Fig 1: CD31 regulates EBF via its effects on EC metabolism.a EC were pre-treated with the glucose analog 2-DG (5 mM, 2 h) then MHC-1- or ICAM–stimulated for 4 h prior to TEER measurements. The bar graph shows the mean of measurement collected in three separate experiments of identical design ± SD. One-way Anova with Tuckey post-hoc test. 2DG MHC vs all ***p = 0.0003. b Glut-1 expression by EC following MHC stimulation (2 h) was evaluated by qRT-PCR. Data are mean ± SD. Two-tailed Student’s T test. MHC vs IsC *p = 0.0412. c, d Representative histograms of antibody-stimulated WT or CD31-deficient EC incubated with 6-NBDG for 2 h prior to analysis. 2-DG was used as a negative control. The graph shows the mean MFI measured in three independent experiments ± SD. One-way Anova with Tuckey post-hoc test. WT IsC vs WT MHC **p = 0.003, cd31-/- IsC vs cd31-/- MHC ns = 0.058. e ATP levels were measured in WT and CD31-deficient EC 4 h after MHC or insulin (1.8 µM) stimulation. Data are mean ± SD. Two-tailed Student’s T test. WT MHC vs cd31-/- MHC ***p = 0.0002, WT insulin vs cd31-/- insulin **p = 0.0062. Extracellular acidification rate (ECAR) of unstimulated and antibody-stimulated WT and CD31-deficient EC is shown in (f–h). The basal and maximal glycolysis and the glycolytic reserve are shown in (i–k). Wells were first injected with anti MHC antibodies at the indicated time point. Isotype-matched and secondary antibodies were used as controls. Further injections followed at the time point indicated (arrows) introducing the indicated compounds into the wells. N = 3 independent experiments. The error bars represent SD. One-way Anova with Tuckey post-hoc test. f WT time 81 vs cd31-/- time 81 **p = 0.0023, WT time 94 vs cd31-/- time 94 ***p = 0.0017; h IsC Glu/Oligo injection vs MHC Glu/Oligo **p = 0.012, IsC Oligo/2DG vs MHC Oligo/2DG *p = 0.027; i WT MHC vs WT IsC ****p < 0.0001, WT IsC vs cd31-/- IsC/MHC *p = 0.0245, WT MHC vs cd31-/- IsC/MHC ****p < 0.0001; j WT IsC vs cd31-/- IsC **p = 0.0056, WT IsC vs cd31-/- MHC **p = 0.0097, WT MHC vs cd31-/- IsC **p = 0.0024, WT MHC vs cd31-/- MHC **p = 0.0041; k WT IsC vs cd31-/- IsC ***p = 0.0002, WT IsC vs cd31-/- MHC ***p = 0.0003, WT MHC vs cd31-/- IsC ***p = 0.00024, WT MHC vs cd31-/- MHC ***p = 0.00021.
Fig 2: Changes in cellular metabolism in Ly6C and Ly6G-expressing cells. (A) Percent of splenocytes expressing Ly6C following Lm infection and (B) Percent of Ly6C+ cells expressing Glut1 over the course of the infection. (C) Percent of Ly6Cint or (D) Ly6Chi cells expressing Glut1. (E) Percent of splenocytes expressing Ly6G following Lm infection and (F) Percent of Ly6G+ cells expressing Glut1 over the course of the infection. Significance was assessed using one-way ANOVA followed by a Dunnett Test for multiple comparison. Data are represented as mean ± SD. High Dose (n = 5) - Control (n = 3). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001.
Fig 3: Markers of cellular metabolism of T cells. Percent of splenocytes expressing (A) CD3+ (T cells) and (B) percent of CD3+ cells expressing Glut1 over the course of the infection. (C) Representative flow cytometric plots of CD3 and Glut1 on T cells on uninfected controls and at day 3 and 14 post infection. Significance was assessed using one-way ANOVA followed by a Dunnett Test for multiple comparison. Data are represented as mean ± SD. High Dose (n = 5) - Control (n = 3). *p < 0.05, **p < 0.01, ***p < 0.005.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Glut1 Antibody [Alexa Fluor® 647]
Available conjugates: Janelia Fluor 549;PE/Atto594;FITC;CoraFluor 1;PE/Cy7;mFluor Violet 450 SE;Alexa Fluor 405;PE/Cy5.5;DyLight 550;DyLight 755;Alexa Fluor 532;Alexa Fluor 350;Alexa Fluor 594;Alexa Fluor 647