Peptidoglycan pgn from staphylococcus aureus

Purified blood neutrophils or sputum cells were resuspended in RPMI medium without phenol red (Gibco, Waltham, MA, USA) at a concentration of 1.5 × 10⁶ cells/ml or 3 × 10⁶ cells/ml, respectively. Prior to stimulation, the cells were incubated for 10 min at 37 °C in the presence of 50 ng/ml tumor necrosis factor-α (TNF-α; Peprotech, Rocky Hill, NJ, USA). Subsequently, the cells were added to a white clear-bottom 96-well plate (Perkin-Elmer, Waltham, MA, USA) and supplemented with luminol (5 mM; Sigma-Aldrich) and one of the following compounds: lipopolysaccharide (LPS) from Pseudomonas aeruginosa (10 µg/ml; Sigma-Aldrich); peptidoglycan (PGN) from Staphylococcus aureus (10 µg/ml; Sigma-Aldrich); N-formyl-methionyl-leucyl-phenylalanine (fMLF) (10^–8 M; Sigma-Aldrich). Phorbol 12-myristate 13-acetate (PMA; 150 ng/ml; Sigma-Aldrich) was used as a positive control. An additional condition without luminol was included to determine the background luminescence. The light signal was measured every minute for a period of 3 h by a Clariostar Monochromator microplate reader (BMG Labtech, Orthenberg, Germany). Analysis was performed by subtracting the background luminescence and determining the maximal luminescence as a measure for maximal ROS production. The maximal ROS production of cells stimulated by one of the compounds was normalized to the ROS production of unstimulated cells.

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.