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. 2016 Mar 15;7(14):17290–17300. doi: 10.18632/oncotarget.8039

Figure 6. [Ruxolitinib + MMF] exposure increases ROS levels and causes release of AIF into the cytosol and AIF localization in the nucleus.

Figure 6

A. SUM149 cells were treated with vehicle control or with, ruxolitinib (1.0 μM), MMF (5.0 μM)] or in combination for 6h and 12h. Fifteen minutes before each time point cells are incubated with diacetate ester dichlorofluorescin (DCFH), 5 μM. The conversion of DCFH to DCF by reactive oxygen and nitrogen species is measured in sextuplicate in a Vector 3 plate reader (n = 3 +/− SEM).B. SUM149 cells were transfected with empty vector control (CMV) or with plasmids to express super-oxide dismutase 2 (SOD2) or thioredoxin (TRX). Twenty four h after transfection cells were treated with vehicle control or with, ruxolitinib (1.0 μM), MMF (5.0 μM)] or in combination for 36h. Cell viability was assessed using a live/dead assay in a Hermes WiScan microscope at 10X magnification (n = 3 +/− SEM). C. Upper: GBM12 cells were treated with vehicle control or with [ruxolitinib (1.0 μM) + MMF (5.0 μM)] for 6h. After 6h cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed to detect the expression of AIF (red fluorescent stain); eIF3A (green fluorescent stain) with images at 60X magnification. Lower: GBM12 cells were treated with vehicle control or with [ruxolitinib (1.0 μM) + MMF (5.0 μM)] for 6h. After 6h cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed to detect the expression of AIF (red fluorescent stain) and HSP70 (green fluorescent stain). The co-localization of AIF with HSP70 was determined by merging the images in Adobe Photoshop CS6 at 9999 dpi.