Fig. 1: Cancer cells transfer mitochondria to fibroblasts through TNTs.
a, Coculture setup with LifeAct A431 cells (red) stained with MitoTracker green and unstained HPFs. This image was created with BioRender.com. b, Immunofluorescence images of the cocultures, counterstained with Hoechst. c, Representative photomicrographs of cocultures of A431 cells prestained with MitoTracker green and HPFs immunostained for COLI (white) and counterstained with phalloidin (red) and Hoechst (blue). TNT-like structures are indicated by white rectangles together with their length (n = 3 A431–HPF cocultures). d, Percentage of MitoTracker-high HPFs in direct or transwell coculture with A431 cells (n = 3 cocultures per setup). e, Percentage of MitoTracker-high HPFs after coculture with A431 cells in the presence of carbenoxolone (CBX) or vehicle (n = 3 cocultures per treatment group). f, RT–qPCR for GJB2 (encoding connexin 26) relative to RPL27 using RNA from A431 cells transfected with control (scrambled) or connexin 26 (Cx26) siRNA, and percentage of MitoTracker-high HPFs after coculture of siCtrl or siCx26 A431 cells (n = 3 cultures per group). g, Holotomographic imaging showing mitochondrial transfer (white arrows) from A431 LifeAct–MitoTracker green cells to HPFs (unstained) (Supplementary Video 1). h, Representative image of a coculture of A431 cells stained with MitoTracker green and HPFs, immunostained for COLI and counterstained with phalloidin and Hoechst (n = 3 A431–HPF cocultures). White arrows point to TNT-like structures. i, Percentage of MitoTracker-high HPFs after coculture with A431 cells in the presence of nocodazole (Noc), dihydrocytochalasin B (Cyto B) or vehicle (n = 3 cocultures per treatment group). j, Western blot analysis for SEC3 and SEC5 using lysates from A431 cells transfected with siCtrl, siSEC3 or siSEC5 (n = 2 cultures per group). Graph shows the percentage of MitoTracker-high HPFs after coculture with MitoTracker-stained control or SEC3–SEC5-knockdown A431 cells (n = 3 cocultures per group). k, Percentage of MitoTracker-high HPFs after coculture with HaCaT or A431 cells (n = 3 cocultures per cell line). l, Representative immunofluorescence images depicting cocultures of MDA-MB-231 and PANC1 cells prestained with MitoTracker green and HPFs immunostained for COLI (white) and counterstained with Hoechst (blue) (n = 3 cocultures per cell line). Graphs show the mean ± s.e.m. An unpaired two-sided Student’s t-test (d–f,i,k) or two-sided one-way ANOVA with Bonferroni post hoc multiple comparison test (j) was used to determine statistical significance. Scale bars, 50 μm (b,g), 20 μm (c) or 25 μm (h,l).