Troglitazone tro

To induce sebaceous differentiation, SebE6E7 sebocytes were treated with 1 μM troglitazone (TRO, PPARγ agonist) (Sigma-Aldrich) with or without 0.1 μM LG100268 (LG, RXR agonist) (Sigma-Aldrich) for 4 days. Where indicated, SebE6E7 sebocytes were treated with 100 nM 5α-dihydrotestosterone (5αDHT) (Sigma-Aldrich) and/or 0.1, 1 or 5 μM all-trans retinoic acid (RA) (Sigma-Aldrich) for 5 (RT-qPCR and stainings) to 12 (colony formation assay) days. For immunological assays, cells were stimulated with 1 μg ml⁻¹ Peptidoglycan (PGN) from Staphylococcus aureus (Sigma-Aldrich) for 72 h or 1 U ml⁻¹ human recombinant IFNγ (ThermoFisher Scientific) for 72 h. Sebaceous organoids were treated with 25 μM RepSox (TGFβ type I receptor/ALK5 inhibitor) (R&D Systems) or 5 μM RA for 6 days.
All chemicals were dissolved in sterile DMSO, except 5αDHT which was dissolved in methanol, and PGN and IFNγ in sterile water. The same concentration of diluent was used as the Vehicle control condition.

Dulbecco’s modified Eagle’s medium (DMEM)-high glucose and collagenase were purchased from Wako Pure Chemicals (Osaka, Japan). Fatty acid-free bovine serum albumin (BSA), isobutylmethylxanthine (IBMX), dexamethasone (Dex), insulin, 3,3′,5-triiodothyronine (T3), and troglitazone (Tro) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum (FCS) was obtained from Trace Scientific Ltd. (Melbourne, Australia). Rabbit anti-serum against UCP1 was a gift from Dr. Teruo Kawada (Kyoto University, Japan). Antibodies against aP2, caspase-9, and the phosphorylated form of Rb were purchased from Cell Signaling Technology (Beverly, MA, USA); those against β-actin, p57, and a-Tubulin were from Sigma-Aldrich; those against p27 and Rb were from BD Biosciences (San Jose, CA, USA); that against PCNA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and those against Ki67 and MCT1 were from Abcam (Cambridge, MA, USA). Anti-rabbit and anti-mouse secondary antibodies conjugated with horseradish peroxidase were obtained from Cell Signaling Technology. Anti-rabbit or anti-chicken secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 were obtained from Thermo Fisher Scientific (Gaithersburg, MD, USA).