Fig. 1: High-throughput screening on multi-cellular murine alveolosphere assay identifies small molecules that rescue TGFβ impaired alveolar type 1 spheroid formation.

A Schematic of assay. Whole distal lung cells from Hopx-GFP mice were plated in 384-well plates, cultured in Matrigel for ~12 days to promote spheroid formation, and imaged live for GFP signal to quantify alveolar type 1 spheroids. B Representative images of live alveolospheres from C57BL/6 control or Hopx-GFP mice treated at time of plating with increasing doses of TGF-β1 with or without 30 µM of TGF-βR1 inhibitor galunisertib. Scale = 500 µM. C Quantification of (B). n = 10-12, individual data points plotted. One-way ANOVA, P = 5.9 × 10⁻⁵. **α < 0.01, Tukey’s HSD (Hopx-GFP mouse; TGF-β1 0.015 ng/mL vs. 2 ng/mL). D Quantification of canonical AT1 markers from alveolospheres described in (B). n = 5–12, individual data points plotted. E Quantification of GFP spheroids vs. GFP total intensity for controls or small molecule library. Red = positive control (30 µM galunisertib). Blue = negative control (no compound addition). Green = called compounds with biological activity. Black = compound without called biological activity. Star = LA-13-ZS70; primary hit for followup. Dotted line indicates Median +3 SD.