Fig 2: BCL9/9l control intestinal Lgr5 expression. a Representative ß-catenin staining of small intestinal sections from Cre-induced WT and VillinCreER Bcl9fl/fl Bcl9lfl/fl mice sampled 4 days post tamoxifen injection. Red arrows indicate nuclear ß-catenin at the base of the crypt. Scale bar = 50 µm. b Proximity ligation assay for E-cadherin: ß-catenin complexes (red) from small intestinal sections of small intestinal sections from mice described in (a). Nuclei stained with DAPI (blue). Paneth cells appear red, but were excluded from analysis. Scale bar = 50 µm. c Quantification of proximity ligation assay, % positive area per crypt (E-cadherin: ß-catenin) was quantified and 10 crypts were scored per mouse, n = 3 per group, one-way Mann–Whitney U test, P = 0.04. Data displayed as mean ±SEM. d qPCR for Cdh1 and Ctnnb1 expression in small intestine of mice described in (a), n = 3 per group, one-way Mann–Whitney U test, P = 0.35 (Cdh1) and P = 0.2 (Ctnnb1). Data displayed as mean ±SEM. e Gene Set Enrichment Analysis of RNAseq data obtained from small intestinal tissue from WT and VillinCreER Bcl9fl/fl Bcl9lfl/fl mice, n = 3 per group. f Representative Lgr5-RNAscope and ß-catenin staining of small intestinal sections from mice described in (a). Red arrows indicate Lgr5-staining at the base of the crypt. Scale bar = 50 µm

Fig 3: Pseudostratified columnar epithelium disruption in trachea of vaped mice.Pseudostratified cell architecture in normal trachea epithelial layer (No Vape) is disorganized by vaping exposure (Vape). (A) Deterioration of Basal Cell (red; Krt5) distribution, diminished Aquaporin 5 channel immunoreactivity (white; Aq5) in surface epithelium, and increased immunolabeling of E-Cadherin (green; E-Cad) in vaped sample relative to No Vape control. (B). Ciliated epithelium (red; a-tubulin) and cytoskeletal tetraspanin (white) marks apical epithelial boundary above disorganized cell layers in Vaped versus No Vape controls revealed by E-Cad (green) and nuclei (blue; DAPI). All images are representative and taken in superior trachea. (C) E-Cadherin (E-Cad) protein levels are significantly increased (**P < 0.01) in tissue samples prepared from lungs of vaped mice (Vaped) relative to normal lung samples (Non-Vaped). Corresponding quantitation of immunoblot is shown representative blot. GAPDH is used as loading control. Independent replicate blots of n = 6 using four non-vaped control mice and 17 vaped mice. Error bars represent SEM.Source data are available for this figure.

Fig 4: Efficacy of candidate compound combinations. MCF10A (left) and MCF10A CDH1-/- (middle) cells were drugged with P5-543 in combination with (A) chloroquine, (B) chlorpromazine, or (C) atorvastatin, then viability was quantified through nuclei counting, and normalization to vehicle controls. Treatment with single inhibitors is also depicted. Although an 8-point serial dilution was assessed, only the three greatest concentrations are shown. Combination index values (CI, right) indicate drug interactions. Synergistic effects are represented by CI > 0.9 (depicted by dotted red line), additive effects by CI values between 0.9 and 1.1, and antagonistic effects by CI < 1.1. Error bars depict +/- 1 standard error of the mean. P-values were calculated using Student’s t-test; * p = 0.05, ** p = 0.01.

Fig 5: Efficacy of compounds inhibiting sphingolipid metabolism and signaling. MCF10A and MCF10A CDH1-/- cells were drugged, then viability was quantified through nuclei counting, and normalization to vehicle controls. Although a nine-point serial dilution was assessed, only the three consecutive compound concentrations that exhibited the greatest difference in viability between CDH1+/+ and CDH1-/- cells are depicted. Where no toxicity was observed, the maximum assessed concentrations are depicted. (A) Cell viability following treatment with inhibitors of sphingolipid metabolism. (B) Cell viability following treatment with inhibitors of S1PRs. Average values were calculated from three biological replicates, with +/- 1 standard error of the mean depicted by error bars. P-values were calculated using Student’s t-test; * p = 0.05.