Fig. 6: Increased PDK4 regulating mitochondrial functionality.

A Graph showing fluorescence intensity of ER-CEpiA obtained from control (EmpVec) and PDK4-transfected cells after histamine (100 μM) treatment. B Graph showing fluorescence intensity of mito-CEpiA obtained from control (EmpVec) and PDK4-transfected cells after histamine (100 μM) treatment. Intensity of ER-CEpiA or mito-CEpiA was normalized at each time point with either ER-GFP or mitoRFP, respectively. ΔF = Intensity at time ‘t’ – Intensity at time ‘0’. F0 = Intensity at time ‘0’, F = Intensity at time ‘t’. ΔF/F0 was plotted against time from mean ± SEM of 3 independent experiments. C Graph represents total ATP production measured by luminescence-based study in SH-SY5Y cells, transfected with indicated constructs under normal (5.5 mM) or high glucose (25 mM) conditions, in RLu (relative luminescence) units. Data represent mean ± SEM of 3 independent experiments. D Plot represents the percentage of ATP generated from glucose dependence, FAO (fatty acid oxidation) and AAO (amino acid oxidation) capacity, mitochondrial dependence and glycolytic capacity of cells transfected with indicated constructs. Data is shown as mean ± SEM of 3 independent experiments. E Cells transfected with indicated constructs were immunoblotted to detect expression of indicated proteins. ► Exogenous (PDK4-RFP), ➤ endogenous. F Graphs represent change in expression of different proteins, as shown in (E). Data represent the mean ± SEM of 3 independent experiments. G Representative flow cytometry histogram plots the intensity of TMRM (250 nM) signals in cells either transfected with the indicated constructs or treated (DMSO or CCCP). H Graph shows quantification of TMRM mean fluorescence intensity measured by FACS, as shown in (G). Data represent mean ± SEM of 3 technical replicates. I Representative flow cytometry histogram plots intensity of MitoTracker Green FM (250 nM) signals in cells either transfected with the indicated constructs or treated (DMSO or CCCP). J Graph shows quantification of MitoTracker Green mean fluorescence intensity with data from (I). Data represent mean ± SEM of 3 technical replicates. K Histogram of TMRM/MitoTracker Green signal ratios with data from (H, J). Data represent mean ± SEM of 3 technical replicates. L Representative flow cytometry histogram plot shows the intensity of DCFDA (1 μM) signals in cells either transfected with the indicated constructs or treated (DMSO or CCCP). M Graph plots data of DCFDA mean fluorescence intensity from (L). Data represent mean ± SEM of 5 technical replicates. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns not significant (estimated via unpaired two-tailed Student’s t-test).