Supplementary MaterialsSupFig1: Supplementary Figure 1 Nucleoid remodelling within A549 and H9C2

Supplementary MaterialsSupFig1: Supplementary Figure 1 Nucleoid remodelling within A549 and H9C2 cardiomyocytes. propidium + ve) of vehicle/DXR treated fibroblasts (3.4M) (n=200). (Aiii) Cytochrome c/DAPI co-labelling of DXR (3.4 M 24 hours), vehicle or H2O2 (80 mM 24 hours) treated fibroblasts. (Aiv) PicoGreen labelling of apoptotic fibroblasts following treatment with staurosporine (1 M 24 hours). (Bi) Histochemical staining of cytochrome c oxidase activity of H9C2 or fibroblasts cells treated with DXR/ vehicle (3.4 LY2109761 cost M 24 hours). (Bii) JC-1 labelling of fibroblasts treated with vehicle/DXR (3.4 M 24 hours) – red J-aggregates are present when mitochondria are polarised. (Biii) Polargraphic oxygen consumption of H9C2s treated with DXR/vehicle (3.4 M 24 hours) or sodium azide (5mM 20 minutes) ($ p = 0.053, **P = 0.005 vs control, n = 3). (Biv) ATP levels measured by luciferase assay of DXR/vehicle (3.4 M 24 hours) or rotenone (10 M, 20 minutes) treated fibroblasts. Error bars +S.D. Size bars 20m. Results are representative of three independent experiments. a.u. = arbitrary units. NIHMS27428-supplement-SupFig3.tif (8.6M) GUID:?BC4F54F9-DB4F-4FED-95AC-CEA5148D6E72 SupFig4: Supplementary Figure 4 Influence of MFN1/2 and OPA1 on mitochondrial morphology. (A) TMRM labelling of wild-type, MFN1 null and MFN2 null MEFs. (B) TMRM labelling of OPA1 or scramble siRNA treated cells. Immunoblotting blot of siRNA treated fibroblasts showing OPA1 and beta-actin protein levels. NIHMS27428-supplement-SupFig4.tif (1.0M) GUID:?CBBD2B24-5DCF-4D8E-B9C1-E1FA4CB224CE Abstract Many anti-cancer drugs, such doxorubicin (DXR), intercalate into nuclear DNA of cancer cells thereby LY2109761 cost inhibiting their growth. However, it is not well understood how such drugs interact with mitochondrial DNA (mtDNA). Using cell and molecular studies of cultured cells we show that DXR and other DNA intercalators such as ethidium bromide, can rapidly intercalate into mtDNA within living cells, causing aggregation of mtDNA nucleoids and altering the distribution of nucleoid proteins. Remodelled nucleoids excluded DXR and maintained mtDNA synthesis, whereas non-remodelled nucleoids became heavily intercalated with DXR, which inhibited their replication leading to mtDNA depletion. Remodelling was accompanied by extensive mitochondrial elongation or interconnection, and was suppressed in cells lacking MFN 1 and OPA1, key proteins for mitochondrial dynamics. In contrast, remodelling was significantly increased by p53 or ATM inhibition, indicating a link between nucleoid dynamics and the genomic DNA damage response. Collectively, our results show that DNA intercalators can trigger a common mitochondrial response, which likely contributes to the marked clinical toxicity associated with these drugs. DXR intercalation into cardiac mtDNA and subsequent damage may contribute to the longer-term tissue dysfunction characteristic of DXR pathology. Supplementary Material SupFig1Supplementary Figure 1 Nucleoid remodelling within A549 and H9C2 Rabbit polyclonal to POLR3B cardiomyocytes. (A) PicoGreen/TMRM labelling of DXR/vehicle treated A549 cells (3.4 M 24 hours). (B) Mitotracker red labelling of mitochondria within DXR/vehicle treated fibroblasts. Note that DXR intercalated into the nucleus is visible in the red channel after fixation. (C) PicoGreen labelling of H9C2 cardiomyocytes treated with DXR/vehicle. (D) Mitotracker labelling of vehicle/ DXR treated H9C2 cells. Bars: 20m except B (10m). Click LY2109761 cost here to view.(2.3M, tif) SupFig2Supplementary Figure 2 Effects of DXR on TFAM, mtSSB, Tid1 and ATAD3. (A) Anti-Tid1/Mitotracker labelling of fibroblasts treated with vehicle/DXR (3.4 M 24 hours). (B) Anti-ATAD/Mitotracker labelling of fibroblasts treated with vehicle/DXR. (C) Anti-DNA (IgM1)/anti-mtSSB labelling of DXR labelling of fibroblasts treated with.