Fig. 8. Distribution of 3F3A-labelled SER tubules in RAS-transformed NIH-3T3 cells is regulated by [Ca²⁺]_cyt. (A) Expression of AMFR and AMF was assessed in NIH-3T3 and RAS-transformed NIH-3T3 (NIH-Ras) cells by western blot using -actin as a loading control. The bar graph shows quantitative immunofluorescence analysis of 3F3A labelling intensity from confocal images of NIH-3T3 and NIH-Ras cells acquired with an open pinhole (^*P<0.005). (B) NIH-3T3 and NIH-Ras cells were immunofluorescently labelled for 3F3A (red) and mtHSP70 (mito; green) and zooms of boxed regions of the merged images presented in the bottom row. Bars, 20 µm; 2 µm (zoom). (C) Average [Ca²⁺]_cyt using Fura-2-AM ratiometric labelling and 3F3A-labelled SER tubule dissociation from mitochondria quantified by the mask overlay approach for NIH-3T3 and NIH-Ras cells. (D) Dissociation of 3F3A-labelled SER tubules from mitochondria was quantified by mask overlay in NIH-3T3 and NIH-Ras cells either left untreated (Control) or treated with 1 µM ionomycin in the presence of 200 µM EGTA or 1 mM [Ca²⁺]_ex, as indicated.