10.1371/journal.pone.0127480.g001 Fig 1  TMEM203 expression drives calcineurin dependent transcription factor activation by elevating the basal cytosolic calcium levels in HeLa cells.
(A) Stably expressed CRTC1-GFP localization was visualized using fluorescent microscope in HeLa-CRTC1-GFP cell line transiently expressing TMEM203–FLAG for 48 hrs. CRTC1-GFP (green) nuclear translocation was induced in cells co-expressing TMEM203-Flag (red). Nuclei (blue) were visualized with Hoechst. Nuclear translocation was inhibited by treatment with 5nM Cyclosporine A or 10nM FK506 for 2 hour prior to fixing the cells. Scale bars = 15 μm. (B) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector. 48 hours later the cells were visualized using fluorescent microscope. Scale bars = 15 μm. (C) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector as indicated. 48 hours later the cells were treated with 5nM Cyclosporine A (CsA) or 10nM FK506 for 2 hours and total cell lysates were prepared. The lysates were subjected to immunoblotting with indicated antibodies. (D) TMEM203-mcherry or mcherry transfected HeLa cells were seeded onto coverslips and single cell Fura-2 fluorescence based calcium measurements were performed. The measurements showed elevated basal calcium levels in TMEM203-mcherry expressing cells. (Mean; +/- SE; n = 64 cells (mcherry); 55 cells (TMEM203-mcherry) from multiple coverslips; p value = 4.06719E-30).