Extended Data Fig. 3: NIK deficiency has no effect on mRNA expression of glycolysis-regulatory genes and glucose uptake but promotes lysosomal localization of HK2.
From: NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity
a, Seahorse analysis of OCR under baseline (no treatment) and maximum or stressed (injection of FCCP) conditions in untreated Map3k14tKO (tKO) or wildtype (WT) naïve CD8 T cells. Data are shown as a representative plot (upper) and summary graphs (lower, each circle represents a well). b, Volcano plot of RNA sequencing analysis of differentially expressed genes in Map3k14tKO OT-I CD8 T cells relative to WT OT-I CD8 T cells, activated with anti-CD3 plus anti-CD28 for 24 hr. c, Immunoblot analysis of the indicated proteins in WT or Map3k14tKO OT-I CD8 T cells stimulated with anti-CD3 plus anti-CD28 for the indicated time periods. d,e, Flow cytometric analysis of glucose uptake using in vitro activated (d) or B16F10 tumor-infiltrating (e) CD8 T cells. f, Flow cytometry analysis of HK2 expression in tumor-infiltrating CD8 T cells of B16F10-bearing wildtype (WT) or Hk2tKO mice, showing the specificity of the HK2 staining. g, Immunoblot analysis of HK2 and the indicated control proteins in the cytoplasmic (Cy) and lysosomal (Ly) fractions of wildtype (WT) and Map3k14tKO CD8 T cells. h, Immunoblot analysis of the indicated proteins in whole cell lysates of wildtype or Map3k14tKO CD8 T cells treated for the indicated time points in the presence (+) or absence (–) of lysosomal inhibitors, E64D plus pepstatin A. i, Summary of h based on densitometric quantification of three independent experiments. Data are representative of two (b,f) or three(a,c-e,g-h) independent experiments. Summary data are shown as mean ± s.e.m. with P values determined by two-tailed Student’s t test.
