Nylon mesh

For RECs isolation from kidney tissues, samples were washed with cold wash buffer (F12 medium containing 5% fetal bovine serum [FBS] and 1% P/S) and minced by sterile scalpel into 0.2–0.5 mm³ sizes to a viscous and homogeneous appearance. The minced tissue was then digested with dissociation buffer including DMEM/F12 (Gibco, USA), 2 mg/ml protease XIV (Sigma, USA), 0.01% trypsin (Gibco, USA) and 10 ng/ml DNase I (Sigma, USA) in 37°C incubator 2 h with gentle agitation. Digested cell suspensions were washed with cold‐wash buffer and passed through 70 μm Nylon mesh (Falcon, USA) to remove aggregates. Cell pellets were collected by centrifuge of 200g and then seeded onto a feeder layer of lethally irradiated 3T3‐J2 cells in modified SCM‐6F8 medium. Human SOX9+ RECs were generated from urine samples and expanded as previously described.²⁹For better visualization of colony growth, td‐Tomato+ RECs derived from mT/mG mice were used. For GFP labelling of cultured SOX9+ RECs, medium containing lentivirus was added to the cell culture together with 10 μg/ml polybrene (1:2000)²⁹ and cell identities were analysed by CytoFLEX LX flow cytometry with appropriate markers before use (SOX9+, ATP1A1− and CDH1−). Analysis was gated using forward and side scatters characteristics and corresponding positive/negative control. Data were analysed using the flow cytometry software, FlowJo (TreeStar).

The bronchoscopic brush with the trachea or bronchus epithelium sample was cut into 1 cm long pieces. After removing the sputum, the brush pieces were directly immerged into dissociation buffer (DMEM/F12 medium with 2 mg/mL protease XIV, 0.01% trypsin, and 10 ng/mL DNase I) and incubated for 1 hour at 37°C with gentle shaking. After dissociation, cell samples were passed through a 70 μm nylon mesh (Falcon, USA) to remove aggregates and washed three times with ice-cold F12 medium supplemented with 5% FBS and 1% Pen/Strep. Viable cell counts were determined using a hemocytometer. Cell pellets were collected by centrifugation at 200 × g and directly plated onto mitomycin-inactivated 3T3 fibroblast feeder cells and cultured under 7.5% CO₂ condition as previously described [9 (link)]. To obtain a single-cell-derived clone, a clone cylinder (Sigma, USA) and high vacuum grease (Dow Corning, USA) were used to pick up a single colony grown up from one cell. Typically, both DASC and TSC are passaged every 4-6 days in a 1 : 7 ratio.

3T3-L1 and C3H10T1/2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in DMEM with 10% FBS mixture at 37°C in a 5% CO₂ environment. Mice SVF was isolated from the iWAT of male C57BL/6J mice aged 6–8 weeks. SVF isolation was performed previously (24 (link), 25 (link)). Briefly, tissues were sheared, digested with 1 mg/ml collagenase in PBS supplemented with 0.5% bovine serum albumin (BSA), and 10 mM Hepes mixture for 30 min, with 300 rpm shaking at 37°C. The suspension was strained through a 40 μM nylon mesh (Falcon), collected by centrifugation. Finally, the adipocytes were then cultured in DMEM/F12 containing 10% FBS mixture and placed inside an incubator at 37°C with a 5% CO₂ environment.
After reaching 100% confluence, 3T3-L1, C3H10T1/2 cells, and mice SVF were stimulated to differentiate a hormone cocktail containing 50 nM insulin, 100 nM T3, 0.125 mM indomethacin, 2 μg/ml dexamethasone, and 0.5 mM IBMX. After 48 h, the adipocytes were moved to the medium containing 50 nM insulin and 1 nM T3. The media was replaced every 2 days before Rb2 treatment. Dimethyl sulfoxide (DMSO) was used as the vehicle treatment. Finally, the mature adipocytes were confirmed by light microscopy and Oil Red O staining, and then used for further analysis.