Panel A shows the diagram illustrating the experimental workflow involving cell coculture and fluorescence-activated cell sorting. The images show results of siRNA knockdowns. Panel B contains four western blots showing protein levels after TIM23 and TOM40 siRNA knockdowns and FACS-isolated cells. Panel C shows representative microscopy images of double-positive cells from a coculture with non-targeting siRNA treated cells. Panel D shows representative microscopy images of double-positive cells from a coculture with TIM23 siRNA treated cells. Panel E shows representative images of double-positive cells from a coculture with TOM40 siRNA treated cells. The images highlight that siRNA knockdowns recapitulate the effect of DHFR-MTX on intercellular mitochondrial transfer.
TIM23 and TOM40 siRNA knockdowns recapitulate phenotypes seen with DHFR-MTX. (A) Schematic of workflow. (B) Western blots of (i) TIM23 siRNA knockdown; (ii) TOM40 siRNA knockdown; (iii) FACS-isolated single-positive cells following TIM23 siRNA knockdown in su9-mScarlet cell line and subsequent 48 h co-culture with su9-mTagBFP2 cell line; (iv) FACS-isolated single-positive cells following TOM40 siRNA knockdown in su9-mScarlet cell line and subsequent 48 h co-culture with su9-mTagBFP2 cell line. (C) Representative images of double-positive cells from a 48 h co-culture of a su9-mTagBFP2 line with a non-targeting siRNA-treated su9-mScarlet line. (D) Representative images of double-positive cells from a 48 h co-culture of a su9-mTagBFP2 line and a TIM23 siRNA-treated su9-mScarlet cell line. (E) Representative images of double-positive cells from a 48 h co-culture of a su9-mTagBFP2 line and a TOM40 siRNA-treated su9-mScarlet cell line. Scale bars 20 μm. DP, double-positive cell; SP, single-positive cell; Cntl, control; NT, non-targeting. Source data are available for this figure: SourceData FS3.