HBECs were isolated from patients who underwent surgery because of lung cancer as described previously [
11]. Only cancer-free tissue was used for cell isolation. The protocol was approved by the institutional review board (ethics committee) of the Landesärztekammer of the State of Saarland, and informed consent was obtained from the patients. Organoids were cultured as described before [
10]. In brief, 80 μl of passage 1 cells (3 × 10
4 cells per ml differentiation media containing 5% Matrigel) were plated in each well of a 96-well plate containing 40 μl per well of a 25% Matrigel (Corning, USA) solution as base layer. 120 μl differentiation media was added at day 2 containing 100 ng/ml of Pam3CSK4, polyinosinic:polycytidylic acid (pIC), ultrapure LPS from
Escherichia coli 055:B5, or flagellin from
P. aeruginosa (Invivogen, USA) and 5% Matrigel. Media was changed every 4 days. The presence of lumina, cilia beating, and diameter were determined under the phase contrast microscope 14, 22, and 30 days after seeding. Organoids from each condition were pooled and embedded in paraffin 30 days after seeding. Paraffin sections (2 μM thickness) were stained with haematoxilin-eosin (H&E). Deparaffinized paraffin sections were treated with BSA (1%) and Tween-20 in PBS (0.1%) and incubated overnight at 4 °C with the primary antibodies for MUC5B (sc-393,952, Santa Cruz, USA, 1/100) and KRT5 (ab-75,869, Abcam, USA, 1/100) in PBS containing BSA (1%). Cells were incubated for 30 min with secondary antibodies (goat anti-mouse FITC, Sigma, Germany; goat anti-rabbit, Cyanine 5, Thermo-Fisher Scientific, Germany). Immunohistochemistry for MUC5AC (ab-3649, Abcam) was performed as described before [
12]. To analyze and merge images, we used ImageJ software (National Institutes of Health). Western blot was performed as described before [
12]. Membranes were probed with monoclonal antibodies directed against MUC5B (sc-393,952, Santa Cruz) and β-actin (#4967, Cell Signaling Technology, USA). The ultrastructure of untreated bronchospheres were analyzed by electron microscopy as described before [
13]. Ultra-thin sections were contrasted with uranylacetate using routine procedures. The analysis was performed using a FEI Technai 12 transmission electron microscope (FEI, Thermo-Fisher Scientific) at 100 kV, equipped with a digital 8-bit camera. Comparisons between groups were analyzed by t test (two-sided) using the software Prism (GraphPad Software, USA). Technical replicates were combined to a single data point for each independent experiment. The results were considered statistically significant for
P < 0.05.