Figure 1.

CPT, DOX, and ETO increase AAV transduction in HeLa cells

HeLa cells were treated overnight with either medium only (no drug control, green circles), 62.5 nM of CPT (blue squares), 50 nM DOX (maroon upward triangles), or 3.13 μM of ETO (pink downward triangles) before drug washout. Treated cells were transduced with AAV-GFP and subsequently cultured in the absence of topoisomerase poisons. GFP fluorescence was measured to track AAV gene expression. (A) Over the first 96 h post transduction, phase and green fluorescence live cell images were taken every 12 h using the Incucyte S3 and 25 images were taken per well. The Incucyte analysis software was used to quantify GFP-positive foci, with the reported count taking an average across the 25 images. Data displayed are the mean value and standard deviation of quadruplicate biological replicates (n = 4) per group. Highlighted time points (24, 36, 48, 60, 72, and 84 h) were analyzed using one-way ANOVA with Dunnett’s multiple comparison test of each drug against the control cells. (B and C) As cells were further passaged, harvested HeLa cells were stained with LIVE/DEAD dye, fixed and run on a flow cytometer to determine the percentage of cell population expressing GFP expression. Cells were analyzed at (B) 6 days post transduction and (C) 19 days post transduction. Data displayed are the percent GFP-positive values for each biological replicate with the mean displayed as a horizontal bar. (B) n = 4 and (C) n = 8. Data were analyzed using one-way ANOVA with Dunnett’s multiple comparison test of each drug against the control cells. In all graphs, significance is displayed as such (ns, no significance; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).