Extended Data Fig. 4. Initial validation of screen hits.

(A) Schematic of screen hit validation strategy. HeLa cells stably expressing GFP-SFTPC-WT and Cas9 were transfected with plasmids containing sgRNAs against specific genes for hit validation. Three guides for each gene were pooled for transfection. Cells underwent puromycin selection for 48 hours to select for transfected cells. (B-C) Knockout pools assessed for cell surface SFTPC enrichment by live cell confocal microscopy (B) and flow cytometry (C). Scale bar = 10 μm. (D) Quantification of full-length cell surface SFTPC as measured by C-terminal antibody and expressed as mean fluorescence intensity. Mean ± SEM, n=3 independent repeats (*p < 0.05, **p < 0.01,***p<0.001; paired two-tailed Studen’s t-test). (E) Relative expression of SFTPC mRNA in GFP-SFTPC-Cas9 control cells and UBE2I, UBA2, and PIAS1 knockout pools (representative result of 3 independent repeats).