Fig 2: Autophagy flux is inhibited in microglia and infiltrating monocytes in acute phase SCI. Young adult male C57BL/6 mice were subjected to moderate contusion injury at T10 and flow cytometry was used to examine autophagy biomarkers at 3 days post-injury. (A) Representative histograms and mean fluorescent intensity (MFI) quantification show the relative production of autophagosomes in CD45intCD11b+ microglia as measured by the Cyto-ID Autophagy Detection Kit. (B-D) Representative histograms and MFI of p62/SQSTM1 (B), LAMP1 (C), and LAMP2 (D) in CD45intCD11b+ microglia. (E) Representative histograms and MFI of lysosomal activity in microglia as measured by the LysoTracker dye. (F) Representative dot plots and quantitative data depict the composition of infiltrating CD45hiCD11b+ monocytes with Cyto-ID positive staining in the spinal cord injury site. n = 5 mice for Sham group and 9 mice for 3 d SCI group. ***p < 0.001. Two-tailed unpaired t-test.

Fig 3: The inhibition of glycolysis reduced chaperone-mediated autophagy and restored mitophagy in W.Ya TFH cells(A–F). Graphs on the left compare TN and TFH cells from untreated W.Yaa (Y) and B6 mice with paired t or Wilcoxon matched-pair signed rank tests, and t tests compare the TN between strains. Graphs on the right compare TFH cells between untreated Ctrl and 2DG-treated (n = 14–34) W.Yaa mice and untreated B6 (n = 5). (A) Lysotracker, (B) DQ-BSA, (C) Cyto-ID, (D) LAMP1, (E) LAMP2, and (F) LAMP2/LAMP1 ratio. Results are shown as MFI measured by flow cytometry.(G). Representative confocal images of CD4+CD44+ T cells from 2DG-treated and control W.Yaa mice stained with LAMP2a, LAMP1, and DAPI (left) and with LC3, LAMP1, and DAPI (right). Scale bars: 5 μm for LAMP2a images on the left and 10 μm for LC3 images on the right.(H) LAMP2a as in (A)–(E).(I) Representative western blot analysis of CD4+CD44+ T cells from 2DG-treated, control W.Yaa, and B6 mice probed for LAMP2a and HSC70 with quantification relative to β-ACTIN. Dunnett’s T3 multiple comparison tests.(J) CD4+CD44+ T cells from B6, control, and 2DG-treated W.Yaa mice stained with LAMP2a, LAMP1, and DAPI at a higher magnification. All images were acquired at the same magnification. Scale bars, 3 μm.(K) Mitophagy in TN and TFH cells from Ctrl and 2DG-treated W.Yaa mice and B6 controls. Representative FACS histograms and MFI quantitation of the mitophagy dye (n = 5–7). Dunnett’s T3 multiple comparison tests compared the groups. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.See also Figures S7-S9.