Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Chronic Porphyromonas gingivalis lipopolysaccharide induces adverse myocardial infarction wound healing through activation of CD8 + T cells

doi: 10.1152/ajpheart.00082.2021

Figure Lengend Snippet: Inhibiting the effector CD8+ T-cell phenotype had no effect on lipopolysaccharide (LPS)-induced macrophage recruitment and activation. A: representative image of M-mode short axis echocardiography images. B: immunohistochemistry for neutrophils [polymorphonuclear neutrophils (PMN); n = 4–16/group] and macrophages (Mac-3; n = 4–16/group) in the infarct area indicated an acceleration in macrophage trafficking with LPS + MI that was not effected by LPS + MI + MHCi. LPS + MI + IgG decreased macrophage recruitment compared with LPS + MI. No effect on neutrophils or collagen (picrosirius red staining; PSR; n = 4–16/group) was observed between the groups. Insets show a ×2 digital zoom. Scale bar: 100 μm; C: confocal imaging of the infarct for Mac-3 and phagocytosis receptor, MertK confirmed immunohistochemistry findings. No differences were observed in overall MertK levels between groups. Percent double-positive Mac3 and MertK staining indicated that there was an increase in macrophage-mediated phagocytosis in the LPS + MI group that remained elevated with LPS + MI + MHCi. LPS + MI + IgG administration decreased the number of Mac3+MertK+ cells (n ≥ 4/group). Scale bar: 40 μm; Females are designated by yellow dots and males by blue dots; multiple group comparisons were analyzed by one-way ANOVA with Student–Newman–Keuls posttest; †P < 0.05 vs. MI; ‡P < 0.05 vs. LPS + MI. MI, myocardial infarction.

Article Snippet: Analysis of MertK on macrophages was performed by incubating hearts with primary antibody targeting Mac3 (Cedarlane CL8943AP; 1:100) overnight followed by Alexa Fluor 546 secondary antibody (Invitrogen; 1:400).

Techniques: Activation Assay, Immunohistochemistry, Staining, Imaging

Journal: Cell Reports Medicine

Article Title: Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing

doi: 10.1016/j.xcrm.2023.101129

Figure Lengend Snippet: Phenylpyruvate upregulated NLRP3 palmitoylation by binding to the PPT1 protein (A) Schematic view of NLRP3 (full length) and the location of the S-palmitoylation sites. (B) ABE assay and immunoblot analysis were used to evaluate NLRP3 palmitoylation in BMDMs treated with or without palmitic acid (n = 3). (C) Immunoblot and statistical analysis showing NLRP3 expression in BMDMs treated with 2BP at the indicated concentrations (0, 50, 100, and 150 μM) for 24 h (n = 3). (D) Immunoblot and statistical analysis showing NLRP3 expression in BMDMs treated with 2BP (100 μM) for 0, 8, 16, and 24 h (n = 3). (E) Immunostaining of the location of LAMP2 (indicating lysosomes), PPT1, and NLRP3 in macrophages. The nuclei were stained with Hoechst dye. Scale bar, 10 μm. (F and G) Immunoprecipitation (IP) and immunoblot analysis were used to detect the endogenous NLRP3 and PPT1 association in macrophages (n = 3). (H) ABE assay and immunoblot analysis were used to evaluate NLRP3 palmitoylation in BMDMs with Ppt1 knockdown (n = 3). (I) Mouse embryo fibroblasts (MEFs) were transfected with WT NLRP3 or the indicated NLRP3 mutants for 24 h with or without knockdown of Ppt1. ABE assay and immunoblot analysis showing palmitoylation levels of the indicated NLRP3 mutants (n = 3). (J) ABE assay and immunoblot analysis were used to determine NLRP3 palmitoylation levels in BMDMs treated with increasing phenylpyruvate concentrations (n = 3). (K) Determination of NLRP3 palmitoylation levels in BMDMs treated with 400 μM phenylpyruvate and then transfected with the Ppt1 WT plasmid or the MUT plasmid for 24 h (n = 3). (L) BMDMs were treated with 400 μM phenylpyruvate with or without 100 μM 2BP and then transfected with or without the Ppt1 MUT plasmid. ABE assay and immunoblot analysis determining NLRP3 palmitoylation levels in the indicated cells (n = 3). Data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.

Article Snippet: Rat anti-mouse LAMP2 , Proteintech , Cat# 65052-1-Ig; RRID: AB_2881468.

Techniques: Binding Assay, Western Blot, Expressing, Immunostaining, Staining, Immunoprecipitation, Knockdown, Transfection, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing

doi: 10.1016/j.xcrm.2023.101129

Figure Lengend Snippet:

Article Snippet: Rat anti-mouse LAMP2 , Proteintech , Cat# 65052-1-Ig; RRID: AB_2881468.

Techniques: Recombinant, Transfection, Lysis, cDNA Synthesis, Real-time Polymerase Chain Reaction, BIA-KA, Enzyme-linked Immunosorbent Assay, Software

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate suppressed osteoclast differentiation through autophagy in vitro. ( a ) TRAP staining images after pre-osteoclasts received different treatments for 5 days. ( b ) Monodansylcadaverine staining images after pre-osteoclasts received different treatments for 5 days. ( c ) Representative images of autophagosomes captured with a transmission electron microscope after pre-osteoclasts received different treatments for 5 days. Western blot assay and relative protein expression of the osteoclast markers TRAF6, c-Fos, MMP-9, MMP-14, and CTSK ( d ) and the autophagic proteins Beclin1, ATG5, LAMP2, LC3-I, LC3-II, and p62 ( e ) after pre-osteoclasts received different treatments for 5 days. (n = 3, mean ± SD, ** p < 0.01, *** p < 0.001).

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: In Vitro, Staining, Transmission Assay, Microscopy, Western Blot, Expressing

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate inhibited osteoclast differentiation through NF-κB-pathway-dependent autophagy in vitro. ( a ) TRAP staining images after pre-osteoclasts received different treatments for 5 days. ( b ) Monodansylcadaverine staining images after pre-osteoclasts received different treatments for 5 days. ( c ) Representative images of autophagosomes captured with a transmission electron microscope after pre-osteoclasts received different treatments for 5 days. Western blot assay and relative protein expression of the osteoclast marker CTSK, the proteins central to the NF-κB pathway (p-IKKα/β, IκBα, and p65) ( d ), and the autophagic proteins Beclin-1, ATG5, LAMP2, LC3-I, LC3-II, and p62 ( e ) after pre-osteoclasts were treated with Bay 11-7082 for 5 days. ( f ) Western blot assay and relative protein expression of the proteins central to the NF-κB pathway after pre-osteoclasts were treated with SR for 5 days. (n = 3, mean ± SD, *** p < 0.001).

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: In Vitro, Staining, Transmission Assay, Microscopy, Western Blot, Expressing, Marker

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate regulated the expression of autophagic proteins in rats. ( a ) Immunohistochemical staining images of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on day 7 in different groups. ( b ) The mean optical density (MOD) of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on days 3, 7, and 14 in different groups. (n = 5, mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001). ( c ) Immunofluorescence staining images of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on day 7 in different groups. The white dashed lines indicate the margin of the tooth root.

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence