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. 2022 Apr 22;13(4):398. doi: 10.1038/s41419-022-04741-9

Fig. 2. Resistant cells show an increased Mitochondria–ER proximity.

Fig. 2

A Images of mitochondria–ER contact sites in sensitive cells (2008, U2OS) and resistant (C13, U2OS-PT) acquired by Tecnai G2 (FEI) transmission electron microscope operating at 100 kV; images were collected by a F114 (TVIPS) CCD camera and the respective magnification of mitochondria–ER contact sites images. The TEM images and experiments are performed by the University of Padova electron microscopy facility. Scale bar 1 µm. B) The morphometric analysis of electron micrographs was performed using ImageJ freehand tool (7 cells per sample, at least 30 images/sample). We used the ER–mitochondria contact coefficient (ERMICC) to measure the extent of physical interaction among mitochondria and ER (as described in the Experimental procedures section). Data are the mean ± SEM of 3 different experiments; *p < 0.05, calculated by a two-tailed unpaired t-test comparing resistant vs sensitive cells. C Images of FRET signal using a modified FRET-based indicator of ER–mitochondria proximity (FEMP). D The respective measure of FRET signal of 2008-C13 and U2OS-U2OS-PT and quantification of the maximum MERC index for the indicated cell lines infected with Adenovirus FEMP. Scale bar 100 µm. Data represents mean ± SEM of 3 independent experiments; *p < 0.05, calculated by a two-tailed unpaired t-test comparing resistant vs sensitive cells. Cells from the raw images were segmented using YFP channel and intensity measures of CFP, YFP and FRET along with corresponding background intensities were calculated. Mean FRET Ratio intensity was then calculated by subtracting the background and normalizing to CFP intensity.