724
2.4. Western blotting.
After treatment with 40 µM curcumin, the cells were washed with ice-
cold 1× phosphate-buffered saline (PBS), collected, centrifuged, and total protein
was extracted using radioimmunoprecipitation buffer. In brief, lysis buffer were
add to re-suspend the pellets, which were subjected to ultrasonication, and then
centrifuged to separate cell debris. Supernatants were kept at – 20°C until use (9,
33). Immunoblotting was carried out using antibodies against β-actin, PKA
(ab5816, abcam, Cambridge, UK), p-CREB (9191s Cell Signaling Technology,
Danvers, MA, USA), CREB (9197, Cell Signaling Technology), p-AMPK (2535s, Cell
Signaling Technology), and AMPK (2532, Cell Signaling Technology) according to
the manufacturers’ protocols.
2.5. Protein Kinase A (PKA) phosphorylation assay.
PKA activity was measured based on transferring 32P-ATP to a targeted
peptide substrate. Relative PKA activity was measured with a liquid scintillation
counter as previously described (34).
2.6. Measurement of reactive oxygen species (ROS) generation.
Intracellular ROS concentrations were assessed using the oxidant-sensitive
fluorescent probe-dichloro-dihydro-fluorescein diacetate (DCFHDA. 3T3L1 cells
were incubated with DCFHDA and then washed with 1× PBS. Fluorescence was
measured spectrofluorometery at wavelengths (excitation 507 nm and emission:
530 nm) as previously described (35).
2.7. Statistical analysis.
Three independent experiments were carried out for the statistical
evaluation. Data are presented as mean ± SEM. All results were generated with
SPSS (Chicago, IL, USA) using analysis of variance (ANOVA) and Least Significant
Difference (LSD) post-hoc tests. Values of P < 0.05 were considered significant.
3. RESULTS
3.1. Curcumin inhibits 3T3L1 adipocyte viability in a dose- and time-dependent
manner.
The cell life survival of curcumin (viability or % inhibition) was measured
with the MTT cell viability assay post-induction and confirmation of adipocyte
differentiation. The results revealed that treatment of 3T3L1 adipocytes for 24 h
with different concentrations of curcumin (5, 10, 20, 40, 60, and 80 µM) in the
culture media resulted in significant (p<0.01) and gradual inhibition in mean cell
viability (94.76% ± 1.01%, 89.65% ± 0.65%, 84.64% ± 0.93%, 71.90% ± 1.09%,
51.51% ± 1.42%, 28.91% ± 6.65%, respectively; vs compared to control cell
(viability of 100%; (Figure 1)).
