Extended Data Fig. 3: Basal-derived GEMM organoids maintain basal identity in vitro. Related to Figs. 1 and 2.

a, Representative flow cytometry data from tracheal basal cells analyzed with a live/dead marker, ITGA6, indicated basal markers (KRT5, NGFR), and/or hillock marker (KRT13). Gating strategy includes progressive gating on cell size, singlets, and live cells (top row), followed by analysis of ITGA6 on Y-axis and indicated control (no primary) or basal marker on X-axis. Percentage of total live cells indicated in each quadrant. Quantification in upper right indicates the percentage of ITGA6⁺ cells specifically that co-express another basal or hillock marker based on mean fluorescence of that marker relative to all live cells. b, Recombination PCR for RPM basal organoids for indicated alleles pre- and post-treatment with TAT-Cre or Ad-CMV-Cre at two concentrations (2.5e7 or 5e7 pfu). *Organoids subject to spinoculation with CMV-Cre virus. Red font = condition selected for subsequent allografting. For gel source data, see Supplementary Fig. 1. c, Co-immunofluorescence staining (Co-IF) of wildtype “WT” basal organoids (collected within ~2 weeks of CMV-Cre transformation) and CMV-Cre-transformed basal-derived organoids (RPM, RPR2, RPMA) for DAPI (nuclei, blue), KI67 (proliferation, orange), and ASCL1 (NE, green). Positive controls (bottom panel) are an RPR2 SCLC lung tumour. Quantification via CellProfiler of KI67 positivity per organoid per genotype (bottom). Number of organoids quantified is labeled. One-way ANOVA with post-hoc Tukey’s correction. Exact p-values in figure. Error is mean ± SD. d, Co-IF of WT basal organoids and CMV-Cre-transformed basal-derived organoids (RPM, RPR2, RPMA): Left panel) DAPI (nuclei, blue), NEUROD1 (neuronal, green), P63 (basal, yellow) and KRT8 (luminal basal, red). Positive control (+) for NEUROD1 is a murine olfactory neuroblastoma tumour and for basal markers is a murine squamous lung tumour. Right panel) DAPI (nuclei, blue), FOXJ1 (ciliated, purple), CCSP (SCGB1A1, club, green) and KRT8 (luminal basal, red). Positive control (+) control for FOXJ1 and CCSP is airway from a normal mouse lung, and for KRT8 is a murine squamous lung tumour. e, Co-IF of WT basal organoids and CMV-Cre-transformed basal-derived organoids (RPM, RPR2, RPMA) for DAPI (nuclei, blue) and POU2F3 (tuft, green). Positive control (+) is a murine olfactory neuroblastoma tumour. f, Co-IF staining of WT basal organoids and RPM basal-derived organoids for DAPI (nuclei) or indicated basal markers. Positive control is murine squamous lung tumour (+). All co-IF scale bars, 150 μm. Staining results in (c-f) are representative of organoids collected from three or more independent experiments per genotype, all with similar results.