Journal: Oncoscience

Article Title: Targeting MD2 in prostate cancer bone metastasis: Mechanistic insights and therapeutic potential

doi: 10.18632/oncoscience.647

Figure Lengend Snippet: ( A ) Detection of HMGB1 in CM from a panel of prostate cancer (CaP) cell lines, including LNCaP control (PCMV vector) and MD2-overexpressing cells (M2 vector). ( B ) HMGB1 levels in CM from DU145 control cells (SCR-siRNA) and MD2-silenced cells (MD2 siRNA). ( C ) Levels of soluble MD2 (sMD2) and HMGB1 in CM from LNCaP cells overexpressing MD2 after treatment with the MD2 inhibitor (MD2-int-1) or vehicle control for 24 h.

Article Snippet: Once tumors were established, as confirmed by IVIS bioluminescence imaging, mice were randomized to receive either vehicle or the selective small molecule MD2 inhibitor, MD2-int-1 (MedChemExpress), for three weeks.

Techniques: Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors

doi: 10.1074/jbc.RA118.006980

Figure Lengend Snippet: Analysis of KDM1A target engagement and proxy markers. A, IC50 determination of KDM1A target engagement by ORY-1001 in MV(4;11) cells treated for 96 h with different doses of ORY-1001 (representative luminescent immunoassay of N = 3, n = 3, mean ± S.D.). B, EC50 determination of H3K4me2 accumulation induced in MV(4;11) cells by 96 h of treatment with ORY-1001 (AlphaLISA; N = 3, n = 2, mean ± S.E. (error bars)). C, EC50 determination of MV(4;11) viability (Alamar Blue assay; representative experiment of N = 5, n = 3, mean ± S.D. (error bars)). D, EC50 determination of LY96 gene expression changes induced in MV(4;11) cells by 96 h of treatment with ORY-1001 (qRT-PCR; N = 3, n = 3, mean ± S.E. (error bars)). Data are normalized to the GAPDH control, and -fold changes were calculated relative to the vehicle control sample.

Article Snippet: Total RNA was extracted using the RNeasy minikit (Qiagen, catalog no. 74106) and cDNA obtained according to standard procedures, using High Capacity RNA-to-cDNA Master Mix (Thermo Fisher Scientific, catalog no. 4390779). qRT-PCR was performed with LightCycler 480 Probes Master (Roche Applied Science, catalog no. 04887301001) and the following predesigned and preoptimized TaqMan Gene Expression Assays from Thermo Fisher Scientific: catalog no. Hs01026734_m1 (FAM dye) for LY96 gene and catalog no. Hs02758991_g1 (FAM dye) for GAPDH gene. qRT-PCR was performed in technical triplicates using the Lightcycler 480 Instrument II (Roche Applied Science).

Techniques: Alamar Blue Assay, Expressing, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Clec7a Worsens Long‐Term Outcomes after Ischemic Stroke by Aggravating Microglia‐Mediated Synapse Elimination

doi: 10.1002/advs.202403064

Figure Lengend Snippet: Clec7a interacts with MD2 in the ischemic brain. A) Immunoprecipitates were subjected to SDS‐PAGE and silver staining. B) Annotated LC–MS/MS spectra of 150‐LGAHLLKIDNSKEFEFIESQTSSHR‐174 from Clec7a and 85‐IELPKRKEVLCHGHDDDYSFCRALK‐109 from MD2 in anti‐Clec7a immunoprecipitates from tMCAO mice. C) Normalized binding curve of Clec7a and MD2. The binding curve yielded a Kd of 2.96 ± 1.11 µ m . The concentration of Clec7a was kept constant at 50 n m while the MD2 concentration was varied from 10 µM to 1.2 nM. n = 3 per group. D) Endogenous Clec7a was coimmunoprecipitated with MD2 after ischemic stroke. The experiments were repeated independently three times. E) The interaction between Clec7a (cyan) and MD2 (pink). The yellow dashed line indicates hydrogen bonding. F) Representative images and analysis of the ischemic penumbra showing the colocalization of MD2 with PSD95, as indicated by the white arrowheads; scale bars, 5 µm. G) Representative images and analysis of the ischemic penumbra showing the colocalization of MD2 with Syn as indicated by the white arrowheads; scale bars, 5 µm. H) Schematic illustrating the in vitro phagocytosis assay in which primary microglia isolated from Clec7a i∆MG or Clec7a fl/fl mice phagocytosed MD2 ko/ko or MD2 wt/wt synaptosomes tagged with pHrodo. I) Representative images of cultured Clec7a i∆MG or Clec7a fl/fl microglia engulfing pHrodo‐conjugated synaptosomes (top: engulfed pHrodo; bottom: merged image of Iba‐1 and pHrodo). Scale bars, 20 µm. Statistics were derived from 18 slices, n = 6 per group. Mice aged 8–10 weeks were used for the experiments shown in this figure. Significance was calculated using either two‐tailed unpaired Student's t ‐test (F and G) or one‐way ANOVA, Tukey's multiple comparisons test (I). Data are presented as mean ± SD. *** p < 0.001.

Article Snippet: Male C57BL/6 mice were purchased from the Shanghai Model Organisms Center, Inc. MD2 ko/ko mice were obtained from Cyagen Biosciences (S‐KO‐03021), Clec7a fl/fl mice were obtained from Gempharmatech (strain no. T009277), and Cx3cr1 CreERT2 mice were obtained from Jackson Labs (IMSR_JAX:02 1160).

Techniques: SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Concentration Assay, In Vitro, Phagocytosis Assay, Isolation, Cell Culture, Derivative Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: The novel diagnostic markers for systemic lupus erythematosus and periodontal disease

doi: 10.3389/fimmu.2025.1614044

Figure Lengend Snippet: The gene-gene interaction network for hub genes were analyzed using GeneMANIA database. Analysis of hub genes related to the function of LY96, EXPH5, RIMS3 and TMEM140. The 20 most frequently changed neighboring genes are shown. Each node represents a gene. The node color represents the possible functions of the respective gene.

Article Snippet: The slices were blocked with fetal bovine serum (FBS) at 37°C for 30 minutes, then, the sections were probed with primary antibodies against TMEM140 (rabbit, 1:100, CSB-PA023710LA01HU, CUSABIO) and LY96 (rabbit, 1:500, 822065, zeobio) overnight at 4 ° C. Next, the sections were re-probed with secondary antibodies to goat anti-rabbit IgG (1:5000, CSB- PA992375 , CUSABIO) for 1 h at room temperature.

Techniques:

Journal: Frontiers in Immunology

Article Title: The novel diagnostic markers for systemic lupus erythematosus and periodontal disease

doi: 10.3389/fimmu.2025.1614044

Figure Lengend Snippet: Expression levels of the hub genes in training and Validation datasets. (A–D) Violin diagrams of LY96 expression levels in GSE61635 , GSE16134 , GSE50772 , GSE10334 ; Comparison was conducted by Wilcoxon rank-sum test. (E–H) Violin diagrams of TMEM140 expression levels in GSE61635 , GSE16134 , GSE50772 , GSE10334 ; Comparison was conducted by Wilcoxon rank-sum test. ***p<0.001.

Article Snippet: The slices were blocked with fetal bovine serum (FBS) at 37°C for 30 minutes, then, the sections were probed with primary antibodies against TMEM140 (rabbit, 1:100, CSB-PA023710LA01HU, CUSABIO) and LY96 (rabbit, 1:500, 822065, zeobio) overnight at 4 ° C. Next, the sections were re-probed with secondary antibodies to goat anti-rabbit IgG (1:5000, CSB- PA992375 , CUSABIO) for 1 h at room temperature.

Techniques: Expressing, Biomarker Discovery, Comparison

Journal: Frontiers in Immunology

Article Title: The novel diagnostic markers for systemic lupus erythematosus and periodontal disease

doi: 10.3389/fimmu.2025.1614044

Figure Lengend Snippet: Expression levels of hub genes in GSE135779 dataset and the expression analysis of hub genes in different cell types within GSE135779 and GSE174609 . (A, B) The expression level of LY96 and TMEM140 in controls and SLE patients in GSE135779 . (C, D) The expression level of LY96 and TMEM140 in 8 clusters of cells in GSE135779 . (E, F) The expression level of LY96 and TMEM140 in 8 clusters of cells in GSE174609 .

Article Snippet: The slices were blocked with fetal bovine serum (FBS) at 37°C for 30 minutes, then, the sections were probed with primary antibodies against TMEM140 (rabbit, 1:100, CSB-PA023710LA01HU, CUSABIO) and LY96 (rabbit, 1:500, 822065, zeobio) overnight at 4 ° C. Next, the sections were re-probed with secondary antibodies to goat anti-rabbit IgG (1:5000, CSB- PA992375 , CUSABIO) for 1 h at room temperature.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: The novel diagnostic markers for systemic lupus erythematosus and periodontal disease

doi: 10.3389/fimmu.2025.1614044

Figure Lengend Snippet: Representative findings on immunohistochemical staining of skin and renal specimens from patients with SLE. LY96 and TMEM140 are upregulated in SLE skin and SLE nephritis tissue. Dotted lines indicate boundaries of dermal-epidermal. The arrows pointed to the regions of pathological alteration. Scale bars = 50 um.

Article Snippet: The slices were blocked with fetal bovine serum (FBS) at 37°C for 30 minutes, then, the sections were probed with primary antibodies against TMEM140 (rabbit, 1:100, CSB-PA023710LA01HU, CUSABIO) and LY96 (rabbit, 1:500, 822065, zeobio) overnight at 4 ° C. Next, the sections were re-probed with secondary antibodies to goat anti-rabbit IgG (1:5000, CSB- PA992375 , CUSABIO) for 1 h at room temperature.

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: The novel diagnostic markers for systemic lupus erythematosus and periodontal disease

doi: 10.3389/fimmu.2025.1614044

Figure Lengend Snippet: Molecular docking pattern of cyclosporine complexed with the two genes (LY96 and TMEM140). (A) LY96. (B) TMEM140.

Article Snippet: The slices were blocked with fetal bovine serum (FBS) at 37°C for 30 minutes, then, the sections were probed with primary antibodies against TMEM140 (rabbit, 1:100, CSB-PA023710LA01HU, CUSABIO) and LY96 (rabbit, 1:500, 822065, zeobio) overnight at 4 ° C. Next, the sections were re-probed with secondary antibodies to goat anti-rabbit IgG (1:5000, CSB- PA992375 , CUSABIO) for 1 h at room temperature.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Cooperation between PU.1 and CAAT/Enhancer-binding Protein ? Is Necessary to Induce the Expression of the MD-2 Gene *

doi: 10.1074/jbc.M109.042580

Figure Lengend Snippet: PU.1 and C/EBPβ are critical co-operative transcription factors for MD-2 expression by HepG2 cells. A, stimulation of the −911 human MD-2 promoter driving the Firefly luciferase gene in HepG2 cells with PMA, 1,25-dihydroxyvitamin D3 (vit D), and incremental doses of IL-6. The −911 human MD-2 promoter activity is increased by PMA but not 1,25-dihydroxyvitamin D treatment (upper panel). A sigmoid dose-response curve was observed after stimulation with incremental doses of IL-6 (lower panel). Data are expressed as Firefly luciferase activity ±1 S.D. (arbitrary units (a.u.)). This represents one experiment in triplicate representative of two independent experiments. B, sequential deletions of the human MD-2 promoter (see Fig. 2 for the restriction enzyme cleavage sites used to generate these mutants; data are expressed as Firefly fluorescence relative to Renilla fluorescence ± 1 S.D., see “Experimental Procedures”). A basal activity of the MD-2 promoter was detected until the −186/+69 deletion. A defective IL-6 inducibility of the MD-2 promoter was detected after the deletion of the promoter region distal to −398. This represents one experiment in triplicate representative of three independent experiments. C, analysis of putative transcription binding sites (see Fig. 2A for details) identified three PU.1 binding sites and one NF-IL-6 in the proximal (−911) MD-2 promoter. Alignment of the proximal MD-2 promoter sequences in human, rabbit, mouse, and torus showed that the two proximal PU.1 sites (positive strand) were conserved among species whether the predicted distal PU.1 binding site (negative strand) was only found in human. Similarly, the NF-IL-6 site (positive strand) was only found in human. D, site-directed mutagenesis of putative transcription factor binding sites (PU.1 and NF-IL-6) in HepG2 cells. These data showed that PU.1 sites are both required for basal MD-2 promoter activity in HepG2 cells. Mutagenesis of NF-IL-6 site indicated that both basal and IL-6-inducible promoter activity were hampered. Data are expressed as Firefly fluorescence relative to Renilla fluorescence ±1 S.D. This experiment is in triplicate and is representative of three independent experiments. E, ChIP assay of PU.1 and C/EBPβ. These data indicated that the PU.1 transcription factor bound to the region containing PU.1 binding sites. PU.1 binding to the MD-2 promoter was enhanced by IL-6 treatment. C/EBPβ bound NF-IL-6 site on MD-2 promoter only after IL-6 treatment of HepG2 cells (C/EBPβ, immunoprecipitation (IP) with anti-C/EBPβ; PU.1, immunoprecipitation with anti-PU.1; IgG, IgG immunoprecipitation with an isotype control). Values were expressed as percent of amplified chromatin obtained after immunoprecipitation with the TF-specific antibody (anti-PU.1 and anti-C/EBPβ; dark column) to input chromatin, normalized to amplified chromatin immunoprecipitated with IgG (white column) ±1 S.D. Data are from one experiment, representative of three. F, PU.1 silencing reduced IL-6-induced MD-2 protein secretion (soluble MD-2) by HepG2 cells. PU.1 small interfering RNA (siRNA) induced a decreased PU.1 content in cell lysates and a decreased amount of secreted soluble MD-2 by HepG2 cells compared with scramble (scr) siRNA. This is one experiment representative of three similar independent experiments.

Article Snippet: Quantitative PCR was performed using the IQ5 thermocycler (Bio-Rad) and the following TaqMan probes ( MD-2 , Hs00209771_m1; PU.1 , Hs00231368_m1; glyceraldehyde-3-phosphate dehydrogenase, Hs99999905_m1) with the universal PCR Mastermix (Applied Biosystems, Foster City, CA).

Techniques: Expressing, Luciferase, Activity Assay, Fluorescence, Binding Assay, Mutagenesis, Immunoprecipitation, Amplification, Small Interfering RNA