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: Journal of pharmacy and pharmacology research

Article Title: Fetuin-A Modulates Tumor Growth and Invasion in a Basal-like Triple Negative Breast Cancer Cell line, MDA-MB-468

doi: 10.26502/fjppr.0103

Figure Lengend Snippet: Overexpression of fetuin-A (Fet-A) in MDA-MB-468 TNBC cell line. MDA-MB-468 cells were transfected with either empty vector (EV) or flag-tagged fetuin-A expression vector (FA). Fetuin-A expression level was determined by QT-RTPCR (panel A), Western blot (panel B) and IHC (panel C) as described in .

Article Snippet: Primary mouse monoclonal anti-human fetuin-A/AHSG antibody (R&D Systems, MAB1184) was used at a dilution of 1:500 in Antibody OP Quanto antibody diluent (Thermo Fisher, TA-125-ADQ).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of pharmacy and pharmacology research

Article Title: Fetuin-A Modulates Tumor Growth and Invasion in a Basal-like Triple Negative Breast Cancer Cell line, MDA-MB-468

doi: 10.26502/fjppr.0103

Figure Lengend Snippet: Enhanced invasion capacity in fetuin-A overexpressing MDA—MB-468 cells. The MDA-MB-468-FA invaded through a bed of Matrigel more readily compared to MDA-MB-468-EV when complete medium (CM) was in the lower compartment of the trans-well (*P<0.05); N=6). Both sub-clones did not invade when SFM was in the lower compartments (controls) (**P<0.001; N=6)

Article Snippet: Primary mouse monoclonal anti-human fetuin-A/AHSG antibody (R&D Systems, MAB1184) was used at a dilution of 1:500 in Antibody OP Quanto antibody diluent (Thermo Fisher, TA-125-ADQ).

Techniques: Clone Assay

Journal: Journal of pharmacy and pharmacology research

Article Title: Fetuin-A Modulates Tumor Growth and Invasion in a Basal-like Triple Negative Breast Cancer Cell line, MDA-MB-468

doi: 10.26502/fjppr.0103

Figure Lengend Snippet: Toll like receptor 4 (TLR4) expression in MDA-MB-468 sub-clones. Surface TLR4 expression levels were determined by flow cytometry in MDA-MB-468-EV (upper panels) and MDA-MB-468-FA (lower panels) in the absence and presence of added fetuin-A (FA)

Article Snippet: Primary mouse monoclonal anti-human fetuin-A/AHSG antibody (R&D Systems, MAB1184) was used at a dilution of 1:500 in Antibody OP Quanto antibody diluent (Thermo Fisher, TA-125-ADQ).

Techniques: Expressing, Clone Assay, Flow Cytometry

Journal: Journal of pharmacy and pharmacology research

Article Title: Fetuin-A Modulates Tumor Growth and Invasion in a Basal-like Triple Negative Breast Cancer Cell line, MDA-MB-468

doi: 10.26502/fjppr.0103

Figure Lengend Snippet: Fetuin-A mediates 2-D and 3-D growth of MDA-MB-468 via TLR4 signaling. The cells were seeded (2 × 10 4 cells/well) in attachment (2-D) ( panel A ) or ultra-low attachment (3-D) ( panel B ) 96-well plates in SFM; SFM + CLI-095 (CLI); Fetuin-A (FA); FA + CLI; complete medium (CM); and in CM + CLI. After 6 days of growth, Alamar blue was added to each well, incubated for 2 h and fluorescence measured as described. *P< 0.05; **P< 0.001; ***P<0.0001; N= 4

Article Snippet: Primary mouse monoclonal anti-human fetuin-A/AHSG antibody (R&D Systems, MAB1184) was used at a dilution of 1:500 in Antibody OP Quanto antibody diluent (Thermo Fisher, TA-125-ADQ).

Techniques: Incubation, Fluorescence

Journal: Journal of pharmacy and pharmacology research

Article Title: Fetuin-A Modulates Tumor Growth and Invasion in a Basal-like Triple Negative Breast Cancer Cell line, MDA-MB-468

doi: 10.26502/fjppr.0103

Figure Lengend Snippet: In A , control chambers had only cells in SFM, while experimental chambers had CLI-095 (28 µM) in SFM (upper chambers) of trans-well assay. The bottom chambers had complete medium (CM). In B , controls had CM only in the bottom chambers while experimental chambers had CM depleted of fetuin-A (CM + HA). Number of invading cells was determined as described in .

Article Snippet: Primary mouse monoclonal anti-human fetuin-A/AHSG antibody (R&D Systems, MAB1184) was used at a dilution of 1:500 in Antibody OP Quanto antibody diluent (Thermo Fisher, TA-125-ADQ).

Techniques: Control