CHUNG, SOONKYU, Ph. D. Mechanisms by Which Conjugated ...

Depletion of NFκB p65 by RNAi Attenuates CLA’s Suppression of Adipogenic Protein
Expression and Glucose Uptake and Activation of ERK1/2
We demonstrated that the perturbation of NFκB activation was sufficient to
prevent or attenuate CLA’s suppression of adipogenic protein expression (Fig 3.7). Using
a second approach, selective depletion of NFκB p65 using siRNA was performed prior to
the treatment with trans-10, cis-12 CLA. As shown in Fig 3.8A, control (non targeting
siRNA) or NFκB p65 siRNA were introduced to the cultures of differentiating adipocytes
on day 6. 72 h post-transfection, cultures were serum starved for 20 h and treated with
either BSA control or 30 µM trans-10, cis-12 CLA for 24 h (Fig 3.8A). To monitor the
transfection efficiency, cultures were transfected with siGLO-RISC-free, a fluorescent
labeled, non-targeting siRNA with impaired ability for RISC formation. Almost all cells
(> 90%) were positive to Cy3-fluoresence of siGLO 24 h post- transfection (Fig 3.8B),
indicating that both adipocytes and non-adipocytes were efficiently transfected (Fig 3.8B).
Specific depletion of NFκB p65 was examined using immunoblotting as shown in Fig 3.
8C. We consistently obtained > 50% NFκB depletion. NFκB p65 protein expression was
severely blunted in the NFκB p65 siRNA transfected cultures compared to untreated or
non-targeting control or lamin-siRNA transfected cultures (Fig 3.8C). In contrast, protein
levels of: 1) NFκB p50, a heterodimeric partner of active NFκB; 2) GAPDH, a
constitutive cytoplasmic protein; 3) β-actin, a cytoskeleton protein; and 4) aP2, a specific
adipocyte marker protein, were unchanged. siRNA-mediated specific knockdown was
validated using lamin siRNA as a positive control (Fig 3.8C).
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