CRISPR/Cas9-mediated knockout of FMNL2 affects lumen formation in MCF10A cells.

<p><b>(A)</b> Simplified structure of the human FMNL2 locus. <b>(B)</b> Cartoon illustrating the FMNL2 protein. Red asterisk (*) indicates the region targeted by Cas9. bp, base pairs; aa, amino acid; GBD, GTPase binding domain; FH, formin homology domain; DAD, diaphanous autoinhibitory domain. <b>(C)</b> Western Blot comparing FMNL1, 2, 3 expression in WT and FMNL2-/- cells. Antibodies were applied as indicated. <b>(D)</b> Colorimetric cell viability assay shows no differences in proliferation between generated cell lines (N = 3, triplicates for each time point, error bars SD, ns non-significant). <b>(E)</b> WT and FMNL2-/- cells were grown in hydrogel for 14 d before staining for F-actin and DAPI. Graphs depict quantifications of successful lumen formation and the overall area covered by spheroids (N = 3, *p ≤0.0001, <i>n</i> = 8, error bars SD, ns indicates no significance).