immunosorbent assay elisa  (BioVendor Instruments)

Journal: Nature

Article Title: Fetal hepatocytes protect the HSPC genome via fetuin-A

doi: 10.1038/s41586-024-08307-x

Figure Lengend Snippet: a , Representative fluorescence images of FetuA and TLR4 on E12.5 fetal liver HSPCs with or without FetuA treatment. Scale bars, 10 µm and 5 µm. The white squares represent the areas enlarged in the bottom row. b , Representative fluorescence images of TLR4 and MYD88 on HSPCs cultured with or without FetuA and TLR4 antibodies. Scale bars, 10 µm and 5 µm. c , Western blots of bZIPs (Fosl1, JunB and Jun), their phosphorylated (p) forms and BLM in E12.5 fetal liver HSPCs cultured with or without FetuA. d , ATAC-seq analysis of E12.5 fetal liver HSPCs with or without FetuA treatment, showing the differential opening and closing peaks and R-loop regulatory genes Smarcc1 , Fanci and Blm . e , DNA-binding factors whose motifs were enriched in the opening regions of panel d . f , Volcano plot showing the differential gene expression. The horizontal dashed line shows the P value threshold ( P = 0.05). The blue and red spots show the downregulated or upregulated genes, respectively, in FetuA-cultured HSPCs. g , Western blots (top) and signal intensity (bottom) of BLM in E12.5 fetal liver Lin – HSPCs cultured with or without FetuA and the bZIP inhibitor SR11032 (data shown as mean ± s.d.). h , i , Representative fluorescence images ( h ) and nuclear signal intensity ( i ) of dRNH1 in E12.5 fetal liver HSPCs cultured with or without FetuA and the BLM inhibitor ML216. The nuclear regions are circled by dotted lines. Scale bars, 10 µm and 5 µm. j , k , Representative comet-tail DNA images ( j ) and percentages ( k ) of Eto induced in E12.5 fetal liver HSPCs cultured with or without FetuA and the BLM inhibitor ML216. Scale bars, 20 µm. l , A working model of the FetuA–TLR4 pathway in protecting the HSPC genome. RPA, replication protein A; RNAP, RNA polymerase. n = 3 independent experiments ( a – k ). The medians (red dashed lines) and quartiles (black dashed lines) are shown ( i , k ). Two-sided binomial test ( e ), two-sided Wald test ( f ), unpaired one-sided Student’s t -test ( g ) and unpaired two-sided Student’s t -test ( i , k ) were used.

Article Snippet: After treatment with or without FetuA (100 μg ml −1 ; 50093-M08H, SinoBiological) and the bZIP inhibitor SR11032 (2 mM; HY-15870, MedChemExpress) for 6 h, mouse Lin – haematopoietic cells were lysed and blotted with BLM antibody.

Techniques: Fluorescence, Cell Culture, Western Blot, Binding Assay, Expressing

Journal: Nature

Article Title: Fetal hepatocytes protect the HSPC genome via fetuin-A

doi: 10.1038/s41586-024-08307-x

Figure Lengend Snippet: a , Scatter-plots showing the consistency of ATAC-seq replicates for each group ( n = 3). b , Fragment size distribution of ATAC-seq data ( n = 3). c , ATAC-seq signals of Blm , Fanci and Smarcc1 in Integrative Genomics Viewer are shown; the red boxes indicate the peaks where the bZIP motifs are located ( n = 3). d , Heatmaps showing the Cut & Tag peak signal intensities ( n = 2). e , Cut & Tag sequencing signals of Jun, JunB and Fosl1 on the Blm gene from the Integrative Genomics Viewer are shown ( n = 2). Cut & Tag for Jun binding to the Blm promoter. f , Western-blots analysis of the BLM protein in LSK cells from E12.5 FL and E16.5 FL ( n = 2). g, h , Western-blots and relative signal intensity of the BLM protein in Lin – cells cultured with or without FetuA at different time points ( n = 3; mean ± s.d.). The n represents independent experiments. Statistical tests: unpaired two-sided Student’s t-test ( h ).

Article Snippet: After treatment with or without FetuA (100 μg ml −1 ; 50093-M08H, SinoBiological) and the bZIP inhibitor SR11032 (2 mM; HY-15870, MedChemExpress) for 6 h, mouse Lin – haematopoietic cells were lysed and blotted with BLM antibody.

Techniques: Sequencing, Binding Assay, Western Blot, Cell Culture

Journal: Nature

Article Title: Fetal hepatocytes protect the HSPC genome via fetuin-A

doi: 10.1038/s41586-024-08307-x

Figure Lengend Snippet: a, b , Representative flow cytometry images and the proportions of each cell cycle stage in HSPCs from E12.5 PL, E12.5 FL and E16.5 FL cells stained with Hoechst 33342 and PyroninY (E12.5 PL, E16.5 FL, n = 3; E12.5 FL, n = 6; S/G2/M phase was compared). c, d , Representative flow cytometry images and the proportions of HSPCs in each cell cycle stage at E12.5 PL, E12.5 FL and E16.5 FL after 5-ethynyl-2’-deoxyuridine (EdU) treatment ( n = 3 per group; S phase was compared). e, f , Representative flow cytometry images and Ki67-positive cell proportions in HSCs and MPPs from E12.5PL, E12.5FL and E16.5FL ( n = 3 per group). g, h , Representative flow cytometry images and the ethyluridine (EU)-positive fraction of HSPCs from E12.5 PL, E12.5 FL and E16.5 FL after EU-treatment (E12.5 PL, E12.5 FL, n = 3; E16.5 FL, n = 6; EU + population was compared). i, j , Representative fluorescence images and nuclear signal intensity of p-RPA in HSPCs from E12.5 PL, E12.5 FL and E16.5 FL ( n = 3). The medians (red dashed lines) and quartiles (black dashed lines) are shown. Scale bar, 10 µm. k, l , Representative fluorescence images and nuclear signal intensity of p-RPA in E12.5 FL-HSPCs treated with or without FetuA ( n = 3). The medians (red dashed lines) and quartiles (black dashed lines) are shown. Scale bar, 10 µm. m, n , Representative flow cytometry images and the proportion of each cell cycle stage in HSPCs cultured with or without FetuA and the BLM inhibitor ML216 ( n = 3). The mean ± s.d. is shown ( b, d, f, h, n ). The n represents individual samples from 3 independent experiments ( a-h ) and independent experiments ( i-n ). Statistical tests: unpaired two-sided Student’s t-test ( b, d, f, h, j, l, n ).

Article Snippet: After treatment with or without FetuA (100 μg ml −1 ; 50093-M08H, SinoBiological) and the bZIP inhibitor SR11032 (2 mM; HY-15870, MedChemExpress) for 6 h, mouse Lin – haematopoietic cells were lysed and blotted with BLM antibody.

Techniques: Flow Cytometry, Staining, Fluorescence, Cell Culture

Journal: Nature Communications

Article Title: Physical and chemical niche of human growth plate for polarized bone development

doi: 10.1038/s41467-025-62711-z

Figure Lengend Snippet: A Experimental procedure for LC-MS/MS. The GP samples from 3 biological replicates were divided into five groups: epiphysis (E), GP-epiphysis (GP-E), GP, GP-metaphysis (GP-M) and metaphysis (M) tissue; B , C Volcano plot showing proteins with differential expression between interfaces and GP tissue/bone tissue (epiphysis and metaphysis). Dotted lines indicate FDR < 0.05 and |log₂FC | > 1. Two-sided moderated t-tests were performed using the limma package with Benjamini–Hochberg correction; SPP1, AHSG, ENPP1 and ALPL have been highlighted in the volcano plot; D Representative images of GP samples immunostained for AHSG (magenta) /calcium (cyan) and SPP1(magenta) /calcium (cyan) at the GP-epiphysis interface; E Representative images of GP samples immunostained for AHSG/calcium, SPP1/calcium, ENPP1 /calcium and ALPL/calcium at the GP-metaphysis interface, scale bar = 20 μm, ( n = 3/group from 3 donors in ( D , E ); F Summary diagram of the macromolecule-based regulatory mechanism of GP-guided polarized long bone elongation; G Representative cryo-TEM and SAED images of prepared ACP after 1 d and 42 d of reaction at 37 °C, scale bar = 200 nm; H Representative images of STEM images, EDS mapping images of Ca, P, N, O and SAED pattern of the ACP particles after 1 d and 42 d of reaction at 37 °C, scale bar = 100 nm/50 nm, ( n = 3 per time point). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: SPP1 (Santa, sc-21742), AHSG (Proteintech, 66094-1-Ig), ALPL (Proteintech, 11187-1-AP), ENPPI (Abcam, ab223268).

Techniques: Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics

Journal: Nature Communications

Article Title: Physical and chemical niche of human growth plate for polarized bone development

doi: 10.1038/s41467-025-62711-z

Figure Lengend Snippet: The inhibitory proteins, SPP1 and AHSG, form a line of defense against mineralization in GP-epiphysis interface. When combining with the mineralization-promoting enzymes, ENPP1 and ALPL at the GP-metaphysis interface, these proteins accelerate the formation of ACP and its transformation to HAp and facilitating the progression of the mineralization front.

Article Snippet: Primary antibodies used were: SPP1 (Santa, sc-21742), AHSG (Proteintech, 66094-1-Ig), ALPL (Proteintech, 11187-1-AP), ENPPI (Abcam, ab223268).

Techniques: Transformation Assay