Fig 2: 16:0 shifts RA signalling and suppresses mammary tumor growth in vivoa) Concentrations of 16:0 in tumors of mice fed denoted diets. Mean±SD (n=6). **p<0.01 by unpaired t-test vs. chow control. b) NaF cells (2×106) were transplanted into the mammary fat pad of NCr athymic female mice fed denoted diets and tumor growth was monitored. Mean±SD (n=6). **p<0.01 by unpaired t-test vs. chow control. c), d) mRNAs for PPARß/d (c) or RAR target genes (d) in mice fed denoted diets. Mean±SD (n=6). *p<0.05, **p<0.01 calculated by unpaired t-test. e) Immunoblots demonstrating expression of PDPK1 and RARß in tumors of mice fed denoted diets. Blots are representative data from 3 individual mice per group. f) Immunohistochemistry analyses demonstrating expression of Ki67 and cleaved caspase 3 in tumors of mice fed denoted diets. Representative images out of three tumors per group are shown. Magnification 400x, scale bars represent 50 µm. g) A model illustrating the mechanism by which SLCFA and ULCFA regulate RA signaling. Left: SLCFA compete with RA and other PPARß/d ligands for binding FABP5, divert RA to RAR and inhibit PPARß/d activation. Right: ULCFA bind to FABP5 and divert RA to RAR. ULCFA are delivered to PPARß/d by FABP5 and activate it.

Fig 3: SLCFA inhibit and ULCFA induce carcinoma cell growtha), b) NaF cells were treated with 16:0, 18:0, 18:2, or 18:1 for 4 days. Proliferation was assessed by MTT assays. c) FABP5 mRNA in normal human mammary epithelial cells (HMEC), NaF, MDA-MB-231 and MCF-7 mammary carcinoma cells, and PC3M prostate carcinoma cells. d) Pdpk1 and Rarb mRNA in denoted cells treated with 16:0 (10 μM, 6 h.). e) Cells were treated with vehicle (−) or RA (1 μM) for 4 days and proliferation assessed by MTT assays. f) Effect of 16:0 (4 days) on proliferation of denoted cells. g) Cells were cultured in CT-FBS, treated with 16:0 in the absence or presence of RA (0.2 μM, 4 days), and proliferation assessed by MTT assays. **p<0.01, by unpaired t-test vs. cells cultured in CT-FBS replenished with RA. h) Effect of 18:2 (4 days) on proliferation of denoted cells assessed by MTT assays. i) Cells were cultured in CT-FBS, co-treated with 18:2 and RA (0.2 μM) for 4 days and proliferation assessed by MTT assays. j), k) Effect of 16:0 (j) or 18:2 (k) on proliferation of NaF cell in the presence or absence of the PPARβ/δ antagonist PT-S58 (5 μM). #p<0.01 vs. untreated control, *p<0.05 vs. PT-S58 treatment without LCFA. Data in all panels are Mean ±SD. *p<0.05, **p<0.01 vs. the respective control. p-values were calculated by unpaired t-test.

Fig 4: 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-1β, TNF-α, and β-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-1β (n=4) and TNF-α (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-1β, TNF-α, and β-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-1β and TNF-α 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-1β, interleukin-1β; TNF-α, tumour necrosis factor-α; LPS, lipopolysaccharide.

Fig 5: FABP5 co-localized with rotenone-induced a-Synuclein aggregates. (A,B) Representative images of immunofluorescent staining showing localization of aSyn (red), FABP5 (green), and DAPI (blue) in Neuro-2A cells. (A) Cells were transfected with pcDNA 3.1, FABP5, aSyn, and aS/F5 respectively, then exposed to DMSO. Scale bars: 10 µm. (B) aS/F5-transfected cells were exposed to 0.1 or 0.5 µM rotenone. Arrows indicate aSyn aggregates in cells treated with rotenone. Scale bars: 10 µm. The region of interest is indicated with a white square and magnified in insets. Scale bars in the higher magnifying images: 2 µm. (C,D) Quantitative analyses of the numbers of aSyn aggregates and aSyn/FABP5 positive aggregates. Results are presented as means ± SEM (n > 40 cells from three respective cell experiments). ** p < 0.01, vs. 0.1 µM rotenone-treated aS/F5 cells; ## p < 0.01, vs. 0.5 µM rotenone-treated aS/F5 cells.