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. Author manuscript; available in PMC: 2016 Aug 1.
Published in final edited form as: Exp Mol Pathol. 2015 Jun 14;99(1):109–115. doi: 10.1016/j.yexmp.2015.06.004

Fig. 4. Both HIV and LPA induced higher binding activity of NFκB.

Fig. 4

A. Nuclei from HRPTCs were extracted following various treatments (as indicated) and EMSA was performed. HIV or LPA added in indicated concentrations. Sphingomyelinase (SMase) 100 mUnits/ml or Hydrogen peroxide (HP-0.5 mM) were loaded in lanes 8 and 10 respectively. Use of cold unlabeled probe, in the binding assay, suggests the specific binding activity of the fluorescent probe. Treatment with LPA synthesis blockers (as indicated) followed by HIV treatment, reduces NFκB binding activity. Lane 10 depicts addition of cold oligomer to the reaction mix, 10 min prior to adding labeled fluorescent probe

C. PDTC (50 μM) and DN NFκB inhibit tubular cell NFκB-Luciferase in HIV milieu. HRPTCs were transiently transfected with either NFκB-luciferase construct (0.5 μg/well, CMV-β-gal (0.5 μg/well), 0.5 μg/well DN-p65, or with IKK-α. After overnight incubation, the transfected cells were treated with PDTC for 4h before transducing cells with HIV for 4h. Cells were lysed and processed for luciferase and β-gal activities. Luciferase activity was normalized with respect to β-gal activity and expressed relative to activity of the control. Data are mean SD of of three different experimental values.

*P< 0.005 compared to relative control and # P< 0.001 compared with HIV treatment alone.

D. PDTC(+ 25 μM, ++ 50 μM) as well as NFκB siRNA inhibited HIV-induced expression of TGF-β, CTGF, Fibronectin, Collagen-1, α–SMA and SNAIL.

HRPTCs were pre-incubated with PDTC (25 or 50 μM) or transfected with NFκB siRNA prior to stimulation with HIV and were incubated for 72h. Cellular lysates were prepared and protein blots were probed for different protein expression.