Fig 2: Inhibiting CPT1A converts excessive FFAs into lipid drops by SOAT1.A Western blot analysis of CDK4, CDK6, CyclinD1, ACADL, and ACADM in HCC cells treated with ETO at the indicated concentration. B CCK8 assay showed that etomoxir (ETO) inhibited cell viability in the HepG2 and HUH7 (means ± SEM, n = 3); **p < 0.01, ns, no significance. C The level of total free fatty acids in HCC cells treated with CPT1A inhibition (means ± SEM, n = 3). ns: no significance. D–E Fluorescence imaging of LDs stained with BODIPY 493/503 (green) treated with pharmacological (avasimibe, 48 h) or genetic (si-RNA, 72 h) inhibition of SOAT1 in HUH7. Nuclei were stained with DAPI (blue). Scale bar, 50 µm. More than 30 cells were analyzed. Data were quantified using ImageJ software; ***p < 0.001, ****p < 0.0001, ns: no significance. F Western blot analysis of SOAT1 in HepG2 and HUH7 treated with ETO at the indicated concentration for 48 h. G–I Cellular LDs level in HCC cells treated with ETO plus AVA. More than 30 cells were analyzed.; *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 3: Fatty acid metabolism-associated gene expression and lipid uptake in T cells. a The cellular mechanism underlying aCD3/F/AN-induced enhancement of lipid metabolism is illustrated. The left half of the schematic cell depicts the untreated condition, and the right half shows treatment with aCD3/F/ANs. aCD3/F/AN treatment is proposed to activate PPARa in T cells, leading to over-expression of fatty acid metabolism-associated proteins, including CD36 (fatty acid translocase), CPT1B, LCAD and MCAD. The increased fatty acid metabolism in mitochondria generates higher energy via the TCA cycle. b The corresponding levels of PPARa protein in T cells (PPARa+/CD3+), measured by flow cytometry. c The levels of CD36 protein in T cells (CD36+/CD3+), measured by flow cytometry. d Protein expression levels of CPT1B, LCAD and MCAD in T cells were measured by western blot. e, f T cells treated with different nanoparticle preparations were incubated with fluorescent lipid BODIPY C16, and the association of fluorescent lipid with T cells was determined by flow cytometry (e) and expressed as mean fluorescence intensity (f) (***P < 0.001). g mRNA expression levels of the fatty acid metabolism-associated genes, CPT1B, LCAD and MCAD, determined by RT-PCR (***P < 0.001)

Fig 4: ZLN005 inhibits lipid accumulation by improving mitochondrial homeostasis. (A) Representative images of lipid droplets detected with Bodipy in HKC cells. Bar = 10 µm. (B) Quantification of lipid droplets fluorescence intensity in each cell. Data are expressed as the mean ± SEM. (n = 17). ** p < 0.01. Level of Tfam (C), Mcad (D) and Lcad (E) mRNA were detected by quantitative RT-PCR. Data are expressed as the mean ± SEM. (n = 3). * p < 0.05, ** p < 0.01. (F) Relative mitochondrial DNA copy number (mtDNA: nDNA). Data are expressed as the mean ± SEM. (n = 5~6). * p < 0.05. (G) Representative images of mitochondrial membrane potential detected by TMRE dye; ATP level revealed by tool plasmid pCMV-AT1.03. Bar = 20 µm. (H) Quantification of TMRE fluorescence intensity in each cell. Data are expressed as the mean ± SEM. (n = 15~20). ** p < 0.01. (I) Quantification of pCMV-AT1.03 fluorescence intensity in each cell. Data are expressed as the mean ± SEM. (n = 15). * p < 0.05; ** p < 0.01. ZLN005 is abbreviated to ZLN.

Fig 5: ZLN005 improves mitochondrial homeostasis and FAO in UUO-induced renal fibrosis. (A) Immunohistochemical staining of PGC-1a, TFAM, LCAD and MCAD was performed to examine kidney mitochondrial homeostasis. Bar = 50 µm. Quantification of PGC-1a (B), TFAM (C), LCAD (D) and MCAD (E) fluorescence intensity. Data are expressed as the mean ± SEM. (n = 3). * p < 0.05, ** p < 0.01. ZLN005 is abbreviated to ZLN and fenofibrate is abbreviated to Feno.