Penicillin streptomycin amphotericin b solution

For the alveolar cell differentiation, cells with 10⁵ cells had seeded on the collagen I-coated 24 well plate. MCDB201 medium (Sigma-Aldrich) supplemented with 5% FBS, Insulin-Transferrin-Selenium (Gibco), 1X penicillin-streptomycin-amphotericin B solution (PSA from 100X stock, Biological Industries), and 25 ng/mL of epidermal growth factor (Corning) were replaced every two days. Cells had differentiated after 7-14 days of culture. For trachea epithelial cell differentiation, cells with 10⁵ cells had seeded on the collagen I-coated 24-well transwell inserts with 0.4 μm pore (Corning). MCDB201 medium supplemented with 5% FBS, Insulin-Transferrin-Selenium, 1X PSA, 0.1 μg/mL of cholera toxin, 30 μg/mL of bovine pituitary extract (Gibco), and 25 ng/mL of epidermal growth factor. Medium in the transwell inserts with 0.4 μm pore and the lower chambers had replaced every two days. As cells reached 100% confluence, cells had maintained under the air-liquid interface by leaving the transwell inserts empty for 10-15 days.

Mouse ES cell lines E14Tg2a.IV were purchased from the American Type Culture Collection (ATCC; https://www.atcc.org:443/). mESCs E14Tg2a.IV, CCE, Smad4 null (BNN)^{77 (link)}, Smad2 null (KT15)^{78 (link)}, Smad3 KD/Smad2 null, and Trim33 null^{25 (link)} lines were maintained on 0.1% gelatin-coated plates in LIF-supplemented medium at 37 °C with 5% CO₂ as previously described^{25 (link),79 (link)}. Basic mESC medium contains Dulbecco’s modified Eagle’s medium (DMEM, Thermofisher), 15% Fetal Bovine Serum (FBS) (ExCell Bio), 1% penicillin-streptomycin-amphotericin B solution (Biological Industries), 1% non-essential amino acids (Thermofisher), 1% L-glutamine (Biological Industries), 1% sodium pyruvate (Sigma), 100 μM β-mercaptoethanol (Sigma), 103 U/mL mouse LIF. HEK293T and Hep G2 cells were cultured in DMEM supplemented with 10% FBS (ExCell Bio) and 1% penicillin-streptomycin-amphotericin B solution. We maintained all cell lines at 37 ^oC in a 5% CO₂ cell culture incubator and tested all cell lines routinely for mycoplasma contamination.

Dulbecco’s Modified Eagle Medium (DMEM) and trypsin-EDTA were obtained from Gibco (Carlsbad, CA, USA). Foetal bovine serum (FBS) was procured from HyClone (Logan, UT, USA). Penicillin-streptomycin-amphotericin B solution was sourced from Biological Industries (Kibbutz Beit Haemek, Israel). Dulbecco’s Phosphate Buffered Saline (PBS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Gefitinib was procured from Selleckchem (Houston, TX, USA). R406, PP2, and the p-Syk antibody were purchased from Calbiochem (San Diego, CA, USA). Olaparib was procured from Selleckchem. Recombinant human EGF was obtained from PeproTech (Rocky Hill, NJ, USA). PARP, γH2AX, p-EGFR, p-JNK, JNK, p-p38, p-ERK, p-Lyn, p-Src, p-STAT1, p-STAT3, and p-STAT5 antibodies were obtained from Cell Signaling (Beverly, MA, USA). EGFR, p38, ERK, Lyn, Fyn, Fgr, Src, Syk, STAT1, and STAT3 antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Antibody against PAR was obtained from BD Pharmingen (San Diego, CA, USA). The β-actin antibody was procured from NOVUS (Littleton, CO, USA).

BEnEpC derived from healthy bovine uterus was purchased from Cell Applications, Inc (San Diego, CA, USA). Cells were seeded in standard sterile 75 cm² tissue culture flask and cultured in Dulbecco’s Modified Eagle Medium (DMEM/F12; Gibco, Thermo Fisher Scientific Inc., Taiwan) supplemented with 10% fetal bovine serum (FBS; Corning Incorporated, USA) and Penicillin-Streptomycin-Amphotericin B Solution (Biological Industries, Germany) at 37°C in a humidified atmosphere of 95% air and 5% CO₂.
Ten-fold serial dilutions of CHX (0.00002 to 2%) and PI (0.00025 to 2.5%) were prepared with ultrapure water. The treatment concentrations were selected based on the MBC of E. coli and T. pyogenes in Experiment 1.

Rat hippocampal neurons were obtained either from one day old new born rats or from 17 days old embryos, as described by Kaech and Banker (2006) (link). Briefly, for embryonic hippocampal cultures the pregnant female was anesthetized, the embryos removed and decapitated. The embryonic or new born hippocampus were removed and treated with papain for 45 min (Sigma–Aldrich), and serially triturated. Cell density at plating was 250,000–500,000 cells/ml. Cells were seeded in attachment\seeding medium [Neurobasal medium, 5% FBS, 2% B27, 1%GlutaMAX (All from Life technologies), 1% Penicillin-Streptomycin Amphotericin B Solution (Biological Industries)]. 24 h (1 DIV) after culturing the seeding medium was replaced with serum-free maintenance\feeding medium (Neurobasal medium, 2% B27, 1% GlutaMAX, 1% Penicillin-Streptomycin Amphotericin B Solution). At 3 DIV 5 μM ara-c (Sigma–Aldrich) was added to prevent glial cell’s proliferation. Half of the maintenance medium was replaced every 3–5 days by astroglial conditioned medium. Hippocampal cultured cells were kept at 37°C in a humidified atmosphere of 5% CO₂. Cultures were kept till 7–21 DIV. All procedures were approved by the Committee for Animal Experimentation at the Institute of Life Sciences of the Hebrew University of Jerusalem.