Supplementary MaterialsAdditional file 1: Figure S1. or colchicine (c and d), at 2?h and 24?h coCculture time. Scale bars, 10?m. 12964_2019_472_MOESM2_ESM.pdf (1.8M) GUID:?D2FBBCF1-4ABF-4584-B420-B8C179395096 Additional file 3: Figure S3. Analysis of mitochondrial morphology. a Representative image of mitochondria networks labeled with MitoT Deep Red in untreated fibroblast. b TIC10 isomer Segmentation of white boxed area in a. c Colour maps of aspect ratio (AR), circularity and roundness as in b. d Mitochondria exchange between unirradiated (MitoT Deep Red) and 6CGy irradiated (MitoT Green) fibroblasts, under untreated, taxol and colchicine conditions. e Single cell analyses of mitochondria shapes of MitoT Green from acceptor cells in d. 12964_2019_472_MOESM3_ESM.pdf (1012K) GUID:?5B833346-5BD6-4A49-B615-E25F0407C2E9 Additional file 4: Figure S4. p105 a Mitochondria exchange between DNACdamaged and healthy fibroblasts. Corresponding to Fig. ?Fig.1e.1e. The absolute values of average aspect ratios (avg. AR). b Comparison of indicated conditions to 2Ch control. Results represent average ARCvalues of 30 cells SD (twoCsided tCtest; ns, not significant, *P?0.05, **P?0.01, ***P?0.005). 12964_2019_472_MOESM4_ESM.pdf (40K) GUID:?269B0468-8670-4063-8D35-C656D8F7E32D Additional file 5: Figure S5. aCf Mitochondria transfer in ATMwt and ATM?/? fibroblasts upon irradiation. Mitochondria transfer was monitored between donor cells labeled with MitoTracker Deep Red (green, indicated with white marker) and 6CGy irradiated acceptor cells labeled with MitoTracker Red (red, indicated with orange marker) after 24?h of coCculture. Nuclei were stained with DAPI. CoCculture TIC10 isomer of ATMwt and irradiated ATMwt fibroblasts (a and b), TIC10 isomer ATMwt and irradiated ATM?/? fibroblasts (c and d), ATM?/? and irradiated ATMwt fibroblasts (e), as well ATM?/? and irradiated ATM?/? fibroblasts (f). g Unilateral transfer of mitochondria from irradiated ATMwt (labeled with MitoTracker Deep Red) to ATM?/? fibroblasts (labeled with MitoTracker Green, indicated with white marker). h Unilateral TIC10 isomer transfer of mitochondria from ATMwt (labeled with MitoTracker Deep Red, indicated with white marker) to irradiated ATM?/? fibroblasts (labeled with MitoTracker Red). SRRF: superCresolution radial fluctuation images. Scale bars, 10?m. 12964_2019_472_MOESM5_ESM.zip (2.1M) GUID:?210D64B0-7659-477D-B53C-183D656B424C Additional file 6: Figure S6. Dynamics of foci resolution in monoC and coCcultured irradiated cells. a Corresponding to Fig. ?Fig.2a.2a. Overlay images show the nucleus location of foci detected by IF. Images were acquired by spinning disc confocal microscopy using a 40x objective. Scale bars, 10?m. b Cell size dynamics of 6CGy irradiated and nonCirradiated, monoC and coCcultured acceptor cells over a right period interval of 24?h. Linked to Fig. ?Fig.2bCc.2bCc. cCf Resolution dynamics of 53BP1 (c, d) and phosphoCATM S1981 (e, f) foci. Foci were visualized by IF, imaged by epiCfluorescence microscopy using a 10x objective. n?=?300 (at first time point) to 5000 (at last time point). g Reduction of 53BP1 foci number in acceptor cells depends on ratio of donorCtoCacceptor cell numbers. Results represent mean??SD (twoCsided tCtest; ns, not significant, *P?0.05, **P?0.01, ***P?0.005). 12964_2019_472_MOESM6_ESM.pdf (201K) GUID:?CD8C04B9-DCBC-46D2-AB38-9138EF7844CB Additional file 7: Figure S7. CoClocalization of H2AX and 53BP1 foci. 6CGy irradiated MiaPaCaC2CGFP cells (acceptor, in green) in monoC and coCculture with untreated MiaPaCaC2 (donor) 24?h after plating. IF images show coClocalization of H2AX and 53BP1 foci. Scale bars, 10?m. 12964_2019_472_MOESM7_ESM.pdf (60K) GUID:?4D29EDEE-31F9-4FB2-8D7A-EE66076137FD Additional file 8: Figure S8. CoCculture conditions profoundly change the association between DSB repair and cell cycling. a Schematic of the Fucci system. b Representative images of MiaPaCaC2 acceptor cells transfected with the Fucci system to TIC10 isomer monitor G1 (red) and G2 (green) phases. Transfected cells were exposed to 6?Gy xCray and subsequently plated for either monoC or coCculture together with untreated MiaPaCaC2 cells. After 24?h, 53BP1 foci were determined. In overlay pictures, the first number indicates the ratio of G1 to G2 fluorescence intensity and the second number indicates the foci number in each nucleus. Scale bars, 10?m. c Storyline of fluorescence strength ideals for G2 vs G1 in each analysed cell. Size of group radius represents amount of foci. d Storyline of G1 to G2 percentage over amount of 53BP1 foci. Pubs represent solitary cell. e Percentage of cell amounts in various cell cycle stages. 12964_2019_472_MOESM8_ESM.pdf (223K) GUID:?50C2F989-C9B5-498F-96CA-E7DB647C413D Extra document 9: Figure S9. DSB restoration is considerably influenced by acceptor cell/donor cell relationships within an ATM activityCdependent way. a ATMwt fibroblasts had been treated with DNACPK inhibitor (DNACPK i) and 6?Gy xCrays. PhosphoCDNA PK S2056 foci had been established at 1?h post irradiation by immunofluorescence microscopy. DMSO utilized.