p38 mapk inhibitor sb203580  (InvivoGen)

Journal: PLOS Pathogens

Article Title: Trichomonas vaginalis extracellular vesicles activate the NLRP3 inflammasome and TLR3-mediated inflammatory cascades in host cells

doi: 10.1371/journal.ppat.1013216

Figure Lengend Snippet: (A) Ect (2 × 10 6 cells) were treated with the PBS control (Ctrl) or TV-EVs (ATCC 50143) for 2, 4, 6, 8, and 10 hours. Subsequently, whole cell lysates were collected and subjected to western blot analysis. The phosphorylation of PI3K, AKT, NF-κB, p38 MAPK, and ERK was detected using specific antibodies. (B) Quantification of protein bands from (A) using Image J. (C) Immunofluorescence analysis was performed to detect the localization of NF-κB in Ect treated with TV-EVs compared with the PBS-treated control (Ctrl) using an antibody against NF-κB (green), followed by visualization under a confocal microscope. Nuclei were stained with DAPI (blue). Scale bar: 10 µm. (D) Ect were pretreated with the PI3K inhibitor, wortmannin, and then co-cultured with TV-EVs isolated from different strains for 4 hours. The cells were collected and analyzed by western blot analysis using anti-p-PI3K, anti-p-AKT, anti-p-p38 MAPK, and anti-p-ERK antibodies. The protein bands were quantified as mentioned above. (E) Ect (4 × 10 4 cells) were pretreated with the PI3K inhibitor wortmannin and then co-cultured with TV-EVs for 4 hours. Immunofluorescence analysis was performed using an anti-NF-κB antibody (green). Nuclei were stained with DAPI (blue). Scale bar:10 µm. (F) Ect (2 × 10 5 cells/well) were pretreated with pathway inhibitors, including NF-κB (BAY 11-7082), p38 MAPK (SB203580), or ERK (PD98059), followed by treatment with TV-EVs isolated from different strains (ATCC 30236, ATCC 50143, and NDMC5) for 8 hours. The culture supernatants were collected and assayed for IL-6, IL-8, and CXCL1 by ELISA. Ect treated with the PBS control (Ctrl) and TV-EVs isolated from different strains (50143-EV and NDMC5-EV) were served as a negative and positive control, respectively. The protein expression of β-actin was used as the internal control for western blot analysis. The quantitative data are expressed as the means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To inhibit the TLR3, PI3K, p38 MAPK, ERK, and NF-κB pathways, Ect (2 × 10 6 cells) were pretreated with 100 μM TLR3 inhibitor 614310 (Sigma-Aldrich, USA), 10 μM PI3K inhibitor wortmannin (Sigma-Aldrich, USA), 10 μM p38 MAPK inhibitor SB203580 (InvivoGen, USA), 50μM ERK inhibitor PD98059 (Sigma-Aldrich, USA) or 100 μM NF-κB inhibitor BAY 11–7082 (Sigma-Aldrich, USA), respectively, for 1 hour in serum-free medium.

Techniques: Control, Western Blot, Phospho-proteomics, Immunofluorescence, Microscopy, Staining, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay, Positive Control, Expressing

Journal: PLOS Pathogens

Article Title: Trichomonas vaginalis extracellular vesicles activate the NLRP3 inflammasome and TLR3-mediated inflammatory cascades in host cells

doi: 10.1371/journal.ppat.1013216

Figure Lengend Snippet: (A) Ect (2 × 10 6 cells) were treated with the PBS control (Ctrl) or TV-EVs (ATCC 50143) for 2, 4, 6, 8, and 10 hours. Whole cell lysates were collected and analyzed by western blot using an anti-TLR3 antibody. Quantification of protein bands using Image J. (B) Ect were pretreated with the TLR3 inhibitor (614310) or siRNA targeting TLR3 (si-TLR3) and then co-cultured with TV-EVs for 4 hours, and the protein expression levels of TLR3, p-PI3K, p-NF-κB, p-ERK, and p-p38 MAPK were analyzed using specific antibodies. (C) Quantification of protein bands from (B) using Image J. (D) Ect (2 × 10 5 cells/well) were pretreated with the TLR3 inhibitor or si-TLR3, then co-cultured with TV-EVs from different isolates for 8 hours. The culture supernatants were collected and analyzed for IL-6, IL-8, and CXCL1 by ELISA. Ect treated with the PBS control (Ctrl) and TV-EVs isolated from different strains (50143-EV and NDMC5-EV) were served as a negative and positive control, respectively. The data are expressed as the means ± SEM from three independent experiments.* p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To inhibit the TLR3, PI3K, p38 MAPK, ERK, and NF-κB pathways, Ect (2 × 10 6 cells) were pretreated with 100 μM TLR3 inhibitor 614310 (Sigma-Aldrich, USA), 10 μM PI3K inhibitor wortmannin (Sigma-Aldrich, USA), 10 μM p38 MAPK inhibitor SB203580 (InvivoGen, USA), 50μM ERK inhibitor PD98059 (Sigma-Aldrich, USA) or 100 μM NF-κB inhibitor BAY 11–7082 (Sigma-Aldrich, USA), respectively, for 1 hour in serum-free medium.

Techniques: Control, Western Blot, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Positive Control

Journal: PLOS Pathogens

Article Title: Trichomonas vaginalis extracellular vesicles activate the NLRP3 inflammasome and TLR3-mediated inflammatory cascades in host cells

doi: 10.1371/journal.ppat.1013216

Figure Lengend Snippet: In THP-1 macrophages, TV-EVs activate the TLR3-mediated NF-κB/NLRP3 pathway, inducing the secretion of IL-8, MIP-1β, CXCL1, and IL-1β. In Ect, TV-EVs activate PI3K signaling to positively regulate the NF-κB, p38 MAPK, and ERK pathways, inducing the secretion of IL-6, IL-8, and CXCL1. Specifically, TV-EV-induced TLR3 overexpression in Ect upregulates PI3K and NF-κB pathways while downregulating the p38 MAPK and ERK pathways. Furthermore, proteomic analysis identifies that TV-EVs induce overexpression of MICB and TRAF3IP2 in Ect, which are positively regulated by TLR3. MICB enhances PI3K-mediated activation of the NF-κB and ERK pathways, while TRAF3IP2 activates PI3K signaling but suppresses the ERK pathway.

Article Snippet: To inhibit the TLR3, PI3K, p38 MAPK, ERK, and NF-κB pathways, Ect (2 × 10 6 cells) were pretreated with 100 μM TLR3 inhibitor 614310 (Sigma-Aldrich, USA), 10 μM PI3K inhibitor wortmannin (Sigma-Aldrich, USA), 10 μM p38 MAPK inhibitor SB203580 (InvivoGen, USA), 50μM ERK inhibitor PD98059 (Sigma-Aldrich, USA) or 100 μM NF-κB inhibitor BAY 11–7082 (Sigma-Aldrich, USA), respectively, for 1 hour in serum-free medium.

Techniques: Over Expression, Activation Assay

Journal: Aging (Albany NY)

Article Title: Fisetin ameliorates vascular smooth muscle cell calcification via DUSP1-dependent p38 MAPK inhibition

doi: 10.18632/aging.206233

Figure Lengend Snippet: Effects of fisetin on p38 MAPK phosphorylation in VSMCs during pro-calcific conditions. Representative Western blots and normalized phospho-p38 and total p38 MAPK protein abundance (n=5) in HAoSMCs treated for 30 min with control (CTR) or calcification medium (Calc.) without and with 1 μM fisetin (Fis). *(p<0.05) significant vs. control group; †(p<0.05) significant vs. Calc.-treated group.

Article Snippet: HAoSMCs were treated for the indicated times with 10 mM β-glycerophosphate and 1.5 mM CaCl 2 (Sigma Aldrich) as calcification medium [ ], 1 μM or the indicated concentrations of fisetin (stock in DMSO, HY-N0182, MedChemExpress), 10 μM of p38 MAPK inhibitor SB203580 (stock in DMSO, 13067, Cayman Chemical) [ ] and 1 μM of p44/42 MAPK inhibitor LY3214996 (stock in DMSO, HY-101494, MedChemExpress) [ ].

Techniques: Phospho-proteomics, Western Blot, Quantitative Proteomics, Control

Journal: Aging (Albany NY)

Article Title: Fisetin ameliorates vascular smooth muscle cell calcification via DUSP1-dependent p38 MAPK inhibition

doi: 10.18632/aging.206233

Figure Lengend Snippet: Role of p38 MAPK in dual-specificity phosphatase 1 knockdown-induced calcific marker expression in VSMCs. Representative Western blots and normalized phospho-p38 and total p38 MAPK protein abundance (n=4, A ) in HAoSMCs transfected for 24h with negative control (Neg.si) or DUSP1 (DUSP1si) siRNA. Relative mRNA expression (n=8) of DUSP1 ( B ), BMP2 ( C ), CBFA1 ( D ), ALPL ( E ) and CDKN1A ( F ) in HAoSMCs transfected for 72h with negative control (Neg.si) or DUSP1 (DUSP1si) siRNA and treated without and with 10 μM p38 MAPK inhibitor SB203580 (SB). *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. Neg.si group; †(p<0.05), ††(p<0.01), †††(p<0.001) significant vs. DUSP1si group.

Article Snippet: HAoSMCs were treated for the indicated times with 10 mM β-glycerophosphate and 1.5 mM CaCl 2 (Sigma Aldrich) as calcification medium [ ], 1 μM or the indicated concentrations of fisetin (stock in DMSO, HY-N0182, MedChemExpress), 10 μM of p38 MAPK inhibitor SB203580 (stock in DMSO, 13067, Cayman Chemical) [ ] and 1 μM of p44/42 MAPK inhibitor LY3214996 (stock in DMSO, HY-101494, MedChemExpress) [ ].

Techniques: Knockdown, Marker, Expressing, Western Blot, Quantitative Proteomics, Transfection, Negative Control

Journal: Aging (Albany NY)

Article Title: Fisetin ameliorates vascular smooth muscle cell calcification via DUSP1-dependent p38 MAPK inhibition

doi: 10.18632/aging.206233

Figure Lengend Snippet: Schematic illustration of mechanisms involved in the protective effects of fisetin during VSMC calcification. Mineral stress with disturbed phosphate and calcium homeostasis may trigger activation of pro-calcific signaling including phosphorylation and activation of p38 MAPK in VSMCs, which leads to a pro-calcific environment causing vascular calcification. Fisetin, a natural flavonol, induces the dual-specificity phosphatase 1 (DUSP1). Fisetin thereby inactivates p38 MAPK signaling through DUSP1 and inhibits further pro-calcific signaling and calcification of VSMCs.

Article Snippet: HAoSMCs were treated for the indicated times with 10 mM β-glycerophosphate and 1.5 mM CaCl 2 (Sigma Aldrich) as calcification medium [ ], 1 μM or the indicated concentrations of fisetin (stock in DMSO, HY-N0182, MedChemExpress), 10 μM of p38 MAPK inhibitor SB203580 (stock in DMSO, 13067, Cayman Chemical) [ ] and 1 μM of p44/42 MAPK inhibitor LY3214996 (stock in DMSO, HY-101494, MedChemExpress) [ ].

Techniques: Activation Assay, Phospho-proteomics