Figure 5.

Functional validation of 206/E7 or 218/E7 as synergistic tumor-suppressing treatments

(A and B) Cell death analysis of NT-, 206/E7-, 206/E7-6Ø-, 218/E7-, or 218/E7-6Ø-transfected HeLa cells (images, upper panel), estimated by flow cytometry with PI and annexin V staining (A, lower panel); quantitation results are as calculated in Figure 1F (B). (C and D) Wound-healing assays for 206/E7 (C) and 218/E7 (D) in HeLa cells, compared with the presence of 6Ø modification. Images are shown in the left panels; quantitation results (right panels) are as measured in Figure 2D. More images used for the analyses are shown in Figures S6E and S6F. (E) Invasion assays of 206/E7- and 218/E7-transfected HeLa cells with Matrigel-based transwells. H&E-stained Matrigel insert (upper left panel), zoomed-in images (lower panel), and quantitation results (upper right panel) are shown. (F) Measurement of HeLa-luc cell size in xenograft mouse model after the serial injection of 206/E7 (as complexed with PEI; indicated as arrows), estimated based on bioluminescence images (photon flux; upper inset). Arrows denote time points of the injection: serial RNA injection started 5 days after implantation (1 × 10⁶ HeLa-Luc cells). (G) Tumor volume measured by caliper 16 days after xenograft in (F). Arrows denote time points of additional injections; red line indicates 206/E7; black line indicates NT. (H–J) The same xenograft mouse model experiments with HeLa-luc cells except for the implantation of 5-fold more cells (i.e., 5 × 10⁶ HeLa-luc cells) to compare the effects of 206/E7 and E6 siRNA, of which injection was started at an earlier time (2 days after implantation) and quantitated (H) based on bioluminescence images (I, after 10 days); tumor volume was measured after 12 days using calipers (J). Of note, there was no significant difference in tumor size between 206/E7 and the control NT treatment early on (F; up to day 8), which was likely due to the low number of HeLa cells used (1 × 10⁶) relative to the other experiments (H–J), which used 5-fold as many HeLa cells (5 × 10⁶). Difference in tumor size between E6 and 206/E7 treatments became unnoticeable 4 days (day 12) after the serial siRNA injections (days 2, 4, 6, 7, and 8). Subsequent 206/E7 treatment (day 13) again showed superior tumor-suppressive activity over E6 siRNA (J; day 22). All p values were from two-sided t tests, relative to NT; ∗p < 0.05; n ≥ 3, repeated with biologically independent samples. Graphs show mean and error bars show SD unless otherwise indicated.