Journal: Medicine

Article Title: Effect of connexin 43 in LPS/IL-4-induced macrophage M1/M2 polarization: An observational study

doi: 10.1097/MD.0000000000037811

Figure Lengend Snippet: Expression levels of Cx43 in LPS-induced macrophage polarization. (A) Total protein levels of Cx43 in RAW264.7 macrophages in the control and LPS groups. (B) Statistical analysis of Cx43. Data are presented as the mean ± SEM (n = 3). * P < .05 vs control groups. (C) Expression levels of Cx43 in RAW264.7 macrophages as detected by immunofluorescence (n = 3). (D) Statistical analysis of Cx43. Data are presented as the mean ± SEM (n = 3). * P < .05 vs control groups. (E) Total protein levels of Cx43 in RAW264.7 macrophages in the control, Gap26 and Gap19 groups. (F) Statistical analysis of Cx43. Data are presented as the mean ± SEM (n = 3). * P < .05 vs control groups. # P < .05 vs LPS groups. (G–H) The total expression levels of CD86 in RAW264.7 macrophages in the control, LPS, LPS + Gap19, and LPS + Gap26 groups (n = 3). (I–J) The total expression level of iNOS in RAW264.7 macrophages in the control, LPS, LPS + Gap19, and LPS + Gap26 groups (n = 3). Data are presented as the mean ± SEM (n = 3). * P < .05 vs control groups. # P < .05 vs LPS groups. Cx43 = connexin 43, iNOS = inducible nitric oxide synthase, LPS = lipopolysaccharide, PI = propidium iodide.

Article Snippet: Next, the membranes were incubated with primary mouse or rabbit antibodies specific for the relative proteins at 4°C overnight: Anti‑CD86 (1:1000, ab39075, Abcam, United Kingdom), anti‑Cx43 (1:1000, ab314908, Abcam, United Kingdom), anti‑iNOS (1:1000, ab178945, Abcam, United Kingdom), anti‑CD206 (1:1000, ab64693, Abcam, United Kingdom), anti‑β‑actin (1:1000, ab8226, Abcam, United Kingdom), anti-Beclin-1 (1:1000, ab207612, Abcam, United Kingdom), anti-LC3B (1:1000, ab192890, Abcam, United Kingdom), and anti‑GAPDH (1:1000, ab8245, Abcam, United Kingdom).

Techniques: Expressing, Immunofluorescence

Journal: Medicine

Article Title: Effect of connexin 43 in LPS/IL-4-induced macrophage M1/M2 polarization: An observational study

doi: 10.1097/MD.0000000000037811

Figure Lengend Snippet: Expression of M1-type polarization markers with Cx43 inhibitors. (A–B) The location and expression levels of CD86 in RAW264.7 macrophages pretreated with Gap19 and Gap26 in the control, LPS, LPS + Gap19, and LPS + Gap26 groups (n = 3). (C–D) Flow cytometry was used to detect the expression frequency of CD86 in RAW264.7 macrophages pretreated with Gap19 and Gap26 (n = 3). Data are presented as the mean ± SEM (n = 3). * P < .05 vs control groups; # P < .05 vs LPS groups. Cx43 = connexin 43, LPS = lipopolysaccharide, PE = phycoerythrin.

Article Snippet: Next, the membranes were incubated with primary mouse or rabbit antibodies specific for the relative proteins at 4°C overnight: Anti‑CD86 (1:1000, ab39075, Abcam, United Kingdom), anti‑Cx43 (1:1000, ab314908, Abcam, United Kingdom), anti‑iNOS (1:1000, ab178945, Abcam, United Kingdom), anti‑CD206 (1:1000, ab64693, Abcam, United Kingdom), anti‑β‑actin (1:1000, ab8226, Abcam, United Kingdom), anti-Beclin-1 (1:1000, ab207612, Abcam, United Kingdom), anti-LC3B (1:1000, ab192890, Abcam, United Kingdom), and anti‑GAPDH (1:1000, ab8245, Abcam, United Kingdom).

Techniques: Expressing, Flow Cytometry

Journal: Mediators of Inflammation

Article Title: Systemic Administration of Calea pinnatifida Inhibits Inflammation Induced by Carrageenan in a Murine Model of Pulmonary Neutrophilia

doi: 10.1155/2020/4620251

Figure Lengend Snippet: Effects of isolated compounds obtained from Calea pinnatifida leaves on p65 (p-p65 NF- κ B) and p38 (p-p38 MAPK) phosphorylation. 3,5-di- O - E -caffeoylquinic acid (3,5-diCQA: 2.5 mg/kg) and 4,5-di- O - E -caffeoylquinic acid (4,5-diCQA: 5 mg/kg) administered 0.5 h before pleurisy induction. Sal: negative control group, animals treated only with sterile saline; Cg: positive control group, animals treated only with carrageenan (1%); Dex: animals treated with dexamethasone (0.5 mg/kg) 0.5 h before pleurisy induction; Indo: animals treated with indomethacin (5 mg/kg) 0.5 h before pleurisy induction. Bars indicate the mean ± SEM of six animals. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: For this protocol, a commercial kit containing monoclonal antibodies specific to phosphorylated mouse p65 protein (phospho-p65 NF- κ B (Ser536) Instant One ELISA kit, eBioscience, San Diego, California, USA) were used.

Techniques: Isolation, Phospho-proteomics, Negative Control, Sterility, Saline, Positive Control

Journal: PloS one

Article Title: Antioxidant and anti-inflammatory effects in RAW264.7 macrophages of malvidin, a major red wine polyphenol.

doi: 10.1371/journal.pone.0065355

Figure Lengend Snippet: Figure 3. Effect of malvidin on LPS induced activation of NFkB in RAW 264.7 macrophages. Cells were pretreated with 0–100 mM malvidin (black bars in B) or 0–100 mM trans-resveratrol (gray bars in B) for 30 min as indicated. Activation of NF-kB was assessed by a luciferase (A) or an alkaline phosphatase (B) reporter assay after 1 mg/mL LPS exposion for 24 h. Values are given as means 6 SEM of 4 independent experiments running in 3 parallels. ** p,0.01, *** p,0.001 compared to untreated control, # p,0.05, ## p,0.01, ### p,0.001 compared to LPS alone. a.u.: arbitrary units. doi:10.1371/journal.pone.0065355.g003

Article Snippet: Antibodies against phosphorylation specific extracellular signal regulated kinase (ERK1/2) Thr 183–Tyr185, ERK1/2, phosphorylation specific p38 MAPK Thr180–Gly–Tyr182, p38-MAPK, phosphorylation specific c-Jun N-terminal kinase (JNK), JNK, phosphorylation specific Akt-1/protein kinase B-a Ser473, Akt1, phosphorylation specific glycogen synthase kinase (GSK)-3b Ser9, NF-kB p65 and phosphorylation specific NF-kB p65(Ser536) were purchased from Cell Signalling Technology, Kvalitex Co. (Budapest, Hungary).

Techniques: Activation Assay, Luciferase, Reporter Assay, Control

Journal: PloS one

Article Title: Antioxidant and anti-inflammatory effects in RAW264.7 macrophages of malvidin, a major red wine polyphenol.

doi: 10.1371/journal.pone.0065355

Figure Lengend Snippet: Figure 10. Effect of malvidin on LPS induced pathophysiological changes in RAW 264.7 macrophages. Well documented effects are indicated by solid lines, whereas effects involving yet unidentified mediator(s) or events are represented by dashed line. Lines with pointed end denote activation, whereas lines with a flat end indicate inhibition. Activating or inhibitory effect of malvidin is indicated by a circled + or 2 next to the line, respectively. LPS induces activating phosphorylation, nuclear translocation and DNA binding of NFkB, induction of ROS production, PARP activation, activation of MAPKs, MKP-1 expression, activation of the phosphatidylinositol-3 kinase-Akt pathway and destabilization of the mitochondrial membrane systems. Malvidin attenuates ROS production, mitochondrial destabilization, and activation of PARP and MAPKs. It also augments Akt activation and MKP-1 expression resulting in diminished activation of NFkB. AP-1: activator protein-1; Akt: Protein Kinase B (PKB); Ask1: apoptotic signal regulating kinase; ERK: extracellular signal-regulated kinase; GSK-3b: Glycogen synthase kinase 3 beta; IkB: inhibitors of NF-kB; IKK: inhibitor of NF-kappa B kinase; IRAK1: interleukin-1 receptor-associated kinase-1; IRAK4: interleukin-1 receptor-associated kinase-4; JNK: c-jun N- terminal kinase; LBP: LPS binding protein; LPS: lipopolysaccharide; MKP-1: MAPK phosphatase -1; MyD88: myeloid differentiation primary response gene 88; NEMO: NF-kB essential modifier; NF-kappa B: nuclear factor kappa B; P: phosphorylated; PARP: poly-(ADP-ribose) polymerase; p65: Transcription factor p65 (RelA); p50: NF-KappaB1; PI3K: phosphoinositide 3-kinase; PTEN: phosphatase and tension homolog deleted on chromosome 10; ROS: reactive oxygen species; TAB: TAK1-binding protein; TAK1: transforming growth factor- b-activated kinase 1; TBK1: TANK binding kinase 1; TF: transcription factor; TIR: Toll-interleukin-1 receptor; TIRAP: TIR domain-containing adaptor protein; TLR4: Toll-like receptor 4; TRAF6: TNF receptor- associated factor 6; TRAM: TRIF-related adaptor molecule; TRIF: TIR domain-containing adaptor inducing IFN-b. doi:10.1371/journal.pone.0065355.g010

Article Snippet: Antibodies against phosphorylation specific extracellular signal regulated kinase (ERK1/2) Thr 183–Tyr185, ERK1/2, phosphorylation specific p38 MAPK Thr180–Gly–Tyr182, p38-MAPK, phosphorylation specific c-Jun N-terminal kinase (JNK), JNK, phosphorylation specific Akt-1/protein kinase B-a Ser473, Akt1, phosphorylation specific glycogen synthase kinase (GSK)-3b Ser9, NF-kB p65 and phosphorylation specific NF-kB p65(Ser536) were purchased from Cell Signalling Technology, Kvalitex Co. (Budapest, Hungary).

Techniques: Activation Assay, Inhibition, Phospho-proteomics, Translocation Assay, Binding Assay, Expressing, Membrane