Fig 1: Oleacein induces a cell cycle arrest in G1/S phase transition. 501Mel cells were treated with OA 20 µM for 72 h. Phosphorylation levels of Histone H3 at pSer10 (A) and Cdk2 at pTyr15 (B) were expressed as fluorescence unit normalized on the corresponding cell amount (Normalized intensity). Data are presented as means ± SD of three independent experiments, each performed in triplicate. Student-t test was performed; ***p < 0.001 compared to the corresponding control (vehicle-treated cells, Ctrl).
Fig 2: Regulation of DNA repair, cell cycle and ER transport in the A226Y mutants. a, b Volcano plots showing genes involved in cell cycle regulation and DNA damage repair. Blue dots represent significantly downregulated genes, red dots represent significantly upregulated genes (N = 3, p < 0.05). c Cell cycle analysis using propidium iodide (50 µg/ml) staining. Representative profiles of A226Y versus WT are shown (N = 6). Cells were incubated (24 h) in serum-free medium as a positive control for G1 arrest, staurosporine (STA, 200 nM, 24 h) used as G2 arrest control. d Levels of CDK2 protein phosphorylated TYR15 (CDK2 pTYR15) as determined by ELISA. CDK2 pTYR15 is elevated in G1/S phase. 1 mM hydroxyurea used as positive control for G1/S arrest (N = 5). e Heatmap of the significantly regulated genes (p < 0.05) involved in ER translocation. f Representative western blot of the ER-firefly luciferase (prolactin signal sequence used as target signal for ER). Three different bands are identified: cytosolic Fluc (uncleaved SRP), ER-localized Fluc (non-glycosylated), ER-localized Fluc (glycosylated). g Ratio of ER-localized/cytosolic localized Fluc as derived from quantification of western blot analysis (N = 3). The full list of cell cycle, DNA repair, ER transport and PQC genes were compiled from available literature and can be found in Supplementary Data 1. ***p < 0.001. a, b, e For transcriptome analysis 4 independent clones of WT-transfected cells and 3 independent clones of A226Y-transfected cells were used; (c, d, f, g) N = number of independent clones analyzed for each genotype; for each clone 3 technical replicates were done. Mean ± SEM is given
Fig 3: Pelorol (PEL) 5–epi–ilimaquinone (EPI) induced a cell cycle arrest in the G1 phase. Phosphorylation levels of Histone H3 at pSer10 and Cdk2 at pTyr15 were expressed as fluorescence units normalized on the corresponding cell amount (normalized intensity). Melanoma cells were treated with PEL 4 µM or EPI 5 µM for 48 h. Data are shown as means ± SD of three independent experiments, each performed in triplicate. Student t-test was performed; * p < 0.05 and ** p < 0.01 compared to the corresponding control (vehicle-treated cells, Ctrl).
Supplier Page from Abcam for Cell Cycle In-Cell ELISA Kit (Fluorescent)