Fig 2: TEC-specific deletion of Neu1 alleviates folic acid-induced renal fibrosis in mice.a Scheme of the experimental approach. The mice were intraperitoneally injected with folic acid (250 mg/kg). b The gross appearance of whole kidneys from control (Ctrl) and Neu1 CKO mice 4 weeks after folic acid injection. Scale bar, 1 mm. c The ratio of left renal weight to body weight (BW). n = 6 mice per group. d, e Creatinine (d) and blood urea nitrogen (e) in serum measured by ELISA. n = 6 mice per group. f Kim1 mRNA levels. n = 4 mice per group. g Hematoxylin and eosin (HE), Masson’s trichrome staining, and immunohistochemistry staining of CD68 and p-NFκB in kidney sections from Ctrl and Neu1 CKO mice 28 days after folic acid administration. n = 3 mice per group. Scale bar, 50 μm. The red arrow indicates positive cells. h–k Quantification of tubular injury score (h), fibrosis area (i), staining of CD68 (j), and p-NFκB (k). n = 3 mice per group. l, m EMT and extracellular matrix associate gene mRNA level. n = 4 mice per group. Gene expression levels were normalized to Gapdh. All statistic data were presented as mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparisons test. All tests were two-tailed.

Fig 3: Treadmill exercise decreased microglial activation in the striatum and subregions of hippocampus of mice with acute METH withdrawal. (A) Immunofluorescence staining of striatal sections from four groups of mice. The bottom left corner of each picture is the enlarged pictures for the CD68 and Iba-1. (B) The number of Iba-1+ cells (left), the number of Iba-1+/CD68+ cells (middle), and the percentage of Iba-1+/CD68+ microglia in Iba-1+ cells (right) from four groups (n = 4–5 each group) in the mouse striatum. The data are expressed as the mean ± SEM, and p < 0.05 was considered a significant difference. (C) Immunofluorescence staining of hippocampal subregion (CA1, CA23, DG) sections from the four groups of mice. The bottom left corner of each picture is the enlarged pictures for the CD68 and Iba-1. (D–F) The number of Iba-1+ cells (left), the number of Iba-1+/CD68+ cells (middle), and the percentage of Iba-1+/CD68+ microglia in Iba-1+ cells (right) from four groups (n = 4-5 each group) in the mouse CA1 (D), CA23 (E), and DG (F) subregions. The data are expressed as the mean ± SEM, and p < 0.05 was considered a significant difference. Iba-1, ionized calcium-binding adapter molecule 1; CT, control; CR, control + running; ME, METH; MR, METH + running.

Fig 4: Immunohistochemistry analysis of CD68+ cells and correlation between CD68-stained area and survival outcome in patients with cancer. To evaluate the infiltration of macrophages into the tumor tissue, tissue specimens were immunostained for CD68 in 31 patients from the patient cohort. (A and B) Representative images of CD68 immunostaining of (A) case no. 7 and (B) case no. 3, both of which are cases of patients with NSCLC. (C and D) Representative images of (C) case no. 7 and (D) case no. 3 showing the CD68-stained areas using the hybrid cell count function. Pearson correlation coefficient was performed to analyze the correlation between the CD68-stained area, and (E) OS and (F) PFS. *P<0.05. Scale bar; 500 µm. PFS, progression-free survival; OS, overall survival.

Fig 5: NEU1 is elevated in mouse fibrotic kidneys.a NEU1 mRNA levels in kidneys of mice subjected to unilateral ureteral ligation (UUO) for 0, 3, 7, 10, and 12 days. n = 4 mice per group. Unpaired two-tailed t-test. Data were presented as mean ± SD. b Western blots of NEU1 levels in kidney from mice subjected to UUO for 0, 3, 7, 10, and 12 days or administered folic acid (FA, 250 mg/kg, i.p.) injection for 4 weeks. n = 3 mice per group. c, d Immunohistochemistry of kidney sections (c) and quantitative results (d) from mice after UUO surgery for 10 days or folic acid injection for 4 weeks. The NEU1 was determined with IOD by Image-Pro Plus 6.0. n = 3 mice per group. Scale bar, 20 μm. Data were presented as mean ± SD. Unpaired two-tailed t-test. IOD, integrated optical density. e–g Immunofluorescence images of NEU1 in kidney from mice subjected to UUO (e, f) or folic acid (g). Na+/K+-ATPase was used as tubular epithelial cell marker, CD68 was used as macrophage marker. n = 3 mice per group. Scale bar, 50 μm. h, i Neu1, Acta2, Vim, Col1a1, Col3a1, and Tgfβ mRNA level in kidney from mice after UUO surgery for 10 days (h) or folic acid injection for 4 weeks (i). n = 6 mice per group. Data were presented as mean ± SD. Unpaired two-tailed t-test. j–s Pearson’s correlation of NEU1 with Acta2, Vim, Col1a1, Col3a1, and Tgfβ mRNA. n = 12, two-tailed Pearson χ2 test.