Fig 2: HIV gp120 up-regulates PIAS3 and STAT1 protein expression in MDDCs. MDDCs were stimulated with gp120 (5 µg/ml) for 18 h. Cell lysates were resolved by 8-12 % SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblot analysis with antibodies specific for: p38, ERK1/2, JNK, p65, p50, IkBa, IKKß, JAK2, STAT-1, -2, -3, SOCS-1, -3, PIAS3, Bcl2, BCLxL, Akt, and PKR. Data from one representative experiment are shown. a Graphs show the level of protein as determined by densitometry (ImageJ software) and calculated relatively to untreated control (C), where each sample was normalized to total actin. The average fold from two-four independent experiments was represented, along with the SD. b Immunoblot analysis with antibodies specific for PIAS3 and STAT1. Actin expression is shown as gel loading control. Values below the lanes show band intensities of the respective bands, normalized to actin expression. Data from one representative experiment out of four analyzed are shown

Fig 3: Quercetin restricts rotavirus infection by inhibiting NF-?B pathway. (A) RV-SA11 (MOI 3)-infected MA104 cells (12 hpi) were treated with DMSO or quercetin (50 µM) 1 h before adding the virus, during virus addition, and 1 h after addition of the virus to assess the effect of quercetin on viral protein expression on the basis of time of addition. Western blot was performed to check VP1 and VP6 along with internal loading control ß-actin. (B) SA11 (MOI 3)-infected MA104 cells (12 hpi) were treated with DMSO or quercetin (50 µM) at 0, 3, and 6 hpi, respectively, to assess VP6 expression keeping ß-actin as loading control. (C) Protein expression and phosphorylation status of chief players of NF-?B signaling pathway such as NF-?B p65, NF-?B p50/p100, I?Ba, and IKKa/ß were evaluated by SDS-PAGE. Cell lysates were prepared by mock-infected or RV-SA11 (MOI 3)-infected MA104 cells for 6 hpi in the presence of either DMSO or quercetin (50 µM). The expression of VP6 and ß-actin served the purpose of infection marker and internal loading control, respectively. The ratio of phospho p65 and basal p65 was plotted in bar graph. (D) pcDNA-NSP1 was transfected in MA104 cells to overexpress RV-NSP1 which triggers the activation of NF-?B p65. Next, the transfected cells were either treated with DMSO or quercetin (50 µM). Consequently, the cell lysates are subjected to Western blot to detect the expression of phospho and basal NF-?B p65, NSP1, and internal loading control ß-actin. Phospho p65 level was normalized against basal p65 and represented in bar graph. (E) MA104 cells are infected RV strain A5-13 (wild-type NSP1) and A5-16 (mutant NSP1), respectively, followed by administration of DMSO or 50 µM quercetin. Cell lysates are run for SDS-PAGE for the detection of phospho and basal NF-?B p65, NSP1, VP6 (infection marker), and ß-actin (loading control). The ratio of phospho p65 and basal p65 was represented by bar graph. (F) TNFa (10 nM) is administered to MA104 cells to induce NF-?B signaling and also co-treated with quercetin (50 µM) to check the effect of quercetin on TNFa-induced NF-?B pathway. Western blot analysis was done to check expression and phosphorylation of NF-?B p65, and ß-actin was checked as internal loading control. Phospho p65 is normalized against basal p65 and plotted in bar graph. (G) BAY11-7082 (10 µM) is added to RV-SA11 (MOI 3)-infected MA104 cells (6 hpi) either alone or co-treated with quercetin (10 µM) to check VP6, NF-?B, IKKa/ß, and ß-actin (loading control) protein levels by Western blot. (H) Plaque assay was performed to assess viral titer from SA11-infected MA104 cells treated with DMSO, quercetin (10 Mm), BAY11-7082 (10 µM), and quercetin co-treated with BAY11-7082, respectively. Phospho p65 and basal p65 ratio is represented in bar graph. Each bar represents mean ± SD of three independent experiments (Unpaired student's t-test, ns denotes p value not significant, *p < 0.05, **p < 0.01).

Fig 4: Kinetics of Nfkb1, Nfkb2, Rel and RelA expression.Quantitative RT-PCR determination of the mRNA content for the Nfkb1 (A), Nfkb2 (B), Rel (c-Rel) (C) and RelA (D) genes in the prostate of non-castrated (NC) and castrated rats up to 7 days after surgery. The fold-change variation with respect to the controls is shown as the mean ± standard variation (n = 3 for each time point). The asterisks indicate p<0.05. The dotted lines in each figure correspond to the fourth-power exponential fitting curve, and are shown together for the sake of direct comparison in E.

Fig 5: Interaction of mutant p50-Tyr350Cys with RelA, p50 and I?Ba. HEK293T cells were transiently (co-)transfected with the indicated vector constructs. DNA amounts were titrated in pre-experiments for each panel to enhance the visibility of protein-protein interactions. Representative examples are shown. (A) Confocal imaging shows most of the ectopically expressed mScarlet-RelA(p65) fusion protein (red) localizing to the cytoplasm. Strong and/or prolonged overexpression of RelA had cytotoxic effects (data not shown). Upon co-expression of wildtype p50 or p50-Tyr350Cys (green), RelA shows a nuclear localization and adapts the homogeneous pattern of wildtype p50 or the aberrant subnuclear accumulation of p50-Tyr350Cys. Nuclei were stained with Hoechst 33342 (blue). Scale bar: 20µm. (B) The empty mScarlet-C1 vector was used as a control. The non-specific localization of mScarlet (red) is not influenced by the wildtype or mutant EGFP-p50 fusion proteins (green). (C) The majority of the nuclear EGFP-fused p50-Tyr350Cys (green) accumulates in high intense spot-like structures, whereas most of the co-expressed mScarlet-fused wildtype p50 (red) remains unaffected and shows its typical homogeneous localization. (D) Co-expressed non-fused I?Ba (not visible), a cytoplasmic inhibitor of NF-?B1 dimers, leads to a virtually complete cytoplasmic retention of wildtype EGFP-p50 (green). The retained EGFP-p50-Tyr350Cys (green) apparently undergoes cytoplasmic decay (as indicated by attenuated fluorescence signals; not shown) whereas a proportion of the mutant protein localizes to the nucleus and adapts a uniform localization (at low abundance) or shows protein aggregation (at higher abundance). The fluorescence intensities were adjusted for better visibility. (E) Increasing amounts of non-fused wildtype p50 (lower molecular weight) were co-expressed with wildtype or mutant EGFP-fused p50 (higher molecular weight) and DNA-binding competition was determined by EMSA. DNA-binding of p50(wt):p50(wt) homodimers, EGFP-p50(wt or mutant):p50(wt) mixed homodimers and EGFP-p50(wt or mutant):EGFP-p50(wt or mutant) homodimers is indicated by low, medium and high band sizes, respectively. An identical gel was prepared with an approximately 2.5-fold running time to separate the DNA-protein bands (lower panel). An intermediate sized band indicates the interaction of wildtype and mutant p50 (right panel). Diverse binding partner preferences are obvious. (F) Cells were transfected with the NF?B-reporter vector alone (100 ng) as background control, plus RelA(p65) (5 ng) to switch on reporter expression (tdTomato, red), and plus EGFP-fused p50 (300 ng) (green) to demonstrate its inhibitory effect on RelA-mediated reporter activity. In the presence of p50 (and thus repressive p50:p50 homodimers) reporter activity is further repressed by increasing relative amounts of mutant p50-Tyr350Cys, whereas p50-Tyr350Cys alone has a dose-dependent attenuating effect. Numbers indicate transfected DNA amounts (ng) (G) Reverse promoter reporter experiment with increasing relative amounts of wildtype p50 alone or in the presence of co-transfected p50-Tyr350Cys (300 ng). The dose-dependent repressive effect of wildtype p50 is further intensified by the co-expressed p50-Tyr350Cys.