Fig 1: Hoxa3 protein treatment modulates p65/NF-?B and HAT/HDAC activation and inhibits acetylation of the TNF promoter.(A) Total and phospho-p65 in Hoxa3-treated vs. mCherry-treated macrophages derived from type II diabetic patients either non-activated (NA) or classically activated (CA) with LPS and IFN-? for 1 hour. (B) Per cent phosphorylated p65/NF-?B (pS536) protein out of total p65 as shown in (A). (C) Total and phospho-p65 in Hoxa3-treated vs. mCherry-treated macrophages derived from type II diabetic patients, either non-activated (NA) or classically activated (CA) with LPS and IFN-? for 6 hours. (D) Per cent phosphorylated p65/NF-?B (pS536) protein out of total p65 as shown in (C). Activation of histone acetyl transferases (HAT, OD value per µg of protein), (E), or histone deacetylases (HDAC, pmol/min/mg), (F), analyzed in nuclear protein samples isolated from Hoxa3-treated vs. mCherry-treated macrophages derived from type II diabetic patients either NA or CA with LPS and IFN-? for 6 hours. HeLa cell nuclear extract was used as a positive control, and Trichostatin A (TSA) used as the control HDAC inhibitor. (G) Representative chromatin immunoprecipitation of the TNF promoter (-601 to -230) using anti-H3K27Ac antibody in mCherry- or Hoxa3-treated macrophages from diabetic patients (1 of 4 biological replicates, showing mean + SD of 2 technical replicates, ***p<0.001). All other experiments shown as mean ± SEM, n = 3 for each population, *p<0.05, **p<0.01, ***p<0.001.
Fig 2: Effects of RAGE-aptamer on LPS-exposed THP-1 cells. (a–d) Each left panel shows the protein levels of TNF-a (a), IL-1ß (b), IL-6 (c), and HMGB1 (d) produced by THP-1 cells. Each right panel shows the mRNA levels of cytokines in THP-1 cells. (e) ROS generation. (f) NADPH oxidase-derived superoxide generation. (g–j) mRNA levels of p22phox (g), Nox2 (h), p47phox (i), and p67phox (j). (k) Total NF-?B p65 (left panel) and p-NF-?B p65 levels (right panel) in THP-1 cells. (l) Viable cell number of THP-1 cells. N = 5 per group. #P < 0.05 and ##P < 0.01 compared with control, respectively. *P < 0.05 and **P < 0.01 compared with LPS+control-aptamer-treated cells, respectively. †P < 0.05 and ††P < 0.01 compared with LPS+vehicle-treated cells, respectively.
Fig 3: NF-?B p65, gene and protein expression of cytokines in serum and PBMCs of mice injected with saline (control), LPS (20 µg/g-BW) plus vehicle (positive control), LPS (20 µg/g-BW) plus 40 pmol/g-BW of control-aptamer, or LPS (20 µg/g-BW) plus 40 pmol/g-BW of RAGE-aptamer. (a) Total NF-?B p65 (left panel) and p-NF-?B p65 levels (right panel) in PBMCs. (b–e) Each left panel shows serum levels of TNF-a (b), IL-1ß (c), IL-6 (d), and HMGB1 (e). N = 7 per group. Each right panel shows gene expression levels of cytokines in PBMCs. N = 5 per group. #P < 0.05 and ##P < 0.01 compared with control, respectively. *P < 0.05 and **P < 0.01 compared with LPS plus control-aptamer-treated mice, respectively.
Fig 4: Effect of CRBST 250 and 500 mg/kg on NF-?B p-65 mRNA (A); Phosphorylated NF-?B p-65/p65 ratio (B); I?Ba mRNA (C); I?Ba (D); Nrf2 (E) and NF?B DNA binding activity (F) in rats with AA-induced UC. Results are shown as the mean ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Normal, normal control rats administered the vehicle; CRBST 500, normal rats administered carbocisteine (500 mg/kg); UC, AA-induced UC rats administered the vehicle; UC/CRBST 250, AA-induced UC rats treated with carbocisteine (250 mg/kg); UC/CRBST 500, AA-induced UC rats treated with carbocisteine (500 mg/kg).
Fig 5: BK channel activation inhibits TNF-a-induced MAPK but not NF-kB-1/p65 or RIG-1 pathways. The treatment of HULEC5a endothelial cells with the BK channel opener NS1619 (30 µM, for 24 h) inhibits the TNF-a-induced (5 ng/mL; 24 h) phosphorylation of the Ca2+-dependent JNK-1/2 (A) and p38 pathways (B), but has no effect on RIG-1 expression or the phosphorylation of ERK-1/2 and NF-kB/p65 (C–F) (n = 3–4, * p = 0.05) as shown by ELISA-based multiplex assays. (G) The inhibition of JNK-1/2 (JNK inhibitor VIII, 20 µM, 24 h) and p38 (SB203580, 20 µM, 24 h) pathways, with or without NS1619, decreases TNF-a-induced (5 ng/mL; 24 h) CCL-2 secretion from HULEC5a cells (n = 3–4, * p = 0.0001), shown by ELISA. n = number of separate cell passages, which are considered biological replicates.
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