Supplementary Figure 6: L1CAM regulates YAP nuclear localization.

(a) Mean IHC scoring summary table of serially sectioned and stained patient samples that contain L1CAM⁺ metastatic cancer cells. Data are from 10 different patients (n = 15, 10, 6, 4, 3, 3, 4, 8, 8, 8 fields of view for patients A-J). Error values are S.D. (b) Single cell image analysis of L1CAM staining intensity with YAP nuclear staining intensity in serially sectioned patient IHC samples. Automated alignment is achieved assigning metastatic cells (L1CAM+, brown circles) to the nearest nucleus in the YAP image (green filled circles). Graphs to the right demonstrate L1CAM staining intensity correlation with YAP staining intensity. P values are calculated by linear regression (n = 159 and 611 cells for upper and lower panels in one field of view shown. Data represent 2 field of views calculated per patient). (c) YAP immunostaining of GFP⁺ H2030-BrM control and L1CAM knockdown cells on capillaries (Collagen IV staining) in brain tissue cultures depicted in Fig. 6c. Data represent 2 independent experiments. (d) YAP immunostaining of GFP⁺ MDA231-LM control and L1CAM knockdown cells spreading on vessels in mouse lungs in vivo, 2 days after tail vein injection depicted in Fig. 6d. Cancer cell GFP is cytoplasmic due to its nuclear export signal sequence (scale bars, 10 μm). Data represent 2 biological replicates. (e) Mean relative luciferase units of indicated H2030-BrM and MDA231-BrM cells in vitro, measured by CellTiter-Glo. YAP^5SA: constitutively nuclear YAP, YAP^S94A: TEAD binding deficient YAP. (n = 3 independent experiments done in three technical replicates). Error bars, S.E.M. See Supplementary Table 1 for statistics source data.