Figure 5.

The biological response to Dex shifts between a narrow range of functional GR levels in 697 cells. A) LCS values at 48 hours post-Dex treatment in 697-shGR2 cells are plotted versus the Doxy concentrations (48 hour pre-treatment prior to Dex). Following the initial experiment using the full 2-fold Doxy serial dilution (open circles), a second experiment was completed with a finer Doxy titration from 7 ng/mL to 17 ng/mL (gray circles). B) LCS values at 72 hours post-Dex-treatment in C7-shGR1 cells pre-treated with 2-fold dilutions of Doxy for 48 hours. LCS values were calculated from a flow cytometric apoptosis analysis using the Vybrant^® Apoptosis Assay Kit #4. The curve fits are theoretical calculations via the following equation:

$$y=\left(\frac{\mathit{Range}}{1+\left(\frac{x}{{ED}_{50}}\right)}s\right)+\mathit{Background}$$

C) Compilation of the 697 cell data. Cell viability (open circles) and Dex-induced GR transcript (gray triangles) and protein expression (black squares) are plotted versus the pre-treatment Doxy concentration used in the experiment with 697-shGR2 cells. Expression levels of GR transcripts and protein shown are plotted as the calculated percentages of the maximum Dex-induced expression values from QRT-PCR (transcripts) and densitometery (protein) performed on the western blot shown in Figure 4. The vertical dotted arrow indicates that a GR expression level above ~20–25% is required for triggering some degree of GC-mediated apoptosis in these cells. The curve fits are theoretical calculations via the same equation used in Figure 5A. The calculated Doxy ED₅₀ values for GR transcripts, GR protein, and cell viability are 10.5 ng/mL, 11.3 ng/mL, and 15.2 ng/mL, respectively.