Fig 2: Effect of NR4A3 overexpression on IL-1ß-induced NF-?B and MAPK pathway activation in rat chondrocytes. (A) A time-based experiment was used to affirm a proper duration of IL-1ß stimulation on rat chondrocytes for the analysis of pathway activation (similar results were obtained from 3 repeated experiments). The NF-?B pathway was evaluated by detecting the phosphorylation of p65, and degradation of IKB-a, while activation of the MAPK pathway was investigated by detecting the phosphorylation of p38, Erk and JNK through Western blot analysis. Significant activation of the NF-?B and MAPK pathways was observed at 10 min, and therefore, 10 min was used as the duration of IL-1ß stimulation in the following experiments. (B) NR4A3-overexpressed or control chondrocytes were incubated with IL-1ß (10 ng/ml) for 10 min, and activation of NF-?B and MAPK pathways was investigated through Western blot analysis (similar results were obtained from three repeated experiments). (C) Nuclear translocation of p65 was visualized by immunofluorescence analysis (similar results were obtained from three repeated experiments); yellow arrows were used to illustrate enhanced nuclear translocation of p65. (D) Activation of NF-?B was also detected by luciferase reporter gene analysis (similar results were obtained from 5 repeated experiments). *P < .05

Fig 3: AS inhibits NF-?B signaling and augments sensitivity to AR antagonist in castrate-resistant PCa cells. (A) Immunoblotting for AR, phospho-p65, total p65, and ß-actin in 22RV1 cells treated with AS (2.5 µM) and Bic (25 µM) alone or in combination in the presence of TNF-a (10 nM). (B) Immunoblotting for phospho-p65 and total p65 in PC3 and 22RV1 cells treated with AS (2.5 µM) and Bic (25 µM) alone or in combination. (C) Schema showing NF-?B luciferase reporter assay (left panel); NF-?B luciferase reporter assay in PC3 cells treated with AS (2.5 µM) and Bic (25 µM) alone or in combination (right panel). Error bars represent mean ± S.E.M. P values calculated from two-sided Student's t test. P values < 0.05 were considered significant.

Fig 4: Regulation of the transactivation activity of NF-?B by SIRT7. (A,B) The transcriptional activity of NF-?B was examined using the dual luciferase reporter assay. Each Sirt7 KD (shSirt7) and control (shCT) NRK-52E cell was transfected with 80 ng pCI-HA-p65 expression plasmid or pCI-HA-control plasmid as well as 20 ng pNF-?B-Luc plasmid, which contains multiple copies of the NF-?B consensus sequence, and 0.8 ng pRL-TK plasmid (n = 4/group). In addition, Sirt7 KD cells were co-transfected with 100 ng pcDNA3-FLAG-Sirt7 expression plasmid or pcDNA3-FLAG-control plasmid (B) (n = 4/group). (C) HEK293T cells were transfected with 2 µg pcDNA3-HA-Sirt1 expression plasmid or pcDNA3-HA-Sirt7 expression plasmid as well as 2 µg pcDNA3-FLAG-p65 expression plasmid. At 24 h after transfection, an immunoprecipitation assay and western blotting were performed. (D) HEK293T cells were transfected with 1 µg pcDNA3-HA-Sirt1 expression plasmid or pcDNA3-HA-Sirt7 expression plasmid as well as 0.5 µg pcDNA3-FLAG-p65 expression plasmid and 0.5 µg pCMVß-p300-myc expression plasmid. After 36 h, an immunoprecipitation assay was performed and p65 acetylation was detected by western blotting. *p < 0.05, **p < 0.01. Data are expressed as the mean ± SEM.

Fig 5: Validation of microarray data. (A) Cellular localization of p65 determined by immunofluorescence. (B) Semiquantitative analysis of immunofluorescence intensity of p65 in nucleus. (C) Expression of the lncRNA LOC541472 in SFMSCs 2 h after stimulation with 10 ng/mL IL-1ß. (D) The efficiency of LOC541472 knockdown using Smart Silencer was confirmed by RT-qPCR. IL-6 gene expression was determined by RT-qPCR (E), and IL-6 protein levels were determined by CBA assays (F). (G) Cellular localization and expression of IL6 and LOC541472, as demonstrated by RNAscope assays. (H) Western blotting of IL6, phosphor-p65, p65, and ß-actin. (I) Quantification of the blots shown in (H). (J) Gene expression of the lncRNA RP3-467K16.4 after stimulation with 10 ng/mL IL-1ß for 2 h. (K) The efficiency of RP3-467K16.4 knockdown using Smart Silencer was confirmed by RT-qPCR. SFMSCs were transfected with Smart Silencer targeting RP3-467K16.4 for 48 h and then incubated with 10 ng/mL IL-1ß for 2 h, and expression of the IL8 gene was measured by RT-qPCR (L), and IL8 protein levels were determined by CBA assays (M). (N) Western blotting of IKKß, IKKa, phosphor-p65, p65, and ß-actin. (O) Quantification of the blots shown in (M). NC negative control, SiL Smart Silencer targeting LOC541472, SiR Smart Silencer targeting RP3-467K16.4, pP65 phospho p65. *P < 0.05 vs. negative control or control group. #P < 0.05 vs. NC + IL-1ß group. N = 3. Scale bars = 100 µm.