Fig 2: MF 6 attenuates the activated astrocyte-related inflammatory response. (a) Confocal microscopy of immunofluorescent GFAP-stained sections (red). (b) Quantification of (a); symptomatic treatment with MF 6 (1 mg/kg) (n>6) significantly decreases the number of GFAP-positive cells in the spinal cord. (c) Schematic diagram of astrocyte primary culture from the cortex of P1-2 mouse pups. (d) Top, confocal microscopy of immunofluorescent GFAP (red) and DAPI (blue) staining of primary astrocytes; bottom, quantification of astrocyte purity (GFAP-positive cells to DAPI positive cells) (n=4). (e) Confocal microscopy of immunofluorescent GFAP (red) and FABP5 or FABP7 (green) staining of primary astrocytes. (f) Immunoblots and densitometry of astrocytes against antibody IL-1ß, TNF-a, and ß-tubulin. Primary astrocytes were treated with LPS (10 µg/mL) for 48 h to simulate inflammatory response, and MF 6 (1 µM) treatment was simultaneously performed. (g and h) Quantification of (e); MF 6 significantly decreases IL-1ß (n=4) and TNF-a (n=4) accumulation in primary astrocytes. (i) Immunoblots and densitometry of astrocytes transfected with FABP5 shRNA (left) and FABP7 shRNA (right), revealing that F5i 3 and F7i 2 are the most efficient. (j) Immunoblots and densitometry of astrocytes against antibody IL-1ß, TNF-a, and ß-tubulin; primary astrocytes were stimulated with LPS and knocked down with FABP5 and FABP7. (k and l) Quantification of (j); knockdown of FABP5 and FABP7 significantly decreases IL-1ß and TNF-a levels in primary astrocytes (n=4). Data are shown as mean ± standard error of the mean (SEM) and were obtained using a one-way ANOVA. **p<0.01, #p<0.05, and ##p<0.01. Cont., control; EAE, experimental autoimmune encephalomyelitis; GFAP, glial fibrillary acidic protein; FABP5, fatty acid protein 5; FABP7, fatty acid protein 7; IL-1ß, interleukin-1ß; TNF-a, tumour necrosis factor-a; LPS, lipopolysaccharide.

Fig 3: Expression of EN1, FABP7, MYB, and VCAN distinguishes ACCs from normal tissues and benign salivary gland tumors. (A–D) Box plot analysis of relative gene expression. Each box represents the quartile distribution range (25-75%). The median is shown by a black horizontal line. The whole range and individual cases are displayed as a black vertical line and scatter dots. The y-axis indicates fold change relative to the average of normal samples’ gene expression. Statistical comparisons between data sets were performed with two-tailed Student’s t tests. P values < .05 were considered significant.

Fig 4: FABP7 expression is poorly correlated with levels of EN1 and MYB in ACC tumors. (A–F) Scatter plot analysis for the strength of a correlation in relative gene expression between two molecules from EN1, MYB, FABP7, and VCAN. Trend lines are included. Trend line equations and R2 values were calculated separately.

Fig 5: FABP7 may predict poor prognosis in salivary ACC patients. (A–H) Kaplan-Meier curves showing overall survival rate of patients with sporadic ACC tumors with a higher or lower mRNA expression of EN1 (A and E), FABP7 (B and F), MYB (C and G), or VCAN gene (D and H). The ACC cohort was divided into two groups according to relative gene expression. Panels A to D compare patients with expression in the top half overall (red line) and the lower half (black line). Panels E to H compare expression in the higher quartile (red line) and lower three quartile (green line) subsets. Circles identify censored data. Statistical comparisons between data sets were made with log rank tests, and each P value is displayed. P < .05 is considered significant.