Figure 1.
Adipocytes display an increased ability to repair DNA double strand breaks as compared to pre-adipocytes.
(A) γH2AX foci formation detected by immunofluorescence using an antibody that specifically recognizes the phosphorylated form of H2AX, in pre-adipocytes and adipocytes cells, exposed for 1 h to 2 pM of CL1γ or 30 min after irradiation at 2 Gy. (B) Kinetics of repair of DNA double-strand breaks as determined by disappearance of γ-H2AX foci after irradiation at 2 Gy (left panel) or after treatment at 2 pM of CL1γ during 1 h (right panel), mean of 3 independent experiments±SEM. (C) Kinetics of DNA DSBs repair determined by pulse-field gel electrophoresis after treatment with CL1γ (40 or 500 pM, 1 h) (left panel) or after γ-irradiation at 80 Gy (right panel). Results were obtained from the quantification of two independent experiments for treatment at 40 pM and three independent experiments for 500 pM and 80 Gy (mean±SEM).
Figure 2.
Adipocyte differentiation is associated with an early increase in DNA-PKcs expression.
(A) Expression of DNA repair proteins (DNA-PKcs, Ku70, Ku80, XRCC4, Rad51 and ATM) or adipocyte differentiation marker (HSL) was analyzed by Western Blots in exponentially growing 3T3F442A fibroblast cells (Ex), in 3T3F442A fibroblast cells grown to confluence (Co) or after the indicated time of culture in adipogenic differentiation medium (B) Similar experiments were performed in exponentially growing pre-adipocytes (Ex), in pre-adipocytes grown to confluence (Co) or during the first two days of culture in adipogenic medium) (C) Expression of DNA-PKcs protein in a murine fibroblast cell line (Balb-C) grown to confluence in the presence or not of insulin in comparison to exponentially growing cells.
Figure 3.
Adipocyte differentiation is associated with an increase of the kinase activity of the DNA-PK complex without modification of the activity of its DNA binding sub-unit Ku.
(A) Two-hundred µg of the cellular extracts were used for the measurement of DNA-PK kinase activity, as described in the Material and Methods Section, in the pre-adipocyte cells (exponentially growing, Ex or at confluence, Co) or during adipocyte differentiation (after 3 and 7 days of culture in insulin containing medium) (mean of 5 independent experiments±SEM). (B) Ku DNA end binding activity was measured by EMSA. 10 µg of extracts obtained from the indicated cells were incubated with a [32P] 25-bp double-stranded DNA probe in the presence of an excess of closed circular plasmid DNA used as a non-specific competitor. The proteins/DNA complexes were resolved on a non-denaturating polyacrylamide gels and the position of the free probe and the Ku/DNA complexes are indicated.
Figure 4.
The increased ability of adipocyte to repair DSBs is strictly dependant on the classical NHEJ pathway.
(A) The kinetic of DSBs repair after 1 h exposure to CLγ1 (500 pM), in presence or not of 50 µM of Wortmaninn, for pre-adipocyte and adipocyte cells, was analyzed by PFGE. Mean of 3 independent experiments±SEM. *Statistically significant by Student's t-test, P<0.05 relative to control without wortmannin; ** statistically significant by Student's t-test, P<0.01 relative to control without wortmannin. (B) DNA-PKcs expression in pre-adipocytes 3T3F442A clones stably expressing shRNA sequences directed against either DNA-PKcs mRNA or shRNA control (upper panel) and kinetic of DSBs repair performed by PFGE after treatment with 500 pM of CLγ1 (1 h) and post incubation in drug-free medium for 6 or 24 h (lower panel)(C) Similar experiments were performed with the two same clones after differentiation into adipogenic medium. Mean of 3 independent experiments±SEM. **Statistically significant by Student's t-test, P<0.01 relative to control; *** statistically significant by Student's t-test, P<0.001 relative to control. The expression of DNA-PKcs was evaluated by Western blots experiments.
Figure 5.
Expression of DNA-PKcs is enhanced during adipogenesis of human pre-adipocytes isolated from sub-cutaneous adipose tissue.
Cells isolated from the stromal vascular fraction of collagenase digested subcutaneous tissue were grown for the indicated times in adipogenic medium as described in the Material and Methods section. (A) Photographs of the cultured cells and (B) Western blots experiments using indicated antibodies. NS, non specific band that show equal loading between the different samples.