CAPER deficiency decreased carbon flux into glycolysis and TCA cycles leading to defective compensatory mitochondrial respiratory responses.

<p>A. Glucose-derived carbon incorporation into Glucose, G6P/F6P and F1/2, 6-BP in cells depleted of CAPER. A graph showing the average percentage of ¹³C labeled metabolites from three biological replicates of cells transfected with siCAPER (dotted line) and ones in cells with siControl (solid line). Cells were harvested at 24 hours after transfection followed by the procedures described in Materials and methods. Error bars represent SEM. B. Graphs showing the ratio of GSH/GSSG from the cells after transfection with either siControl (black solid) or siCAPER (white) in the absence (i) or the presence (ii) of the indicated treatment. A bar represents the mean value of relative GSH/GSSG from triplicate with an error bar (SEM). Data were analyzed by t-test using GraphPad software. C. A microscopic image shows the morphology (i) and crystal violet staining indicating the effect on the proliferation (ii) of cells transfected with either siControl or siCAPER followed by treating with either DMSO or NAC treatments for 24 hours. D. (i) A microscopic image (upper panel) showing the morphology and western blot (lower panel) scoring LC3 isoforms of cells transfected with either siControl or siCAPER followed by treating with either DMSO or 10 mM F1, 6-BP treatments for 24 hours. (ii) A microscopic picture (upper panel) and its corresponding quantitative graph (lower panel) of crystal violet staining shows the effect on the proliferation of cells transfected with either siControl or siCAPER followed by treating with either DMSO or 10 mM F1, 6-BP for 24 hours. E. Decreased glucose-derived carbon incorporation into the shown metabolites in cells depleted of CAPER. A graph showing the average percentage of ¹³C labeled metabolites from three biological replicates of cells transfected with siCAPER (dotted line) and ones in cells with siControl (solid line). Cells were harvested at 24 hours after transfection followed by the procedures described in Materials and methods. Error bars represent SEM. F. (i) A heat map (right panel) showing the relative amount of the indicated enzymes analyzed by qRT-PCR by Log ₂ (FC: fold change) (left colum-24 hour samples) and by Log ₁₀(p values) (p values adjusted for multiple comparisons using Benjamini-Hochberg method) (right column). Scale of red (positive values) and green (negative values) are represented as a color code on bottom of the figure. (ii) ChIP assays showing the presence of CAPER on the c-Myc binding sites of the promoter of Hk1. G. Graphs showing amount of total NAD (NADt), NAD, NADH in the cells transfected with siControl and siCAPER followed by treating NAM (left) or glucose (right). G0 and G25 indicate that cells were cultured in media containing either 0 or 25mM glucose, respectively. Average of raw values from 3 replicates was plotted and error bars represent SEM. H. A graph showing that ATP content per cell at the indicated time after transfection with siCAPER (solid white) compared to siControl (solid black).