Figure 3.

Figure 3.

H3-DPC levels are increased in vivo in tdp1 mutant fish line and in RPE1 cells with TDP1 deficiency. (a) Western blot showing H3-DPC levels in tdp1 mutant embryos in combination with sprtn knockdown and CPT (10 µM, 1 h) treatment. Total DPCs were isolated from 2-day-old embryos, separated by SDS–PAGE (DPC equivalent of 200 ng DNA per well) and detected with H3-specific antibody. (b) Quantifications of H3-DPCs from four biological replicates with mean (± s.d.) fold change to endogenous H3-DPCs in WT embryos. (c) Western blot analysis of H3-DPCs in zebrafish embryos and (d) corresponding quantification after FA treatment (5 mM for 30 min) (n = 4). (e) Dot blots showing H3-DPCs after silencing TDP1 and/or SPRTN before and after CPT exposure in RPE1 cells (50 nM CPT, 1 h) and DNA loading controls. Equivalent of 200 ng DNA of total DPCs was loaded per sample. (f) Quantification of H3-DPC analysis in RPE1 cells (n = 3). (g) Dot blots showing H3-DPCs after silencing TDP1 and/or SPRTN before and after FA exposure (1 mM FA, 20 min) in RPE1 cells and DNA loading controls and (h) corresponding quantification (n = 3). Results are presented as mean ± s.d. with statistically significant changes, determined using an unpaired Student's t-test, indicated with *p < 0.05, **p < 0.01, ***p < 0.001 and ^#p < 0.0001.