Kgm 2

Primary keratinocytes were isolated from the tails of young and old Atg7^F/F or atg7ΔKC mice as described before [16 (link)] (young: <8 Months; old: >16 Months). The cells were cultured with keratinocyte growth medium-2 (KGM-2; Lonza, Basel, Switzerland) and experiments were performed without further passaging.

The human alveolar basal epithelial cell line (A549; ATCC number, CCL-185) and human bronchial epithelial cell line (BEAS-2B; ATCC number, CRL-9609) were used for the exposure. Before adding the cells, 24-well polyethylene terephthalate transparent membrane inserts (ThinCert™, Greiner Bio One International AG, Kremsmünster, Austria) (pore size =0.4 μm) were preconditioned (wetted) for 4 h +/− 15 min with the cell culture medium. For culturing A549 and BEAS-2B cells, F12k (Life Technologies, Zug, Switzerland) and KGM-2 (LONZA, Basel Switzerland) media were used, respectively. Cells were thawed from the stock pool and cultured in 75 cm² flasks. Confluent cell cultures were trypsinized after washing twice with phosphate buffered saline (PBS) without Ca²⁺ and Mg²⁺. The number of cells was counted using an electronic cell counter (CASY® Scharfe System, Roche Innovatis AG, Reutlingen, Germany). In brief, 50,000 A549 cells/insert and 60,000 BEAS-2B cells/insert were seeded for 24 h. Then, 18 ± 2 h before exposure, the culture medium was removed from the apical side of each insert, and cell monolayers were washed with PBS with Ca²⁺ and Mg²⁺.

miR-155 mimics and a scrambled control were obtained from Thermo Scientific. KCs were grown to 50–60% confluence. Fifty microliters Lipofectamine 2000 (Invitrogen) was combined with 5 mL Opti-MEM medium (Thermo Scientific) and 50 μL of the miR-155 mimics or scrambled control oligos. After incubation at room temperature for 30 min, the solution was added to 20 mL KGM-2 (Lonza) and transferred to the cells. KCs were then incubated for 24 h and used for skin models.