A3160501

The HEK293T cells (a kind gift from Drs. Jae Lee and Soo Lee at University at Buffalo, SUNY) were cultured at 37 °C and 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (Gibco™ 11965092) supplemented with 10% fetal bovine serum (Gibco™ A3160501). To initiate transfection, 20,000 cells were seeded in Nunc™ Lab-Tek™ II chambered coverglass (8 wells). After 24 h, Lipofectamine 2000 reagent (Thermofisher 11668030) was used to transfect the cells with 0.5 μg plasmid, according to the manufacturer’s protocol. The cells were imaged after 20−24 h of transfection. Colocalization experiments were carried out with 0.5 μg plasmid for each construct. Table S5 provides a complete list of plasmids used for protein expression in HEK293T cells.

The 3D organoid cultures were performed as described previously [21 (link)]. Freshly isolated AT2 cells (6000/transwell) were mixed with MLg2908 cells (2 × 10⁵/transwell, ATCC CCL-206™, USA) and suspended in a 1:1 mixture of growth factor reduced Matrigel (BD Biosciences) and organoid medium (DMEM/F12 + 2 mM l-Glutamine [MP Biomedicals Europe, Illkirch, France] + 10% Active FBS [A31605-01, Gibco] + 1% ITS [41400-045, Gibco] + 1% Pen/Strep and 10 μM TGF beta inhibitor [Ab120163, Abcam, Cambridge, UK]). The cell mixture was seeded on top of polyester membrane cell culture transwell inserts at 45 μL/transwell (Costar 3470: 0.4 μm pore size, 0.33 cm² area, Corning) and incubated for 30 min to allow the matrix to solidify. We added 600 µL of organoid medium to the bottom of the transwell and replaced half (300 μL) of the medium on alternate days. Cultures were grown at 37 °C and 5% CO₂ for 4–12 days. Then, 10 µM of PKA inhibitor H89 (371963, Millipore, Bedford, MA, USA) was dissolved in the culture medium and applied to cells. For LPS treatment, 1 µg/mL of LPS (L6529, Sigma) was dissolved in the culture medium and applied. H89 and LPS were added to the medium on alternate days.