Journal: The Journal of Clinical Investigation

Article Title: Enhanced glycolytic metabolism supports transmigration of brain-infiltrating macrophages in multiple sclerosis

doi: 10.1172/JCI124012

Figure Lengend Snippet: (A) Micrographs of MCT-4+ cells (green) with expression of EMMPRIN (red) in a perivascular cuff (scale bar: 50 μm); insets show magnified cells (original magnification, ×200). 3D reconstruction image confirmed the overlap between MCT-4 and EMMPRIN channels (scale bar: 10 μm). Image are representative of 4 mice. (B) Immunoblot (IB) with upregulation of multiple glycosylated forms of EMMPRIN in D16 EAE spinal cords as compared with naive controls. n = 3 mice per group analyzed. (C) IB confirmed the interaction between a 50-kDa glycosylated form of EMMPRIN upon co-IP with MCT-4 as well as in unbound fractions. n = 2 independent experiments; protein was pooled from duplicate treatments. (D) Relative transcript levels (RT-PCR, comparative ΔΔCt method) show siRNA-mediated knockdown of EMMPRIN in transfected BMDMs. (E) IB for MCT-4 and loading control Na-K+ ATPase in the membrane fractions of EMMPRIN-knockdown BMDMs shows reduced levels of MCT-4 in the knockdown cells. All assays were performed in triplicate or quadruplicate for 3 independent experiments unless otherwise indicated. All graphs show the mean ± SD. Means were compared using a 2-tailed Student’s t test. **P < 0.01.

Article Snippet: The membranes were then blocked with either 5% milk or Startblock T20 buffer (Thermo Fisher Scientific; for co-IP blots) for 1 hour and then incubated with the following antibodies overnight at 4 o C: rabbit anti-EMMPRIN (42–58 kDa, ab108317, Abcam, 1:1000); rat anti-EMMPRIN (50 kDa, OX114, MCA2283, 1:500, Bio-Rad Laboratories); rabbit anti–MCT-4 (SC-50329, Santa Cruz Biotechnology, 1:500); rabbit anti–HIF-1α (NB100-134, Novus Biologicals, 1:1000); anti–rabbit LDHA (NBP1-48336, Novus Biologicals, 1:1000); anti-GFAP (Z0334, Dako, 1:400); anti–Na-K + ATPase (ab76020, Abcam, 1: 10,000); and β-actin (ab20272, Abcam, 1:5000) in 1% skim milk.

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: The Journal of Clinical Investigation

Article Title: Enhanced glycolytic metabolism supports transmigration of brain-infiltrating macrophages in multiple sclerosis

doi: 10.1172/JCI124012

Figure Lengend Snippet: (A) Live/dead assay as measured by calcein-AM (live cells; gray) and PI (dead cells; red) staining in macrophages in different conditions. (B) Lactate levels in supernatants of LPS-stimulated BMDMs in the presence or absence of 200 and 400 μM CHCA. (C) ECAR measurements of LPS-stimulated cells treated with 400 μM CHCA. Results are representative of 2 independent experiments run in quadruplicate. (D and E) Graphs depict (D) glycolysis and (E) glycolytic capacity in LPS and LPS plus CHCA conditions. Values were normalized to micrograms of protein. Means compared with 2-tailed Student’s t test. (F) IB of LPS-stimulated BMDMs shows a reduction in EMMPRIN, LDHA, and HIF-1α within 12 hours of CHCA treatment. (G) IB shows different glycosylated forms of EMMPRIN when lysates from CHCA-treated inflammatory BMDMs were “pulled” with MCT-4 antibody (co-IP: MCT-4). The MCT-4 band is shown for specificity of the co-IP antibody. (H) Graph represents the relative change in protein levels of MCT-4 and EMMPRIN (>100 kDa; high glycosylated or multimeric form) in co-IP conditions upon CHCA treatment in LPS-stimulated BMDMs. Graph represents 2 experiments, with protein pooled from triplicate treatments each. (I) Representative bright-field images of BMDMs transmigrated in LPS and LPS plus CHCA conditions in a Boyden chamber setup. Scale bar: 50 μm (original magnification, ×20 for insets). (J) Graph represents the average number of cells that migrated across the chamber in different conditions. Results are representative of 2 independent experiments run in quadruplicate. Graphs show the mean ± SD. Means were compared using 1-way ANOVA with Tukey’s post hoc test unless otherwise indicated. *P < 0.05 and **P < 0.01.

Article Snippet: The membranes were then blocked with either 5% milk or Startblock T20 buffer (Thermo Fisher Scientific; for co-IP blots) for 1 hour and then incubated with the following antibodies overnight at 4 o C: rabbit anti-EMMPRIN (42–58 kDa, ab108317, Abcam, 1:1000); rat anti-EMMPRIN (50 kDa, OX114, MCA2283, 1:500, Bio-Rad Laboratories); rabbit anti–MCT-4 (SC-50329, Santa Cruz Biotechnology, 1:500); rabbit anti–HIF-1α (NB100-134, Novus Biologicals, 1:1000); anti–rabbit LDHA (NBP1-48336, Novus Biologicals, 1:1000); anti-GFAP (Z0334, Dako, 1:400); anti–Na-K + ATPase (ab76020, Abcam, 1: 10,000); and β-actin (ab20272, Abcam, 1:5000) in 1% skim milk.

Techniques: Live Dead Assay, Staining, Co-Immunoprecipitation Assay