FIGURE 6.
TMEM16 proteins are essential for RVD in HEK293 cells. A, swelling (Hypo)-induced changes in calcein fluorescence in control HEK293 cells and cells treated with siRNA for TMEM16A or suramin (100 μm) or DIDS (100 μm); mean curve ± S.E. (n = 10). B, summary of the rate of fluorescence recovery per second, indicating recovery of the cell volume from hypotonic swelling. Treatment with siRNA for TMEM16A or exposure to various inhibitors of ICl,swell inhibited recovery of the cell volume, i.e. RVD. C, hypotonic cell swelling and recovery from cell swelling of cells treated with (i) scrambled RNA, (ii) TMEM16A-RNAi, or (iii) coexpressing P2Y2 receptors/TMEM16A, as determined by FACS analysis of 30,000 cells per experiment. Experiments were performed in triplicate. D, summary of volume, i.e. fluorescence recovery after hypotonic cell swelling of HEK293 cells treated with siRNA for different TMEM16 proteins, as determined by FACS analysis of 30,000 cells per experiment. E, apoptotic cell shrinkage induced by staurosporin (2 μm) under normotonic conditions in cells treated with two different batches of siRNA for TMEM16A (si16A-a and si16A-b), or when treated with scrambled RNA (FACS analysis of 30,000 cells). Mean ± S.E., (n) = number of cells measured. *, significant recovery from cell swelling of cells treated with scrambled siRNA (paired t test). #, significant difference when compared with control (unpaired t test).