Journal: Current protocols in molecular biology

Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo

doi: 10.1002/cpmb.46

Figure Lengend Snippet: gRNAs are inserted into AAV plasmid containing U6 promoters and cardiac troponin T (cTNT) promoter-driven Cre recombinase. Annealed oligos corresponding to the targeting portions of gRNA1 and gRNA2 are cloned between AarI and SapI sites, respectively. The entire construct is contained within AAV inverted terminal repeat sequences, allowing for packaging into AAV capsids. Examples of gRNAs designed against Gata4 are shown.

Article Snippet: AAV-U6gRNA1-U6gRNA2-TnT-Cre Vector (Addgene #87682) gRNA sequences from Basic Protocol 1/DNA oligos Thermocycler AarI enzyme and buffer (ThermoFisher ER1581) SapI enzyme and buffer (NEB R0569L) 37°C incubator 37°C shaking incubator Agarose (GeneMate E-3120-500) 50× TAE (Boston BioProducts BM250) Ethidium bromide (Sigma E7637) DNA loading dye (NEB B7024S) Gel electrophoresis chamber UV transilluminator (365 nm) Gel purification kit (Invitrogen K210012) Quick ligation kit (NEB M2200L) Chemically competent cells such as ThermoFisher MAX Efficiency Stbl2 Competent Cells (Cat# 10268019).

Techniques: Plasmid Preparation, Clone Assay, Construct

Journal: Scientific Reports

Article Title: Activation of GPR35 protects against cerebral ischemia by recruiting monocyte-derived macrophages

doi: 10.1038/s41598-020-66417-8

Figure Lengend Snippet: Pamoic acid (PA) activates Akt and p38 MAPK signaling in the ischemic brain. ( A ) PA treatment increased the concentration of pAkt at 24 hours after the MCAO, One-Way ANOVA, F (2/18) = 9.515, **P = 0.0039 (Bonferroni multiple comparison tests), values are min to max. n = 7. ( B ) PA treatment increases the pGSK-3β at 24 h after the MCAO, one-Way ANOVA, F (2/11) = 4.652, *p = 0.0332 (Bonferroni multiple comparison test) values are min to max, (n for Sham = 4, n for MCAO = 4, n for MCAO + PA = 6), ( C ) PA treatment increased the p38 MAPK at 24 h after the MCAO, One-Way ANOVA, F (2/19) = 9.089, *P = 0.0221 (Bonferroni multiple comparison test), values are min to max, (n for Sham = 4, n for MCAO = 4, n for MCAO + PA = 6). ( D ) PA treatment increased the activity of p38 MAPK 48 h after the MCAO, One-Way ANOVA, F (2/9) = 7.598, *P = 0.0290 (Bonferroni multiple comparison tests), values are min to max, n = 4. ( E,F ) The effect of PA was lost when combining with Triciribine, One-Way ANOVA, F (2/17) = 19.21, **P = 0.0029, ***P = 0.0001 (Bonferroni multiple comparison tests), values are means ± s.e.m, (n for MCAO = 7, n for MCAO + PA = 8, MCAO + PA + Triciribine =5).

Article Snippet: The ELISA of p38 MAPK and GSK 3β was performed using the InstantOne p38 MAPK ELISA kit (Invitrogen, ref. no. 85-86023-11) and InstantOne GSK 3β ELISA kit (Invitrogen, ref. no. 8586173-11).

Techniques: Concentration Assay, Activity Assay

Journal: PloS one

Article Title: A novel chromone derivative with anti-inflammatory property via inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway.

doi: 10.1371/journal.pone.0037168

Figure Lengend Snippet: Figure 3. Effects of DCO-6 on the LPS-induced activation of MAPKs and NF-kB in RAW264.7 cells. Cells were treated with various concentrations of DCO-6 in the absence or presence of LPS for 6 h. (A–B) The protein levels of total and phosphorylated p38, JNK, ERK and IKKa, IKKb, IkBa were determined at least three times, and the representative data are shown. (C) Cytoplasmic and nuclear proteins were extracted and assayed by immunoblotting analysis. Expressions of b-actin and LaminB1 were shown as loading controls. Bands from (A–C) were analyzed by densitometry. Quantitative data are shown. *P,0.05 vs LPS control. (D) Nuclear proteins were extracted and assayed by EMSA. A 75-fold excess of unlabelled oligonucleotide probe and mutant probe were used as controls. doi:10.1371/journal.pone.0037168.g003

Article Snippet: Precipitation were washed twice and lysed in the lysis buffer containing Triton X-100 as nucleoprotein. p38 MAP Kinase Activity Assay Cells were stimulated by 500 ng/ml LPS for 6 h and the protein was collected. p38 MAP Kinase activity was detected by p38 MAP Kinase Assay Kit (Cell Signaling Technology).

Techniques: Activation Assay, Western Blot, Control, Mutagenesis

Journal: PloS one

Article Title: A novel chromone derivative with anti-inflammatory property via inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway.

doi: 10.1371/journal.pone.0037168

Figure Lengend Snippet: Figure 4. Effects of DCO-6 on p38 MAPK activation induced by different TLR ligands in RAW264.7 cells. (A) Cells were treated with DCO-6 in the absence or presence of indicated TLR ligands for 6 h. Whole cell lysates were prepared for Western blotting analysis. The protein levels of total and phosphorylated p38 were determined at least three times, and the representative data are shown. (B) Cells were stimulated by 500 ng/ml LPS for 6 h and the protein was collected. Phosphorylated p38 MAPK was immunoprecipitated. The immune complexes were used for testing the effects of DCO-6 and SB203580 on kinase activities. Representative data are shown. Bands from (A–B) were analyzed by densitometry. Quantitative data are shown. *P,0.05 vs control. doi:10.1371/journal.pone.0037168.g004

Article Snippet: Precipitation were washed twice and lysed in the lysis buffer containing Triton X-100 as nucleoprotein. p38 MAP Kinase Activity Assay Cells were stimulated by 500 ng/ml LPS for 6 h and the protein was collected. p38 MAP Kinase activity was detected by p38 MAP Kinase Assay Kit (Cell Signaling Technology).

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Control

Journal: PloS one

Article Title: A novel chromone derivative with anti-inflammatory property via inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway.

doi: 10.1371/journal.pone.0037168

Figure Lengend Snippet: Figure 5. Effects of DCO-6 on the production of intracellular ROS and the formation of TRAF6-ASK1 complex in RAW264.7 cells. (A) Cells were incubated in the absence or presence of indicated TLR ligands for 6 h. Intracellular ROS production was detected by DCF fluorescence using flow cytometry. (B) Cells were treated with various concentrations of DCO-6 in the absence or presence of LPS. Intracellular ROS production was detected as mentioned above. Data are shown as means 6 S.D. of three independent experiments. *P,0.05 vs LPS control. (C) Cells were treated with various concentrations of DCO-6 in the absence or presence of H2O2 for 6 h. Whole cell lysates were prepared for Western blotting analysis. The protein levels of total and phosphorylated p38 were determined at least three times, and representative data are shown. (D) The interaction between TRAF6 and ASK1 was measured by coimmunoprecipitation assay. Representative data are shown. doi:10.1371/journal.pone.0037168.g005

Article Snippet: Precipitation were washed twice and lysed in the lysis buffer containing Triton X-100 as nucleoprotein. p38 MAP Kinase Activity Assay Cells were stimulated by 500 ng/ml LPS for 6 h and the protein was collected. p38 MAP Kinase activity was detected by p38 MAP Kinase Assay Kit (Cell Signaling Technology).

Techniques: Incubation, Fluorescence, Flow Cytometry, Control, Western Blot, Co-Immunoprecipitation Assay

Journal: PloS one

Article Title: A novel chromone derivative with anti-inflammatory property via inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway.

doi: 10.1371/journal.pone.0037168

Figure Lengend Snippet: Figure 6. Protective effect of DCO-6 against LPS-induced septic shock in mice. BALB/c mice were administered LPS (10 mg/kg) and DCO-6 (10 or 20 mg/kg) or vehicle (olive oil) intraperitoneally. (A) Survival rate. (B) Serum cytokine levels of IL-1b and IL-6 were measured by ELISA 3 h after LPS injection. Data are shown as means 6 S.D. of three independent experiments. n = 10. *P,0.05 vs model control. (C) Peritoneal macrophages were isolated from the mice 3 h after LPS injection. Whole cell lysates were prepared, and the protein levels of total and phosphorylated p38 was detected by Western blotting analysis. Traces shown are representative of three independent experiments. Bands from (C) were analyzed by densitometry. Quantitative data are shown. *P,0.05 vs model control. (D) Peritoneal macrophages were isolated from the mice 3 h after LPS injection and incubated with DCFH-DA. DCF fluorescence distribution was detected by flow cytrometry. Data were analyzed by Cell Quest software. *P,0.05 vs model control. doi:10.1371/journal.pone.0037168.g006

Article Snippet: Precipitation were washed twice and lysed in the lysis buffer containing Triton X-100 as nucleoprotein. p38 MAP Kinase Activity Assay Cells were stimulated by 500 ng/ml LPS for 6 h and the protein was collected. p38 MAP Kinase activity was detected by p38 MAP Kinase Assay Kit (Cell Signaling Technology).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Control, Isolation, Western Blot, Incubation, Fluorescence, Software

Journal: Journal of advanced research

Article Title: SAP deficiency aggravates periodontitis possibly via C5a-C5aR signaling-mediated defective macrophage phagocytosis of Porphyromonas gingivalis.

doi: 10.1016/j.jare.2022.10.003

Figure Lengend Snippet: Fig. 2. SAP deficiency aggravates periodontitis and inflammation. (a) Micro-CT 3D reconstruction images of the maxilla of wildtype and SAP-KO mice. (b) CEJ-ABC distance was analyzed from Micro-CT images (n = 5). (c) Histological images of H&E and TRAP staining, and representative CD34 and CD45 immunohistochemistry images of the periodontium in wildtype and SAP-KO periodontitis mice. Quantitative analysis of CEJ-ABC distance (d) and osteoclasts (e) from histological images (n = 5). Quantitative analysis of CD34 (f) and CD45 (g) positive cells from immunohistochemistry images (n = 3). (h-j) Inflammatory cytokine mRNA expression in the periodontium of wildtype and SAP-KO periodontitis mice (n = 3). (k-m) Protein level expression of inflammatory cytokines detected by ELISA (n = 5). Red dot line, CEJ level; Black dot line, ABC level; Blue double arrow line, CEJ-ABC distance. Yellow ‘‘R”, tooth root; Yellow ‘‘B”, alveolar bone. Black arrow, osteoclasts. Data are presented as mean ± SD. The significant difference among the groups, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The protein level of SAP in the human gingival lysate was analyzed using the human SAP ELISA kit (Cusabio, Wuhan, China) and in mouse periodontal tissue lysate, liver lysate, and serum was analyzed using the mouse SAP ELISA kit (SEB539Mu, Cloud-Clone Corp, Wuhan, China).

Techniques: Micro-CT, Staining, Immunohistochemistry, Expressing, Enzyme-linked Immunosorbent Assay