Fig 1: In vitro NIR-II photothermal ferrotherapy.a Confocal laser scanning microscopy (CLSM) images of different types of cancer cells and normal cells after incubation with HSN ([pTBCB] = 50 µg mL-1) or PBS for 24 h. ROS was indicated by green fluorescence from DCF-DA staining. Nuclei were stained with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) and indicated by blue fluorescence. 4T1: murine mammary carcinoma cell line; MCF-7: human breast adenocarcinoma cell line; HepG2: human hepatocellular carcinoma cell line; NIH/3T3: murine fibroblast cell line; NDF: normal human dermal fibroblast cell line. b Western immunoblot analysis of expression levels of ferroptosis-related proteins (ACSL4, FPN-1, GPX4) in a panel of cancer cell lines. MDA-MB-231 (231): human breast adenocarcinoma cell line; PC12: rat pheochromocytoma cell line; HeLa: human cervical adenocarcinoma cell line; SKOV3: human ovarian adenocarcinoma cell line. Source data were provided in Source Data File. c CLSM images of 4T1 cells after incubation with PBS, HSN, HSN with apoptosis inhibitor DEVD (100 µM), HSN with ferroptosis inhibitor deferoxamine (DFO) (100 µM) for 24 h, respectively. [pTBCB] = 50 µg mL-1. Expression of Cas-3 was indicated by immunofluorescence staining (green fluorescence), and lipid peroxidation was stained with a red-fluorescent probe BODIPY 665/676. Cell viabilities (d) and relative GSH levels (e) of 4T1 cells after incubation with HSN0 or HSN at various concentrations for 24 h with or without 1064 nm photoirradiation (1 W cm-2, 6 min). [pTBCB] = 50 µg mL-1. f Western immunoblots analysis of expression levels of ferroptosis and apoptosis related proteins in 4T1 cells in d and e. Plus and minus symbol indicated with and without photoirradiation, respectively. Source data were provided in Source Data File. g Proposed molecular mechanisms of HSN-mediated NIR-II photothermal ferrotherapy. GSSG glutathione disulfide, AA arachidonic acid, AA-CoA arachidonyl-CoA, LH phospholipid. Error bars indicated standard deviations of three independent measurements.
Fig 2: Transcriptome analysis of siRNA mediated ACSL4 knockdown in the pig intramuscular preadipocytes. (A) Realtime PCR showed ACSL4 gene was dramatically downregulated after siRNA knockdown. (B) Western blot indicated ACSL4 protein was dramatically reduced after siRNA knockdown compared to control. (C) Differentially expressed genes after ACSL4 knockdown as shown by the volcano plot. (D) KEGG pathway analysis of the differentially expressed genes after ACSL4 knockdown. Data are presented as the mean ± SEM. Comparisons were performed by unpaired two-tailed Student’s t-tests. *** indicated p < 0.001.
Fig 3: In vivo NIR-II PA imaging-guided photothermal ferrotherapy.a Time-course NIR-II PA images of tumor region on living mice bearing 4T1-xenograft tumor after intravenous administration of HSN or HSN0 ([pTBCB] = 250 µg mL-1, 200 µL per mouse, n = 3). Wavelength: 1064 nm. b Quantification of PA amplitudes in a (n = 3). c Biodistribution study of mice in a at 24 h post-administration of HSN or HSN0 (n = 3). d Surface tumor temperature of 4T1 tumor-bearing mice upon 1064 nm photoirradiation (1 W cm-2, 6 min) at 4 h after intravenous administration of HSN or HSN0 ([pTBCB] = 250 µg mL-1, 200 µL per mouse, n = 3). e Tumor growth curves of mice after photothermal ferrotherapy and monotherapies (n = 3). P-values were calculated by Student’s two-sided t-test. **P < 0.01, ***P < 0.001 (n = 3). f Scheme of photothermal depths of tumor. g H&E staining and immunofluorescent staining (Cas-3, LPO, and ACSL4) images of tumor sections at different photothermal depths after monotherapies or photothermal ferrotherapy. Cas-3, LPO, and ACSL4 staining was indicated with green, red, and yellow false colors, respectively. h–k Quantification of cell death percentage (h), Cas-3 expression (i), LPO extent (j), and ACSL4 expression (k) of tumor sections at different photothermal depths after monotherapies or photothermal ferrotherapy. Upwards or downwards arrows indicated the increased or decreased percentage at 9 mm relative to 2 mm. Error bars indicated standard deviations of three independent measurements.
Fig 4: The pig ACSL4 expression pattern in different tissues and during adipogenic differentiation. (A) The ACSL4 gene expression in different pig tissues including heart, liver, spleen, lung, kidney, fat, and skeletal muscle. (B) The mRNA expression levels of pig ACSL4 during adipogenic differentiation of intramuscular preadipocytes. (C) The protein levels of pig ACSL4 during adipogenic differentiation of intramuscular preadipocytes as shown by Western blot. Data are expressed as means + SEM.
Fig 5: Overexpression of pig ACSL4 gene increased adipogenesis and lipid deposition in pig intramuscular preadipocytes. (A) Western blot showed that ACSL4 protein increased after adenovirus mediated overexpression. (B) Oil Red O staining showed that ACSL4 overexpression stimulated lipid deposition. (C) Realtime PCR showed that adipogenic genes FASN, ACACB, C/EBPa increased significantly after ACSL4 overexpression. Bars are presented as the mean ± SEM. Comparisons were performed by unpaired two-tailed Student’s t-tests. * indicated p < 0.05 and ** indicated p < 0.01.
Supplier Page from Abcam for Anti-FACL4 antibody [EPR17587-42]