Fig. 8

Spheroid density of the HCA₃ expressing esophageal squamous cell carcinoma cell line Colo680N affected by HCA₃ agonists and dyn mutants. Colo680N cells express HCA₃. a cAMP inhibitory response induced by the HCA₃ agonists 3HO and 3HDec was detected and sensitive to PTX. b Only 25 μM 3HO but not 100 μM 3HDec induced an ERK activation in Colo680N cells. c, d Colo680N cells were cultured in 96-well ultra-low attachment plates (Nexcelom) in absence and presence of 3HO and 3HDec (c) or after transfection with dyn-2 wt, K44A or R399A (d) for the indicated time. Acquisition of images and analyses of the average spheroid volume was performed every 24 h using a Celigo Image Cytometer. c Presence of 3HO, but not 3HDec, caused significantly larger Colo680N spheroids. Hoechst 33342 (total number of cells) and propidium iodide (PI, dead cells) staining revealed higher pixel counts for spheroids in presence of 3HO but the ratio of dead cells to total cells did not differ. d The size of Colo680N spheroids was affected by the presence of dyn-2 mutants K44A and R399A. c, d Data is shown as average spheroid volume in μm³ (box and whiskers min to max of n = 3 independent experiments, each carried out in 8 replicates). ^#P ≤ 0.1; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001