Supplementary Figure 5: L1CAM mediated re-wiring of transcriptional programs.

(a) Heat map of differential gene expression with unsupervised clustering. TRAP RNA sequencing results of H2030-BrM control and L1CAM knockdown cells in BSCs described in Fig. 5a are shown as normalized row z-scores. (b) Top five gene signatures from the TRAP data in (a) ranked based on GSEA normalized enrichment scores (NES). FDR, false discovery rate. Data in (a-b) are from 2 biological replicates. (c) Relative mean expression of YAP target genes ANKRD1, ITGB2, and CTGF in H2030-BrM cells transiently expressing the indicated constructs. YAP^WT: YAP Wildtype; YAP^5SA: constitutively nuclear YAP mutant; YAP^S94A: TEAD binding deficient YAP mutant (n = 4 independent experiments in triplicate). (d) Relative mean expression of MRTF-A target genes in indicated cell lines. MKL1: gene name for MRTF-A. Data are from n = 3 biological replicates. Error bars in (c-d): S.E.M. (e) Fluorescence and bright field images of MDA231-BrM cells in 2D culture 24 hours after addition of small molecule stabilizer with indicated treatments. LatA: Latrunculin A (scale bar, 50μm). Data represent 2 biological replicates. (f) Immunoblots showing RFP protein accumulation in indicated cells with indicated treatments. GFP, loading control. Data represent 2 biological replicates. (g) Immunoblot analysis of phosphorylation of Hippo pathway kinases LATS1 and MST1 in cells plated on collagen for 1 hour. GAPDH loading control. Data represent 3 biological replicates. (h) Mean relative luciferase units in indicated cell lines measured by CellTiter-Glo in monolayer cultures in vitro. Error bars, S.E.M. (n = 3 independent experiments in triplicate). (i) Images of indicated H2030-BrM cells in BSCs (scale bars, 10 μm). Data represent 2 biological replicates. Dot plots: roundness index. Red lines, median (n = 45, 53 and 53 cells for Control, shYAP#1 and shYAP#2 cells pooled from 2 independent experiments). P values are calculated using Mann-Whitney test. (j) YAP immunoblots in indicated cells with or without doxycycline treatment. TetON-shYAP: Doxycycline inducible YAP shRNA construct. Data represent 2 biological replicates. See Supplementary Figure 8 for unprocessed blots. See Supplementary Table 1 for statistics source data.