Figure 1.

Genome-wide RNAi screen implicates Krtap5-5 in cancer cell interaction with an endothelial monolayer. (a) Schematic of RNAi screen. E0771 transduced with a genome-wide RNAi library were subjected to Human Umbilical Vein Endothelial Cell (HUVEC) monolayer attachment. Unattached cells were collected and expanded over three rounds. After the 4^th round, clonal cells were generated by limiting dilution and tested functionally, and the inserted shRNA was identified by sequencing. (b) Electric Cell-substrate Impedance Sensing (ECIS) monitors real-time disruption of an endothelial monolayer by invading cancer cells¹⁰. (c) Clonal E0771 lines derived from the RNAi library screen (L1A, L1B) show reduced endothelial monolayer disruption relative to control cells with a non-silencing shRNA. The plot depicts real-time disruption of endothelial monolayer after addition of cancer cells (data point intervals, 15 min). *** p<0.001, vs control; mean ± SD. (d) Expression of Krtap5-5 mRNA in E0771 cells relative to actin. Mean ± SEM. (e) Krtap5-5 mRNA in RNAi library screen-derived L1A and L1B cells. ** p<0.01, *** p<0.001, vs controls; mean ± SEM. (f) shRNA targeting sites relative to the Krtap5-5 mRNA. P2, P3 shRNAs were used to generate de novo pooled knockdown cell lines. ORF = open reading frame. (g) Krtap5-5 mRNA in P2 and P3 cells. ** p<0.01, *** p<0.001, vs controls; mean ± SEM. (h) P2 and P3 cell disruption of an endothelial monolayer as in panel c. *** p<0.001, mean ± SD. (i) Krtap5-5 amino acid sequence highlighting cysteine residues.