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

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Genome-wide differential genetic profiling characterizes colorectal cancers with genetic instability and specific routes to HLA class I loss and immune escape

doi: 10.1007/s00262-011-1147-7

Figure Lengend Snippet: List of inflammatory- and macrophage markers-related gene-specific probes differentially expressed ( P < 0.05) between MSI and MSS/HLA-positive (control) colorectal cancers

Article Snippet: The following mouse mAbs were used to stain tumour infiltrates: GRT2 (anti-CD45), produced in our lab [ 31 ]; OKT3 (anti-CD3 hybridoma, ATCC, Teddington, UK), OKT8 (anti-CD8 hybridoma, ATCC), anti-CD4 (Clone RPA-T4, Becton–Dickinson Biosciences (BDB), San Jose, CA), anti-perforin (Clone δG9, BD Biosciences), anti-CD64 (Clone 10.1, BD Biosciences), anti-CD206 (macrophage mannose receptor; Clone 19.2, BD Biosciences), anti-CD163 (macrophage scavenger receptor; Clone Ber-Mac3, MBL, Woburn, MA) and anti-CD56 (Clone 123C3, Dako, Barcelona, Spain.

Techniques: Significance Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Genome-wide differential genetic profiling characterizes colorectal cancers with genetic instability and specific routes to HLA class I loss and immune escape

doi: 10.1007/s00262-011-1147-7

Figure Lengend Snippet: Immunohistological results of microarray data validation in CRC tissue samples. a MSI tumour (CRC-6), b MSS/HLA-positive (control) tumour (CRC-19) and c MSS/HLA-negative tumour (CRC-14). A higher infiltration by CD8+ cells was observed in MSI tumour versus control CRC, whereas no CD8+ cells were observed in MSS/HLA-negative CRC. d Elevated expression of CD3 complex ζ-chain in MSI tumour (CRC-1) compared with e Control tumour (CRC-19) and f MSS/HLA-negative sample (CRC-14). Perforin staining in paraffin-embedded samples was higher in g MSI tumours (CRC-6) than in h Control tumours (CRC-16) and i MSS/HLA-negative CRCs (CRC-11). No differences were observed in infiltration by M1 (CD64) and M2 (CD163) macrophages in CRC samples. j, m MSI tumour (CRC-1); k, n MSS/HLA-positive sample (CRC-19); l MSS/HLA-negative CRCs (CRC-11) and o (CRC-12)

Article Snippet: The following mouse mAbs were used to stain tumour infiltrates: GRT2 (anti-CD45), produced in our lab [ 31 ]; OKT3 (anti-CD3 hybridoma, ATCC, Teddington, UK), OKT8 (anti-CD8 hybridoma, ATCC), anti-CD4 (Clone RPA-T4, Becton–Dickinson Biosciences (BDB), San Jose, CA), anti-perforin (Clone δG9, BD Biosciences), anti-CD64 (Clone 10.1, BD Biosciences), anti-CD206 (macrophage mannose receptor; Clone 19.2, BD Biosciences), anti-CD163 (macrophage scavenger receptor; Clone Ber-Mac3, MBL, Woburn, MA) and anti-CD56 (Clone 123C3, Dako, Barcelona, Spain.

Techniques: Microarray, Positive Control, Expressing, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Genome-wide differential genetic profiling characterizes colorectal cancers with genetic instability and specific routes to HLA class I loss and immune escape

doi: 10.1007/s00262-011-1147-7

Figure Lengend Snippet: Summarized results of immunohistochemical study of leucocyte infiltration in CRC samples

Article Snippet: The following mouse mAbs were used to stain tumour infiltrates: GRT2 (anti-CD45), produced in our lab [ 31 ]; OKT3 (anti-CD3 hybridoma, ATCC, Teddington, UK), OKT8 (anti-CD8 hybridoma, ATCC), anti-CD4 (Clone RPA-T4, Becton–Dickinson Biosciences (BDB), San Jose, CA), anti-perforin (Clone δG9, BD Biosciences), anti-CD64 (Clone 10.1, BD Biosciences), anti-CD206 (macrophage mannose receptor; Clone 19.2, BD Biosciences), anti-CD163 (macrophage scavenger receptor; Clone Ber-Mac3, MBL, Woburn, MA) and anti-CD56 (Clone 123C3, Dako, Barcelona, Spain.

Techniques: Immunohistochemical staining

Journal: Journal of Thrombosis and Haemostasis

Article Title: Off‐target effects of oral anticoagulants – vascular effects of vitamin K antagonist and non‐vitamin K antagonist oral anticoagulant dabigatran etexilate

doi: 10.1111/jth.15289

Figure Lengend Snippet: Short‐term oral anticoagulant effects on atherosclerotic lesions. (A) Schematic overview of experimental setup of the animal experiment. (B) Apoe −/− mice were treated for 6 weeks with WTD without or with warfarin or dabigatran, after which animals were sacrificed. Aortic arches of Apoe −/− mice on WTD without or with warfarin or dabigatran showed no significant difference on plaque size at 6 weeks of treatment. (C) MAC3+ cells were significantly increased in lesions of warfarin‐treated mice compared with control ( p < .05) and dabigatran ( p < .01). (D) Six weeks of warfarin treatment significantly increased ucMGP in atherosclerosis lesions compared to control and dabigatran. a, adventitia; d, aorta descendens; h, heart; i, intima; l, lumen; m, media; ucMGP, uncarboxylated matrix Gla protein; WTD, Western type diet

Article Snippet: The following antibodies were used: anti‐uncarboxylated matrix Gla‐protein (1:400; IDS, Boldon, UK), anti‐bone morphogenetic protein 4 (BMP4; 1:50; Santa Cruz Biotechnology, Dallas, TX), anti‐8‐hydroxyguanosine (8OHdG; 1:300; Meridian Life Science, TN), anti‐MAC3 (1:50, Bioscience, Merck KGaA), and anti‐integrin ß 3 (ITGB3 1:100, Sigma‐Aldrich).

Techniques: Western Blot