Fig. 5

Tacle extract recapitulates Hes and Nar effects in vitro. A Real time OCR measurement in closed chambers performed in H929 cells, 48 h after 2 mg/mL Tacle treatment. Histogram bars report multiple key parameters, including basal respiration, spare capacity, maximal respiration, leak state and ATP production, calculated after consecutive injections of Oligomycin (2 µM), FCCP (0.5 µM) and Antimycin A (2 µM). B Cell viability assessed by CTG assay, 48 h after treatment with increasing doses of Tacle extract or vehicle. Live cells are represented as percentage of vehicle-treated cells. *p < 0.05. C Colony formation assay performed in AMO cells exposed for 10 days to Tacle extract (2 mg/mL), or vehicle as control. Histogram bars reported the mean ± SD of colony formation units of three independent experiments. Representative images of colonies at day 10 are also shown. *p < 0.05. D Flow cytometry evaluation of Annexin V-positive AMO cells, after 48 h of Tacle extract treatment. Data are representative of at least three independent biological replicates (n = 3). *p < 0.05. E q-RT-PCR analysis of SREBF1 and c-MYC mRNA expression levels in AMO cells, 48 h after Tacle exposure. Histogram bars represent the average ± SD of mRNA expression levels after normalization with GAPDH and ΔΔCt calculation method. *p < 0.05. F WB analysis of SREBP1 and c-MYC in AMO cells, 48 h after Tacle treatment. GAPDH or α-tubulin were used as loading control. *p < 0.05. G Triglycerides levels were determined using Triglycerides Glo assay in AMO cells after 2 mg/ml Tacle treatment; luminescence was evaluated at 48 h. *p < 0.05. H Fluorescence microscopy analysis of lipid droplets in AMO cells labeled with LipidTOX Red probe, 24 h after exposure to Tacle (2 mg/ml); representative images (100 × magnification) are reported. Histogram bars represented the average ± SD of the number of lipid droplets in at least 100 cells from three different fields