recombinant human a2m  (MedChemExpress)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel role for alpha-2-macroglobulin (A2M) as a disease modifying protein in senile osteoporosis

doi: 10.3389/fcell.2023.1294438

Figure Lengend Snippet: A2m is downregulated in Lin - LEPR + SSPCs from elderly vs. mature mice. (A) Heat map of the top 1000 expressed transcripts in Lin − LEPR + SSPCs enriched from the BM of mature (3-month) vs. elderly (24-month) male mice based on RNA-Seq analysis. Colors correspond to per-gene z-score computed across each row. (B) PCA analysis of data from (A) . (C) Gene sets contributing to PC1 and PC2 based on data from (B) . (D) Volcano plot showing Log 2 fold-change (FC) values for DEGs and their corresponding p -values in Lin − LEPR + cells from (A) . (E) Transcripts per million counts (TPM) of A2M in Lin − LEPR + cells from mature vs. elderly mice. Plotted data are mean ± SD and p -value by paired Student’s t-test. (F) Top gene ontology (GO) terms based on p -value identified by GSEA of DEGs in Lin − LEPR + cells from (D) . (G) Most significantly changed Reactome pathways based on DEGs from (D) .

Article Snippet: Alternatively, BM-MSCs were cultured in AIM or OIM alone or supplemented with 0.5 mg recombinant human A2M (rhA2M)/mL (R&D Systems, #1938-PI), cultured for a total of 7 or 5 days, and then stained as above.

Techniques: RNA Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel role for alpha-2-macroglobulin (A2M) as a disease modifying protein in senile osteoporosis

doi: 10.3389/fcell.2023.1294438

Figure Lengend Snippet: A2M gain and loss-of-function skews lineage bifurcation of human BM-MSCs. (A,B) Immuno-blots of BM-MSC cell extracts confirming expression of A2M (A) and LRP1 (B) protein expression. (C) Bar graphs showing cell yield and population doublings of BM-MSCs at 5d post-transfection with a scrambled or A2m -specific siRNA. Data are mean ± SD of triplicates and p -value by unpaired Student’s t-test. Insert is immuno-blot of cell extracts from scrambled or A2m -specific siRNA transfected BM-MSCs. (D) Proliferation index (right panel) calculated based on CFSE dilution in BM-MSCs from (C) via flow cytometry. Histogram (left panel) shows dilution of CFSE at 5d post-transfection. (E,F) Quantification of osteogenic (E) and adipogenic (F) differentiation of BM-MSCs from (C) at 21d and 14d post-induction, respectively. Cell monolayers were stained with AdipoRed™ or Alizarin Red S to quantify fat and mineral deposits, respectively. Plotted data are mean ± SD of replicates ( n = 8) from two independent experiments and p values by unpaired Student’s t-test. (G–J) Adipogenic (G,I) and osteogenic (H,J) differentiation of BM-MSCs following co-culture with trypsin (G,H) or rhA2M protein (I,J) . Photomicrographs show monolayers stained with AdipoRed™ at 7d post induction (G,I) and Alizarin Red S at 5d post induction (H,J) . Plotted data are mean ± SD of technical replicates ( n = 8) and p -values by Student’s t-test.

Article Snippet: Alternatively, BM-MSCs were cultured in AIM or OIM alone or supplemented with 0.5 mg recombinant human A2M (rhA2M)/mL (R&D Systems, #1938-PI), cultured for a total of 7 or 5 days, and then stained as above.

Techniques: Western Blot, Expressing, Transfection, Flow Cytometry, Staining, Co-Culture Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel role for alpha-2-macroglobulin (A2M) as a disease modifying protein in senile osteoporosis

doi: 10.3389/fcell.2023.1294438

Figure Lengend Snippet: Expression profiling of A2m in BM and phenotypic impacts of its loss of function in mice. (A–F) Body composition phenotypic assays of BMD (A) , bone mineral content (B) , bone area (C) , body length (D) , lean (E) and fat mass (F) in 8 female and 7 male 13-week-old homozygous mutant (HOM) A2m tm1b(NCOM)Mfgc mice and 226 female and 209 male wild type mice. Data are from the International Mouse Phenotypic Consortium. p -values determined by unpaired Student’s t-test. (G) Methylation status of 27,578 unique CpG sites in human BM-MSCs from young (≤25 years old) vs. elderly (≥50 years old) donors (GSE17448) and used to quantify the extent of A2M promoter methylation plotted as the intensity ratio between methylated and unmethylated alleles (β-values). Values range from 0 to 1 with 0 being unmethylated. ** p < 0.01 by unpaired Student’s t-test. (H) Color coded visualization of tSNE analysis of single cell RNA-Seq of bone marrow niche cells. ( n = 17,347 cells). P1, P2, P3, and P4 are subpopulation clusters of LEPR+ SSCs wherein P1, P3, P4 represent osteogenic skewed populations, and P2 represents adipogenic-skewed cells. Expression landscape of Lepr , Lrp1 , and A2m are shown. O1, O2, and O3 represent osteoblast and V1 and V2 endothelial cells. Data and visualization taken from publicly available online source of single cell RNA seq analysis of the bone marrow niche cells called niche explorer ( https://compbio.nyumc.org/niche/ ).

Article Snippet: Alternatively, BM-MSCs were cultured in AIM or OIM alone or supplemented with 0.5 mg recombinant human A2M (rhA2M)/mL (R&D Systems, #1938-PI), cultured for a total of 7 or 5 days, and then stained as above.

Techniques: Expressing, Mutagenesis, Methylation, RNA Sequencing