Fig. 4: PDK4 localizes at MAM junction under ER stress.

A Schematic representation of MAM (in red). B SH-SY5Y cells transfected with KDEL-GFP, treated with DMSO or TG (0.5 µM for 3 h), followed by staining with MitoTracker Deep Red FM. Cells were fixed and immuno-stained to detect PDK4. Enlarged views of the areas within the white boxes are shown (inset). Overlapped regions are marked with red arrowheads for black and white insets, and white arrowheads for colored insets. Scale bar: 5 µm. C Graph with data from (B) shows quantification of KDEL-GFP and MitoTracker Deep Red FM overlap; co-localization measured from the binarized images represented as relative fold change (overlap index) ± SEM. Data was calculated from ∼80 cells from 3 independent experiments. D Graph represents raw pixel count of overlapped KDEL-GFP and MitoTracker Deep Red FM and PDK4 signals (left). Graph (right) represents co-localization measured from the binarized images as relative fold change (overlap index) ± SEM. Data was calculated from ∼80 cells from 3 independent experiments. E U2OS cells transfected with KDEL-GFP, treated with DMSO or TG (0.5 µM for 3 h), followed by staining with MitoTracker Deep Red FM. Cells were fixed and immuno-stained to detect PDK4. Enlarged views of the areas within the white boxes are shown (inset). Overlapped regions are marked with red arrowheads for black and white insets, and white arrowheads for colored insets. Scale bar: 5 µm. F Graph with data from (E) represents KDEL-GFP and MitoTracker Deep Red FM overlap; co-localization measured from the binarized images represented as relative fold change (overlap index) ± SEM. Data was calculated from ∼20 cells from 3 independent experiments. G Graphs with data from (E) subjected to similar analyses and quantification as (D). Data was calculated from ∼20 cells from 3 independent experiments. H Different cellular fractions obtained from DMSO and TG (0.5 µM for 3 h) treated SH-SY5Y cells (as described in “Materials and methods”) were subjected to immunoblotting to check the expression of indicated proteins. MFN2, VDAC1 and PTDSS1 were used as MAM markers; whereas calnexin and vinculin were used as ER and cytosolic markers, respectively. MFN2 and VDAC1 served as mitochondrial markers, as well. WCL whole cell lysate, Cyto cytosol, Mito mitochondria. I Graphs represent the indicated protein levels (H) and represented as the mean ± SEM of 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 (estimated via unpaired two-tailed Student’s t-test).