Journal: Cellular signalling

Article Title: DNA Topoisomerase IIα contributes to the early steps of adipogenesis in 3T3-L1 cells.

doi: 10.1016/j.cellsig.2016.07.002

Figure Lengend Snippet: Fig. 2. Expression levels of class IA PI3K catalytic isoforms during differentiation and effect of their selective inhibition on differentiation and Topo IIα expression. A. Representative Western blots of whole cell extracts from 3T3-L1 cells, day 0 (D0) and up to 7 days (D7) of differentiation, blots probed with anti-PI3K p110α, β, δ and anti-β-actin as a loading control. B. Representative photos of 3T3-L1 cells fixed and stained with Oil Red O on day 0 (D0) or day 7 (D7) of differentiation after incubation for the first 4 days with the selective PI3K inhibitors for p110α (A66), p110β (TGX-221) or p110δ (PI3065), LY294002 or DMSO (photos of plates on top, microscope 10× magnification on bottom), C. Quantification of Oil Red O staining from day 7 (D7) plates normalised to DMSO. D. Representative Western blots of 3T3-L1 whole cell extracts obtained at day 0 (D0) or day 1 (D1) of differentiation in the presence of PI3K inhibitors or DMSO, probed with anti-Topo IIα, anti-ribosomal protein S6 (RS6), anti-phosphoS240/S244-ribosomal protein S6 (pRS6) and β-actin as loading control, E. Quantification of Topo IIα protein band densitometry from day 1 (D1) normalised to β-actin and then to DMSO. All quantifications were from a minimum of 2–3 independent experiments, shown as means ± SDs. Bands of interest on Western blots are highlighted with * if multiple.

Article Snippet: The following antibodies were used: anti-PI3K p110α (4249), anti-Poly(ADP-ribose) Polymerase (PARP) (9542), anti-ribosomal protein S6 (2217) and anti-phospho-S240/S244 ribosomal protein S6 (5364) from Cell Signaling Technology; anti-DNA Topoisomerase IIα (ab52934) from Abcam; anti-PPARγ (MA5-14889) from ThermoFisher; anti-DNA Topoisomerase IIβ (HPA024120) from Sigma-Aldrich and anti-β-Actin (sc-69879), anti-PI3K p110β (IgM, sc376492), anti-PI3K p110δ (sc-7176), anti-cyclin A (sc-596) from Santa Cruz Biotechnology.

Techniques: Expressing, Inhibition, Western Blot, Control, Staining, Incubation, Microscopy