Journal: Cells

Article Title: Protective Effects of SIRT6 Overexpression against DSS-Induced Colitis in Mice

doi: 10.3390/cells9061513

Figure Lengend Snippet: SIRT6 regulated expression and phosphorylation of TAK1. ( a ) The expression and phosphorylation levels of TAK1 were determined by immunohistochemistry. ( b , c ) The statistical results of ( a ). n = 3–6, ** p < 0.01.

Article Snippet: Sections then were blocked in 5% rat serum and incubated overnight at 4 °C with the diluted biotinylated primary antibody (1:500; rat-anti-mouse; anti-TAK1, #ab109526, Abcam, Cambridge, England, UK; anti-p-TAK1, #4508, Cell Signaling Technology, Danvers, MA, USA; anti-c-Jun, #ab40766, Abcam; anti-p-Jun, #ab32385, Abcam; anti-NF-κB p65 antibody, #ab32536, Abcam; anti-NF-κB p65 (phosphor S536), #ab86299, Abcam; anti-NF-κB p65 (acetyl K310) antibody, #ab19870, Abcam).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus -Induced Osteomyelitis

doi: 10.2147/JIR.S307316

Figure Lengend Snippet: The development of immune cells in bone marrow and spleen were not affected due to DCAF1 deficiency in macrophages. ( A ) The genotype of DCAF1 myeloid cell-conditional KO mice was determined by PCR analysis. ( B ) The expressions of DCAF1 in the T cells and macrophages of DCAF1 fl/fl Lyz2 cre/+ mice were determined by Immunoblotting analysis. ( C–D ) The populations of B lymphocytes, T lymphocytes, macrophages, and neutrophils in the spleen from DCAF1 fl/fl Lyz2 +/+ (WT) or DCAF1 fl/fl Lyz2 cre/+ mice were analyzed by Flow cytometer. Representative FACS plots were shown. In this study, the data were shown as the mean ± SEM. The data were acquired from three independent experiments. The significance of data in ( C–D) was calculated by Student’s t -test. ns indicates no statistical significance.

Article Snippet: The primary antibody against DCAF1 was purchased from Abcam (ab1766, Shanghai, China).

Techniques: Western Blot, Flow Cytometry

Journal: Journal of Inflammation Research

Article Title: Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus -Induced Osteomyelitis

doi: 10.2147/JIR.S307316

Figure Lengend Snippet: Pro-inflammatory cytokines were elevated in macrophages with DCAF deficiency. ( A–B ) The mRNA and protein levels of pro-inflammatory cytokines in primary BMDMs induced by LPS were determined by qRT-PCR and specific ELISAs, respectively. ( C ) The expressions of osteoclasts-specific marker genes were detected by qPCR. ( D ) The efficiency of osteoclast generation from WT and DACF1-deficient BMDMs were measured by TRAP staining. ( E ) The mRNA levels of pro-inflammatory cytokines in WT and DCAF1-deficient osteoclasts induced by LPS were determined by qRT-PCR. The data were presented as fold change as compared to the internal control. In this study, the data were presented as the mean ± SEM. The significances of differences comparisons between the responses group in the two genotypes were determined by a two-tailed Student’s t -test. Three independent experiments were operated to acquire the results. * P < 0.05; ** P < 0.01.

Article Snippet: The primary antibody against DCAF1 was purchased from Abcam (ab1766, Shanghai, China).

Techniques: Quantitative RT-PCR, Marker, Staining, Two Tailed Test

Journal: Journal of Inflammation Research

Article Title: Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus -Induced Osteomyelitis

doi: 10.2147/JIR.S307316

Figure Lengend Snippet: DCAF1 deficiency in macrophages induced severe symptoms in S. aureus -induced osteomyelitis. ( A ) The body weight was monitored every two days after the mice were injected with S. aureus (10 6 CFU) (n = 10). ( B ) After the injection of S. aureus , bacterial burdens were enumerated from the infected femurs and liver on day 14 (n=10). ( C ) The cortical bone loss (mm 3 ) and reactive bone formation (mm 3 ) were determined by μCT three-dimensional analysis. ( D ) After the injection of S. aureus , inflammatory tissues were collected and the mRNA levels of inflammatory cytokines were determined by qRT-PCR on day 8. In this study, the data were shown as the mean ± SEM. ( E ) Peripheral blood was collected and the secretion of these inflammatory cytokines were determined by ELISA on day 8. ( F ) The expressions of RankL and Opg inflammatory tissues were detected by qPCR. Three independent experiments were operated to acquire the results. The significance of data in ( A–E ) was calculated by Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001..

Article Snippet: The primary antibody against DCAF1 was purchased from Abcam (ab1766, Shanghai, China).

Techniques: Injection, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus -Induced Osteomyelitis

doi: 10.2147/JIR.S307316

Figure Lengend Snippet: DCAF1 deficiency induced p38 hyperactivation. ( A–B ) BMDMs were isolated from WT or DCAF1 fl/fl Lyz2 cre/+ mice. Next, LPS was applied to stimulate BMDMs. The phosphorylation and total proteins of NF-kB and MAPKs signal-related markers were determined by Immunoblotting analysis. ( C ) BMDMs were pre-treated with p38 inhibitor (Doramapimod,100 nM) for 2 h prior to incubating with LPS. Next, the mRNA levels of inflammatory cytokines were determined by qRT-PCR. In this study, the data were presented as the mean ± SEM. The data were acquired from three independent experiments. The significance of data in ( C ) was calculated by Student’s t -test. * P < 0.05.

Article Snippet: The primary antibody against DCAF1 was purchased from Abcam (ab1766, Shanghai, China).

Techniques: Isolation, Western Blot, Quantitative RT-PCR

Journal: Pharmaceuticals

Article Title: Morroniside Inhibits Inflammatory Bone Loss through the TRAF6-Mediated NF-κB/MAPK Signalling Pathway

doi: 10.3390/ph16101438

Figure Lengend Snippet: Morroniside inhibits the osteogenic differentiation of BMSCs by inhibiting the TRAF6-mediated NF-κB/MAPK signaling pathway in vivo. ( A ) TRAF6 expression was detected using immunohistochemistry. ( B ) Positive-expression area of TRAF6 in femur tissue. ( C − E ) TRAF6, OCN, and Runx2 mRNA expression levels in tibia tissue. ( F ) The expression of TRAF6-mediated expression of NF-κB/MAPK signaling-pathway-related proteins (p-p65, TRAF6, p-ERK, and p-p38) were detected using Western blotting assays. ( G − I ) The levels of TRAF6, p-p65, and p-p38 were quantified using Image J and normalized to GAPDH. ( J ) The expression of the osteogenic differentiation proteins Runx2 and OCN was detected using Western blotting assays. ( K − M ) The levels of p-Erk, Runx2, and OCN were quantified using Image J and normalized to GAPDH. Data are presented as the mean ± SD of three experiments. ** p < 0.01 versus control group; # p < 0.05, ## p < 0.001 versus LPS group. Abbreviations: ALN, sodium alendronate.

Article Snippet: After washing with BSA and sealing with 5% BSA, BMSCs were incubated with primary mouse anti-NF-κB p65 monoclonal antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) at room temperature for 30 min. Immunoreactivity was assessed using Alexa594-conjugated streptavidin, and BMSCs were counterstained with 10 mg/mL DAPI.

Techniques: In Vivo, Expressing, Immunohistochemistry, Western Blot, Control

Journal: Pharmaceuticals

Article Title: Morroniside Inhibits Inflammatory Bone Loss through the TRAF6-Mediated NF-κB/MAPK Signalling Pathway

doi: 10.3390/ph16101438

Figure Lengend Snippet: Morroniside inhibits the osteogenic differentiation of BMSCs by inhibiting the TRAF6-mediated NF-κB/MAPK signaling pathway in vitro. ( A ) The expression of osteogenic differentiation proteins Runx2 and OCN was detected using Western blotting. ( B , C ) The expression of Runx2 and OCN was quantified using Image J ( https://imagej.nih.gov/ij/ ) and normalized to GAPDH. ( D ) The expression of TRAF-mediated NF-κB/MAPK signaling-pathway-related proteins (p-p65, p65, TRAF6, p-Erk, Erk, p-p38, and p38) was detected using Western blotting assays. ( E − H ) TRAF6, p-p65/p65, p-ERK/ERK, and p-p38/p38 were quantified using Image J and normalized to GAPDH. ( I ) immunofluorescence staining of NF-κB p65 in BMSCs, with counterstaining using DAPI, as indicated (100 μm). Data are presented as mean ± SD of three experiments. ** p < 0.01 versus control group; ## p < 0.01 versus LPS group.

Article Snippet: After washing with BSA and sealing with 5% BSA, BMSCs were incubated with primary mouse anti-NF-κB p65 monoclonal antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) at room temperature for 30 min. Immunoreactivity was assessed using Alexa594-conjugated streptavidin, and BMSCs were counterstained with 10 mg/mL DAPI.

Techniques: In Vitro, Expressing, Western Blot, Immunofluorescence, Staining, Control

Journal: Heliyon

Article Title: Effect of procyanidins on lipid metabolism and inflammation in rats exposed to alcohol and iron

doi: 10.1016/j.heliyon.2020.e04847

Figure Lengend Snippet: Primer sequences.

Article Snippet: The membranes were blocked with TBST containing 10% skimmed milk for 2 h at room temperature and then probed with the primary antibody against NFκB p65 (1:1000) (Santa Cruz Biotechnology, California, US), IκB (1:1000) (Santa Cruz Biotechnology, California, US), β-actin (1:5000) (Easybio, Beijing, China), and Histone H3 (1:5000) (Easybio, Beijing, China) overnight at 4 °C.

Techniques: Sequencing

Journal: Heliyon

Article Title: Effect of procyanidins on lipid metabolism and inflammation in rats exposed to alcohol and iron

doi: 10.1016/j.heliyon.2020.e04847

Figure Lengend Snippet: Effect of procyanidins on NFκB, IκB protein, and mRNA expression levels in rat liver tissue. (A) The above graphical representations show that NFκB mRNA expression is increased in the model Group B but lowered after procyanidin treatment. (B) The IκB mRNA expression levels are increased in the procyanidin treated groups. (C) NFκB protein levels increased in model Group B as compared to the procyanidin groups (D) IκB levels are increased in procyanidin treatment groups. (E) and (F) Western blot analysis was used to detect the expression levels of NFκB p65 and IκB protein in liver tissue. 1: Control Group A; 2: Model Group B; 3: Low dose procyanidin Group C; 4: High dose procyanidin Group D ( ∗ P < 0.05 vs Control; # P < 0.05 vs Model). The full, uncropped images of the western blots are presented in Supplementary Figure 2.

Article Snippet: The membranes were blocked with TBST containing 10% skimmed milk for 2 h at room temperature and then probed with the primary antibody against NFκB p65 (1:1000) (Santa Cruz Biotechnology, California, US), IκB (1:1000) (Santa Cruz Biotechnology, California, US), β-actin (1:5000) (Easybio, Beijing, China), and Histone H3 (1:5000) (Easybio, Beijing, China) overnight at 4 °C.

Techniques: Expressing, Western Blot, Control