NaOH (0.2 M). Radioactivity was assessed in 300 µl cell lysate with a β-counter (Perkin Elmer) and normalized to the protein content. For glucose treatment, cells differentiated in medium with 4.5 mg/ml glucose were incubated overnight in medium with 2.5 mg/ml glucose and thereafter in medium containing 6 mg/ml glucose^{29 (link)}. Control cells received 6 mg/ml mannose instead.

Cell proliferation of responder T cells was assayed as previously described^{14 (link)}. After purification, T_regs were added to autologous CD4+CD25− cells, CD4+CD25−CD7+ cells, or CD4+CD25−CD7− cells (T_reg: responder T-cell ratio of 1:84). Cells were cultured (5% CO₂, 37 °C) with anti-CD3 and anti-CD28 T-cell expander (bead: cell ratio of 1:2; Dynal Biotech, Oslo, Norway) along with recombinant IL-2 (30 U/ml; Peprotech, NJ, USA) for 5 days. Similarly, CD4+CD25− cells, CD4+CD25−CD7+ cells, and CD4+CD25−CD7− cells were cultured without T_regs under identical conditions for control purposes. Over the final 18 h, these cells were pulsed with 3H-thymidine (0.25 μCi/well) and then harvested with the help of a multichannel harvester. The amount of cellularly incorporated 3H-thymidine was assessed with a β-counter (Perkin Elmer, Shelton, CT, USA). The proliferative inhibition percentage was calculated as follows: (1 − CPM [with T_regs]) ÷ (CPM [without T_regs]). Experiments were performed in triplicate.

¹⁴C-labeled substrate oxidation to ¹⁴CO₂ was performed as described earlier^{51 (link)} with minor modifications. Cells at ~80–90% confluency were treated with rapamycin (100 nM)/torin1 (250 nM) for 24 h. Cells were then serum starved for 2 h and 0.5 μCi of radiolabeled 1-¹⁴C palmitate (BRIT, Mumbai, India) or 0.5 μCi of [¹⁴C(U)]d-glucose (Perkin-Elmer, Waltham, MA, USA) was added to 1.5 ml of media. A Whatman chromatography paper cut to the size of a 6-well plate was soaked in 3 M NaOH and placed on the lid of the plate, such that each well is covered. The cells were incubated at 37 °C for 2 h. Following the incubation, 500 μl of 70% perchloric acid was added to each well and the released CO₂ was trapped in the Whatman paper for 1 h. The filter paper was left for drying overnight and the ¹⁴C levels were estimated using a β-counter (Perkin-Elmer). Cells were scrapped and pelleted down after a brief centrifugation at 5000 r.c.f for 5 min at 4  °C. The pellet was washed two times with 1× PBS and lysed using 0.5 N NaOH, vortexed and centrifuged to isolate the cell lysate at 18 000 r.c.f. for 10 min at 4 °C. Experiments were carried out in triplicate and normalized to total cellular protein.

3T3-L1 adipocytes were cultured overnight in serum-free DMEM in 12-well plates and then washed in phosphate-buffered saline (PBS) and incubated in glucose-free DMEM for 1 h. Subsequently, 18.5 kBq of 2-deoxy-d-³H-glucose (PerkinElmer, San Jose, CA, USA) and 0.1 mM 2-deoxyglucose were added. After 30 min, the reaction was terminated with cytochalasin B (20 μM), and the cells were washed with ice-cold PBS. Cells were lysed for 10 min in 0.1% sodium dodecyl sulfate (SDS), and then aliquots of lysates were used for liquid scintillation counting and determination of protein concentration by BCA protein assay (Thermo Scientific, Rockford, IL, USA). Radioactivity was measured using a β-counter (PerkinElmer). Radioactivity was normalized to protein concentration and is presented as a percentage of basal level.