Fig. 7

Influence on Cytochrome C and ER stress marker and flow cytometric quantification of apoptosis/necrosis. A–C To investigate the effect of the test substances on cytochrome C distribution and specific ER markers, cells were treated for 2 × 72 h and stained with Alexa Flour® 546 anti-Cytochrome C Ab, Alexa Flour® 594 anti-Calnexin Ab [red], and Alexa Flour® 647 anti-ATF4 Ab (scale bar: 50 μm). Nuclei were stained with DAPI. A, B Representative single channel (A) and merged (B) images are shown, n = 3 independent experiments. Images were taken on a Zeiss Axio Observer 7 microscope. C Quantification of fluorescence intensity using ImageJ software. n = 3 independent experiments. Given is the percentage increase in fluorescence intensity in relation to controls; *p < 0.05, **p < 0.01 vs. control, #p < 0.05 vs. monotherapy; ##p < 0.01 vs. monotherapy, One-way ANOVA. (D) Flow cytometry for detecting early (Yo-Pro-1 +) and late apoptotic (Yo-Pro-1 + /PI +) or necrotic (PI +) cells. Results show data from at least 3 independent experiments. Mean + SD. ****p < 0.0001 vs. control, #p < 0.05 vs. monotherapy; ###p < 0.001 vs. monotherapy, One-way ANOVA (Tukey’s multiple comparison test)