Fig 7. Quantitation of cell viability and demise upon Yan 7874 exposure in CHO cells.

(A) Concentration-response relationship of Yan 7874. (B) The impact of the inhibition of caspases and protein synthesis. Ctrl CHO cells (not expressing orexin receptors) were cultured on 96-well plates for 24 after which they were treated with the compounds as indicated. After another 24 h, the cells were stained with Hoechst for total cell number, FDA for living cells, and PI for necrotic cells. (A) The values for total and living cells were normalized to the control, where essentially all cells are alive (see Fig 6) and thus the specific fluorescence is maximal. PI-stained cells could not be normalized in this way, since the specific PI fluorescence in control cells was essentially 0, and thus small deviations in the background subtraction would have introduced much noise. Therefore, the values were normalized to the PI fluorescence (or PI/Hoechst fluorescence ratio) in cells treated with 30 μM Yan 7874. "FDA/Hoechst" stands for the FDA reading divided by the Hoechst reading, indicating the proportion of living cells of all cells. "PI/Hoechst" similarly gives the proportion of necrotic/secondary necrotic cells among all cells. N = 6. (B) The effect of the pan-caspase inhibitor Q-VD-Oph and the protein synthesis inhibitor CHX on the response to Yan 7874 (10 μM). For the sake of clarity, only the PI/Hoechst fluorescence ratios, normalized to the control response to 10 μM Yan 7874 (in the absence of Q-VD-Oph or CHX), are shown. The ratios were always calculated separately for each independent sample before averaging. N = 8.