Fig. 7.

Growth and density of HCA₃-expressing CHO-K1 spheroids. CHO-K1 cells stably expressing HCA₃ (Eurofins Discover X) and CHO-K1 control cells were cultured in 96-well ultra-low attachment plates in absence and presence of 3HO and 3HDec (a, b) or after transfection with dyn-2 wt, K44A or R399A for the indicated time (c). Acquisition of images and analyses of the average spheroid volume was performed every 24 h using a Celigo Image Cytometer. a Presence of 3HO, but not 3HDec, caused significantly larger HCA₃-expressing CHO-K1 spheroids. Total (Hoechst 33342 staining) and dead cell (propidium iodide (PI) staining) pixel counts for spheroids in presence of 3HO and 3HDec were significantly higher than in absence of HCA₃ agonists but the ratio of dead cells to total cells did not differ. b CHO-K1 control cells spheroids were smaller in presence of 3HO or 3HDec. Total (Hoechst 33342 staining) and dead cell (propidium iodide (PI) staining) pixel counts for spheroids were significantly lower in presence of 3HDec. The ratio of dead cells to total cells did not differ. c The size of HCA₃-expressing CHO-K1 spheroids was differentially affected by the presence of dyn-2 mutants whereas no effect was observed on spheroid growth of CHO-K1 control cells. a-c Data is shown as average spheroid volume in μm³ (box and whiskers min to max of n = 4 independent experiments, each carried out in 6 replicates). ^#P ≤ 0.1; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001