Fig. 4

Impact of DCF on TNFα and TNFR1 interaction. a Concentration time courses of pIKK, non-phosphorylated NFκB–IκBα complex in the cytoplasm and free, non-phosphorylated NFκB in the nucleus were simulated for TNFα concentrations between 0.1 and 1000 ng/ml. In the experiment 10 ng/ml were applied. The first maximum of pIKK and the first two maxima of nuclear NFĸB are indicated by dots. The different TNFα levels have a large effect on the peak height and position of pIKK relative to NFκB–IκBα complex and nuclear NFκB that remain almost unchanged for higher TNFα concentrations and exhibit a minorly reduced and delayed response for lower TNFα concentrations. b–e dSTORM imaging of TNFR1 in the plasma membrane of HepG2 cells. b Respresentative dSTORM image of TNFR1 labeled via indirect immunocytochemistry on the plasma membrane of HepG2 cells (inset: brightfield image) (scale bar 5 µm). c Magnification of the inset indicated in b (scale bar 500 nm). d Number of TNFR1 clusters on the cell membrane at the indicated time points before and after induction with TNFα (gray: non-stimulated cells, black: TNFα-stimulated cells, blue: negative control with secondary antibody only). e Number of TNFR1 clusters for cells pre-treated with DCF (light red) followed by induction with TNFα (red) (number of cells measured under each condition n ≥ 13). Stimulation with TNFα shows no significant change to the according unstimulated population. Pre-treatment with DCF shows no significant changes in regard to untreated cells. Box plots in d and e indicate the median (line in box), lower and upper quartile (box), the mean value (square), and the data range (asterisks). Whiskers represent 1.5× the interquartile distance. Statistical analysis was performed using the Kolmogorov–Smirnov test with significance level α = 0.05