Fig. 3.

AKT-mTOR–HIF–1α axis in regulating glycolysis and tolerogenic cytokine production in NCoR1 depleted DCs

A. Representative western blots showing the phosphorylated and total protein levels of AKT, mTOR and HIF-1α in control and NCoR1 depleted DCs after 6h CpG stimulation. Ratios of phosphorylated to total protein levels were further normalized to the housekeeping control β-actin. Corresponding bar plots depicted the densitometry analysis of the western blots. (n = 3–4)

B. Bar graph with dots showing the relative mRNA expression of Hif-1α in 6h CpG activated control and NCoR1 depleted tolerogenic DCs. (n = 7)

C. Heatmap showing the differentially expressed genes of HIF-1α pathway observed in the RNAseq data of control and NCoR1 KD 6h CpG stimulated DCs. (n = 3)

D. IGV snapshot depicting the NCoR1 binding at the distal genomic regions of Hif-1α gene loci in unstimulated and 6h CpG activated control DCs.

E. Line graph and box and whisker plots with dots depicting the levels of ECAR along with glycolysis and glycolytic capacity respectively upon pre-injection with either rapamycin (20nM) or KC7F2 (10μM) in control and NCoR1 KD DCs after 6h CpG activation, as measured using Seahorse extracellular flux analyzer. (n = 6)

F. Scatter plots (percent positive cells) and bar graphs (MFI) showing the cytokine levels of IL-10 and IL-27 upon treatment with rapamycin (20nM) and KC7F2 (10μM) in CpG activated control and NCoR1 depleted tolerogenic DCs. (n = 7–8)

*p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001 and ****p ≤0.0001. p-value has been calculated using two tailed paired student’s t-test. Error bars represent SEM.