Supplementary Materials Supporting Information supp_293_17_6544__index. in baseline cellular metabolism between control and KLF4-expressing cells. However, when mitochondrial function was impaired, KLF4 significantly increased spare respiratory capacity and levels of reactive oxygen species in the cells. To identify the biological effects of these changes, we analyzed proliferation and survival of control and KLF4-expressing cells under stress conditions, including serum and nutrition deprivation. We found that following serum starvation, KLF4 altered cell cycle progression by arresting the cells at the G2/M phase and that KLF4 protected cells from nutrition deprivationCinduced death. Finally, we demonstrated that methylation-dependent KLF4-binding activity mediates mitochondrial fusion. Specifically, the downstream targets of KLF4-mCpG binding, guanine nucleotide exchange factors, serve as the effector of KLF4-induced mitochondrial fusion, cell cycle arrest, and cell protection. Our experimental system provides a robust model for studying the interactions between mitochondrial morphology and function, mitochondrial dynamics and metabolism, and mitochondrial fusion and cell death during tumor initiation and progression. (11) order SB 525334 have revealed that mice lacking KLF4 developed profound heart failure in response to stress. In our previous studies, we found that KLF4 binds to methylated DNA in in Fig. 1and Fig. S2) and confirmed that the observed mitochondrial fusion order SB 525334 in KLF4-expressing cells was not due to cell morphology change, a flat or more spreading cell phenotype. Open in a separate window Figure 1. KLF4 promotes mitochondrial fusion in GBM cells. ATP synthase staining of control (showed fragmented mitochondrial staining; and indicated fused mitochondria. normal fluorescence microphotograph. and superimposed images of mitochondrial staining from 40 adjacent confocal microphotographs. 20, 30, and 20 m in the slightly overexposed photograph of ATP synthase staining showed the spread out the cytoplasma into control cells (20 m. percentage of cells with fused and fragmented mitochondrial staining in control and KLF4-expressing cells. analysis of average mitochondrial length by ImageJ. and analysis of the average numbers of branches (immunoblotting analysis of the expression level of mitochondrion-specific proteins Tom 20 and ATP synthase following KLF4 expression (***, 0.001). We quantified the percentage of cells with fused mitochondria (network-like) and fragmented mitochondria (dotted) based on the ATP synthase staining. Mitochondria in 90% of the KLF4-expressing cells were fused together. In contrast, 90% of the control cells showed punctate staining around the nucleus (Fig. 1 0.001). To further analyze the mitochondrial network, we compared the number of branches and junctions order SB 525334 of the network using ImageJ. KLF4-expressing cells showed an extensively branched mitochondrial network (Fig. 1, and mitochondrial fusion induced by KLF4 as shown by staining with additional mitochondrial markers, including MitoTracker Red ((time course of mitochondrial fusion induced by KLF4. As early as 16 h following KLF4 expression, mitochondrial fusion has formed in U87 cells. Sixteen hours after Dox withdrawal, no mitochondrial network has been observed. parent U87 cells were treated with doxycycline for 48 h, and there order SB 525334 was no mitochondrial fusion formed in U87 cells. 20 m. control. To determine the time course of KLF4-induced mitochondrial fusion, we treated Itgb1 U87 cells with Dox at different time points and found that as early as 16 h following Dox treatment, there was a distinct mitochondrial fusion (Fig. 2glucose uptake using 2-[3H]deoxyglucose showed no difference between control and KLF4 expression cells. lactate assay showed that KLF4 did not stimulate glycolysis in U87 cells. G6PD assay showed no difference in the activity of the G6PD enzyme between control (glucose oxidation analysis using d-[U-14C]glucose indicated that there was a slight but order SB 525334 not significant decrease in oxidative glucose phosphorylation in KLF4-expressing cells. ATP assays indicated no significant increase in ATP level in KLF4-expressing U87 cells. and Fig. S4). However, KLF4 dramatically increased the spare (or reserve) respiratory capacity of U87 cells (Fig. 4 0.001), consistent with our Seahorse XF analysis. Open in a separate window Figure 4. KLF4 increases the spare respiratory capacity and ROS of GBM cells. representative seahorse experiments of control (KLF4 did not change the basal OCR of U87 cells. KLF4 significantly increased the spare respiratory capacity of U87 cells. compared with control, ATP production was not significantly increased in KLF4-expressing cells. KLF4 significantly increased the nonmitochondrial oxygen consumption in U87 cells. flow cytometry using the dye H2DCFDA to detect ROS. H2O2-treated cells were used as a positive control. Nonstained cells were negative control (compared with control, the H2DCFDA intensity was three times higher.