Figure 7
At 24 h post-small interfering (si)RNA transfection, vesicular overexpressed in cancer prosurvival protein 1 (VOPP)1 knockdown induces oxidative stress, preceding the activation of apoptosis. (a) A representative set of images from VOPP1 knockdown in squamous cell carcinoma (SCC)-9, 24 h after transfection, analyzed by the reactive oxygen species (ROS) sensor 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA). Green color indicates the intensity of the ROS sensor's signal, whereas blue color from 4′,6-diamidino-2-phenylindole (DAPI) serves as a reference to identify cells. All images were processed and scaled identically. At 24 h post-siRNA transfection, an increase in the percentage of cells with augmented ROS signal levels is directly evident. This coincides with a time point from previous experiments, wherein VOPP1 protein expression was reduced, but caspase activation and cell death had not yet occurred. (b) The percent CM-H2DCF-DA-positive cells per field in five independent experiments are presented along with a positive control, 100 μM H2O2 for 20 min is shown. Error bars represent the 95% confidence interval. Both VOPP1 targeting siRNAs induced a significant increase (P<10−10, Student's t-test) in percent ROS-positive cells. (c) Analysis of mitochondrial integrity 24 h after VOPP1 knockdown using MitoTracker Red CMXRos®. In the top portion, a representative set of images is shown, with all images scaled identically as indicated on the color bar. A decrease in signal intensity is visually evident from the data. In the bottom portion, the synthesis of two independent experiments showing the distributions of per cell signal intensities of the MitoTracker Red CMXRos® probe extracted using the CellProfiler analyses suite; n represents the total number of cells measured in each condition. The horizontal line represents the median per cell intensity of the control condition and the boxed percentage indicates what proportion of each condition was less than this value. The Kolmogorov–Smirnov tests (KS test) indicate a significant (P<10−10, marked by asterisks) downward shift in the distribution of signal with VOPP1 knockdown. (d) The same data from (c) is represented in standard bar graph format with 95% confidence intervals. Again, a significantly decreased signal is observed with VOPP1 knockdown (P<10−10 marked by asterisks, Student's t-test). This analysis indicates loss of mitochondrial integrity at a time point coincident with VOPP1 protein expression loss, but before caspase activation and cell death occur.
