Fig 2: cAMP enahnaces the IR-induced phosphorylation of IKKβ and nuclear translocation of p65. (A) Reh cells were treated with or without forskolin (80 μM) for 30 min before irradiation (10 Gy). Cells were harvested at the indicated times and subjected to Western blot analysis with the indicated antibodies. The figure shows 1 representative blot of 4 experiments. (B) Reh cells were treated as in A, harvested at the indicated times and subjected to immunoblot analysis with the indicated antibodies. The figure shows 1 representative blot of 4 experiments. (C) Reh cells were treated as in A. One half of each sample was used for preparation of total cell lysate and the other half was subjected to cellular fractionation to obtain nuclear lysate. The lysates were then analyzed by immunoblotting with the indicated antibodies. The figure shows 1 representative experiment of 3. (D) Reh cells were cultured in the absence or presence of Bay 11-7082 (5 μM) for 90 min before treatment with or without forskolin (80 μM) for 30 min. Cells were then exposed to 10 Gy IR and harvested after 2 h. One half of each sample was used for preparation of total cell lysate and the other half was subjected to cellular fractionation to obtain nuclear lysate. The lysates were then analyzed by immunoblotting with the indicated antibodies. The figure shows 1 representative experiment of 3.

Fig 3: Knockdown of p65 relieves the inhibitory effect of forskolin or 8-CPT-cAMP on IR-induced cell death. Cells transfected with control siRNA or p65 siRNA were treated with or without forskolin (80 μM) or 8-CPT-cAMP (200 μM) for 30 min before exposure to IR (10 Gy). After 20 h, cells were analyzed for PI uptake by FACS (n = 4). The p values were calculated relative to cells treated with IR only: Reh, *p < .01, **p < .04. TK6, *p < .03, **p < .05. The histograms in the right panel depict percent inhibition of IR-induced cell death by forskolin or 8-CPT-cAMP in cells transfected with control siRNA or p65 siRNA.

Fig 4: The NF-κB–SHP2–ERK and IL-6–JAK–SHP2 pathways are concomitantly increased in HBV-associated liver tissues(A) Whole tissue lysates were prepared from 17 pairs of HBV-infected tumors (T) and matched non-neoplastic tissues (N), and analyzed by Western blot with anti-SHP2 antibodies. Vinculin served as a loading control. (A) NF-κB-p65, SHP2, p-ERK, ERK, IL-6, p-JAK, JAK, p-STAT3 and STAT3 levels were assessed by Western blotting in T/N tissues prepared as described in A. β-Actin was used as a loading control. (B) The whole tissue lysates described in (A) were assayed to determine the levels of p-STAT3 and NF-κB-p65. Columns represent the means of three independent experiments; bars indicate standard deviations (* P < 0.05). The numbers denote tissue numbers.

Fig 5: miR-132 does not regulate PA-induced TLR signaling. THP-1 cells were transfected with an inhibitor negative control or inhibitor of miR-132 at 20 nM for 48 h before being treated with 200 μM PA or 1 μg/mL LPS (as a positive control) for 12 h. (A) The mRNA levels of MyD88, IRAK1, and TRAF6 were examined by quantitative RT-PCR; (B) Nuclear and cytosolic fractions were subjected to Western blot analysis using antibodies specific for NF-κB p65, histone H1, and β-actin. Histone H1 and β-actin were used as a loading control. All results are representative of three independent experiments. * p < 0.05 compared with the inhibitor negative control-transfected cells, and # p < 0.05 compared with the miR-132 inhibitor-transfected cells. NS, no significance.