Fig 2: U0126 blocks ERK activation, endomesoderm formation, and gastrulation. a–e Control embryos. f–j U0126-treated embryos. a, f Confocal z-sections using anti-phospho-ERK (cyan) to visualize activated ERK. The gray dotted lines indicate the outline of the embryo and yellow arrows the accumulation of phosphorylated ERK (pERK)-positive cells above background levels. b–e, g–j Confocal z-sections using phalloidin (green) to show f-actin filaments and propidium iodide (red) to visualize the nuclei. a, b, f, g Blastula stages [24 hours post fertilization (hpf)]. c, h Late gastrula stages (48 hpf). d, i Early planula (72 hpf). e, j Late planula (96 hpf). All images are lateral views with the animal/oral pole to the top. The insets in a and f correspond to animal pole views. Ratios in g–j indicate the number of embryos displaying the phenotype shown in the image to the total number of analyzed embryos

Fig 3: Ex vivo 2 × 2 × 2 mm3 tissue explants retain tissue morphology, tissue microenvironment, cell proliferation and cell signaling pathways. (A) Schematic diagram of the work flow. 2 × 2 × 2 mm3 tissue explants cut from the xenograft tumors of 3 HNC patients (SE-Pt#1, SE-Pt#2, and SE-Pt#3) were cultured for 24, 48, and 72 h in 400 µl DMEM media. At 0, 24, 48, and 72 h, explants were fixed, embedded and stained. (B) 2 × 2 × 2 mm3 tissue explants retain tumor morphology and tumor microenvironment. The tissue explants were stained for hematoxylin and eosin (H&E) and IHC staining was done for epithelial tumor cell marker, cytokeratin 14, fibroblast cell marker, vimentin and a-SMA (Magnification = 20×, bars = 100 µm). (C) 2 × 2 × 2 mm3 tissue explants retain cell proliferation and cell signaling pathways. The tissue explants were stained for cell proliferation marker Ki67 and cell signaling molecules p-PRAS40, p-ERK and p-S6 (For p-PRAS40, p-ERK and p-S6, magnification = 20×, bars = 100 µm and for Ki67, magnification = 40×, bars = 50 µm). (D) Graphs showing quantification of Ki67 positive cells; p-PRAS40, p-ERK and p-S6 positive areas in all three patient xenograft tumors at 0, 24, 48, and 72 h, respectively. Images were captured by pannoramic scanner and analysis was done by HistoQuant™ software.

Fig 4: Effects of NAC treatment on LARS2 inhibited ERs and AKT/ERK cell signaling pathway. (a, b) Protein levels of GRP78, CHOP in KGN and mGC cells transfected with si-LARS2 followed by NAC, as determined by western blot. (c, d) Protein levels of AKT/ERK signaling pathway in KGN and mGC cells transfected with si-LARS2 followed by NAC, as determined by western blot. The data are presented as mean ± SD from three independent experiments. *P < 0.05.

Fig 5: miR-122-5p promoted angiogenesis through enhancing fatty acid utilization in ECs. (A) Target genes of miR-122-5p were predicted by miRanda, miRDB, and TargetScan. Among the possible miR-122-5p target genes, 6 genes were negative regulators of angiogenesis; (B) Expression levels of the predicted negative regulators of angiogenesis from (A) were detected in ECs transfected with NC or the miR-122-5p mimic; (C and D) The miR-122-5p mimic (C) increased fatty acids uptake and (D) showed no significant effect on glucose uptake; (E) The miR-122-5p mimic increased the expression levels of CD36 and CPT1A in cultured ECs; (F and G) The miR-122-5p mimic increased fatty acid oxidation in ECs as detected by seahorse. Only palmitic acid (PA) was provided in the detection medium. Oligomycin, FCCP, and antimycin A act as ATP synthase inhibitor, mitochondrial uncoupler, and complex III inhibitor, respectively; (H) CD36 knockdown abolished the effects of the miR-122-5p mimic on upregulation of VEGF, p-VEGFR2/VEGFR2, p-Akt, Akt, and p-ERK/ERK contents in ECs; (I) CD36 knockdown decreased the effects of the miR-122-5p mimic on the promotion of wound closure as detected by scratch assay; (J and K) CD36 knockdown decreased the effects of the miR-122-5p mimic on (J) promoting tube-like network formation in ECs and (K) aortic ring sprouting in cultured aortic rings. Scale bars: 500 µm (I and K); 200 µm (J). n = 3 in (F) and (G) and n = 5 in other figures. Values are presented as mean ± SEM. Data are analyzed using the unpaired Student t test in (B–G) and two-way ANOVA followed by unpaired t tests in H–K. * p < 0.05; ** p < 0.01. 2-NBDG = 2-(N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino)-2-deoxyglucose; Akt = protein kinase B; ANOVA = analysis of variance; CD36 = fatty acid translocase; Cmyc = cellular myelocytomatosis oncogene; CPT1 = carnitine palmitoyl-transferase 1; E2f1 = E2F transcription factor 1; EC = endothelial cell; ERK = extracellular signal-regulated kinase; FCCP = carbonilcyanide p-triflouromethoxyphenylhydrazone; miR = miRNA; NC = negative control; OCR = oxygen consumption rate; Sgk1 = serum/glucocorticoid regulated kinase 1; si = small interference; Smad7 = SMAD family member 7; Tgfbr1 = transforming growth factor ß receptor 1; Tsp1 = thrombospondin-1; VEGF = vascular endothelial growth factor; VEGFR2 = VEGF receptor-2.