Figure 4

Conserved pathways facilitate Tdp1 entry into human and mouse mitochondria following H₂O₂ stress. (a) Left panel: Immunoblot of human fibroblast mitochondrial lysates without or with knockdown of ERK1 and P38 and in the absence or presence of 1 µM H₂O₂ for 1 hour. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H₂O₂ for 1 hour. Tdp1 levels were normalized to complex IV and fold changes are shown relative to levels in cells treated with nontargeting siRNA. (b) Left panel: Immunoblot of Rho-zero MEF mitochondrial lysates without or with knockdown of ERK1 and P38 and in the absence or presence of H₂O₂ treatment. Mitochondrial complex IV was used as a loading control. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H₂O₂ for 1 hour. (c) Left panel: Immunoblot of human fibroblast mitochondrial lysates without or with knockdown of ERK1 and P38 and in the presence of H₂O₂ treatment. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H₂O₂ for 1 hour. (d) Upper panel: Immunofluorescent staining of P38 and ERK1 in human fibroblasts before and after 5 minutes with 1 µM H₂O₂ or 200 nM rotenone treatment. Lower panel: Immunofluorescent staining of Tdp1 in human fibroblasts treated with non-targeting siRNA or TIM23 siRNA with or without 1 µM H₂O₂ for 1 hour. (e) Upper panel: Immunoblot of lysates from purified mitochondria of cultured MEFs expressing wt or S81A HA-tagged Tdp1 cDNA in the presence or absence of treatment with 1 µM H₂O₂ for 1 hour. Lower panel: Graph of fold change in mitochondrial Tdp1 across three independent experiments following treatment with 1 µM H₂O₂ for 1 hour. Tdp1 levels were normalized to complex IV levels. Error bars represent one standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001. Mitochondrial complex IV was used as a loading control. Scale bar = 10 µm.