Fig. 4: KIRREL1 regulates Hippo signaling through recruiting SAV1 to the cell–cell contact sites.

a Dual inhibition of KIRREL1 and SAV1 has no additive effect on GTIIC-GFP^PEST reporter. HEK293A GTIIC-GFP^PEST Cas9 cells with inhibition of KIRREL1, SAV1 or both were subjected to flow cytometry analysis. b Dual depletion of KIRREL1 and SAV1 has no additive effect on expression of YAP target genes. Cells were plated at medium density (0.5 × 10⁶ cells/well) in 12-well plate and subjected to qRT-PCR analysis 48 h of post cell plating. Data represent mean ± SD, n = 4, error bars denote the SD between four biological replicates; Unpaired two-tailed t-test was used to determine the statistical significance, ***p value < 0.001, ns—0.0978. c Overexpression of KIRREL1 promotes localization of SAV1 to the cell-cell contacts. HEK293A cells were co-transfected with plasmids encoding GFP-tagged SAV1 and Myc-tagged KIRREL1 and subjected to immunofluorescence assay using anti-Myc and anti-GFP antibodies. DAPI was used to mark the nuclei. Scale bar 10 µm. d Knockout of KIRREL1 enhances YAP activity in neighboring cells. HEK293A GTIIC-GFP^PEST Cas9 cells and HEK293A GTIIC-GFP^PEST Cas9 cells transduced with lentivirus expressing RFP and control or KIRREL1 gRNAs were mixed at 1:10 ratio, co-cultured for 2 days, and subjected to FACS analysis. The signal of GTIIC-GFP^PEST in RFP-negative parental cells is plotted. e Active YAP induces expression of KIRREL1 mRNA. HEK293A cells stably expressing vector or YAP 5SA mutant were plated at medium density (0.5 × 10⁶ cells/well) in 12-well plate and subjected to qRT-PCR analysis 48 h of post cell plating. LATS2 and NF2 were used as positive control. The data represent mean ± SD, n = 4, error bars denote the SD between four biological replicates; Unpaired two-tailed t-test was used to determine the statistical significance, ***p value < 0.001. f KIRREL1 mRNA expression is regulated by cell density. HEK293A cells were plated at high (1 × 10⁶ cells/well), medium (0.5 × 10⁶ cells/well), or low density (0.25 × 10⁶ cells/well) in 12-well plate and subjected to qRT-PCR analysis 48 h of post cell plating. CTGF, CYR61, and ANKRD1 were used as positive control. The data represent mean ± SD, n = 4, error bars denote the SD between four biological replicates; Unpaired two-tailed t-test was used to determine the statistical significance. Source data for Fig. 4b, e, f are provided as Source Data file.