Journal: Cell reports

Article Title: Macrophage Exosomes Resolve Atherosclerosis by Regulating Hematopoiesis and Inflammation via MicroRNA Cargo

doi: 10.1016/j.celrep.2020.107881

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Mouse Macrophages / Monocytes Purified (Clone MOMA-2) (rat IgG2b) , Cedarlane , Cat# CL89154; RRID: AB_10086520.

Techniques: Western Blot, Binding Assay, Flow Cytometry, Purification, Recombinant, SYBR Green Assay, Red Blood Cell Lysis, Reverse Transcription, Blocking Assay, Plasmid Preparation, Sterility, Transfection, Bicinchoninic Acid Protein Assay, Subcloning, RNA Sequencing, Qubit Protein Assay, Cell Culture, Software

Journal: PLoS Genetics

Article Title: Pregnancy-Induced Noncoding RNA ( PINC ) Associates with Polycomb Repressive Complex 2 and Regulates Mammary Epithelial Differentiation

doi: 10.1371/journal.pgen.1002840

Figure Lengend Snippet: (A–C) RNA was isolated from FACs sorted mammary populations including, virgin luminal (VL) and basal (VB) as well as pregnant luminal (PL) and basal (PB). (A) qPCR showed Krt8 expression was enriched in the luminal populations (VL and PL), (B) Krt14 was enriched in the basal populations (VB and PB) and (C) mPINC was enriched in the pregnant luminal population, (D–F) MECs were FACs sorted into luminal and basal populations using CD24 and CD29. The luminal population was selected and further sorted into mature luminal (ML), luminal progenitors (LP), and alveolar progenitors (AP) using CD14 and ckit. (D) FACs dot plots showing CD24 and CD29 (left panel) as well as CD14 and ckit (right panel) from virgin MECs. (E) qPCR showed Ly6a and Wnt4 enriched in the ML population and Elf5 in the LP population thus verifying the purity of each population. (F) mPINC was enriched in the luminal progenitors and alveolar progenitors. Data represent mean ±SD (n = 3). Target genes were normalized to Gapdh .

Article Snippet: Mammary epithelial cells were subsequently resuspended at a density of 1×10 7 cells/ml and stained with anti-mouse CD24 PE (Stem Cell Technologies, 1∶100), anti-CD49f FITC (Stem Cell Technologies, 1∶100), anti-mouse CD24 APC (Biolegend, 1∶100), anti-mouse CD29 Pacific Blue (BioLegend, 1∶100), anti-mouse CD14 FITC (eBiosiences, 1∶80) and anti-mouse ckit PE (Clone ACK4, Cedarlane Laboratories, 1∶50).

Techniques: Isolation, Expressing

Journal: bioRxiv

Article Title: Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis

doi: 10.1101/365809

Figure Lengend Snippet: mMSCs were treated daily with either DMSO or 5-aza-dC for 12 days continually with basal media alone or with the introduction of the adipogenic cocktail (ACT) starting at day 3 A) Timeline diagram of treatment for mMSCs. B) Western blot shows treatment with 5-aza-dC increases Thy1 protein levels and decreases protein levels of the adipogenic marker, Fabp4 versus cells only receiving the adipogenic cocktail (ACT). C-D) RT-qPCR results show that treatment with 5-aza-dC significantly increases Thy1 mRNA and decreases Fabp4 mRNA levels. Relative percentages were normalized to Media DMSO for Thy1 mRNA and ACT DMSO for Fabp4 mRNA levels. E) Immunofluorescent images depict reduced FAPB4 (Green) expression and increased Thy1 (Red) expression in cells receiving 5-aza-dC versus those receiving ACT alone. Cell nuclei are stained with DAPI and depicted in blue. Scale bars in white represent 200μm. *p<0.5, **p<.01, ***p<.001.

Article Snippet: The cells were then incubated with anti-mouse Thy1.2-PE conjugated antibody, 1:500, (BD Biosciences, San Jose, CA) for 1 h on ice.

Techniques: Western Blot, Marker, Quantitative RT-PCR, Expressing, Staining

Journal: bioRxiv

Article Title: Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis

doi: 10.1101/365809

Figure Lengend Snippet: 3T3-L1 cells were treated daily with either DMSO or 5-aza-dC for 7 days continually with basal media alone or with the introduction of the adipogenic cocktail (ACT) starting at day 3. A) Timeline diagram of treatment for 3T3-L1s. B) Western blot showing treatment with 5-aza-dC resulted in an increase in Thy1 total protein levels in both media alone and adipogenic cocktail exposed samples. ACT samples also had a decrease in Fabp4 protein levels when treated with 5-aza-dC, shown by a representative western blot. C-D) RT-qPCR shows treatment with 5-aza-dC increases Thy1 mRNA and decreases Fabp4 mRNA levels in ACT samples. Relative percentages were normalized to 100% to media DMSO for Thy1 mRNA or 100% of ACT DMSO for Fabp4 mRNA levels. *p<05, **p<.01, ****p<.0001

Article Snippet: The cells were then incubated with anti-mouse Thy1.2-PE conjugated antibody, 1:500, (BD Biosciences, San Jose, CA) for 1 h on ice.

Techniques: Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis

doi: 10.1101/365809

Figure Lengend Snippet: 3T3-L1 cells were treated daily with either DMSO or 5-aza-dC for 7 days with the introduction of the adipogenic cocktail (ACT) starting at day 3. A) Representative histogram of Thy1 surface levels measured by flow cytometry using a CD90.2-PE conjugated antibody. B) ACT samples had a decrease in Thy1 mean fluorescence intensity (MFI) that was partially attenuated by 5-aza-dC treatment. C) ACT samples had a decrease in Thy1+ cells compared to Media alone, while ACT cells treated with 5-aza-dC had an increase in the Thy1+ population. D) Immunofluorescent images show decreased staining of Thy1 (Red) with ACT compared to pre-adipocytes (media alone) and increased Fabp4 (Green). Thy1 staining was sustained when ACT samples were treated with 5-aza-dC, along with a decreased staining of Fabp4. Cell nuclei are stained with DAPI and depicted in blue. Scale bars in white represent 200μm. **p<.01, ***p<.001, ****p<.0001

Article Snippet: The cells were then incubated with anti-mouse Thy1.2-PE conjugated antibody, 1:500, (BD Biosciences, San Jose, CA) for 1 h on ice.

Techniques: Flow Cytometry, Fluorescence, Staining

Journal: bioRxiv

Article Title: Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis

doi: 10.1101/365809

Figure Lengend Snippet: 3T3-L1 cells were treated daily with either DMSO or 5-aza-dC for 7 days with the introduction of the adipogenic cocktail (ACT) starting at day 3. A) Schematic of Thy1 gene showing Thy1-CGI1 and CpG sites between exon 1 and 2. B) Average percent DNA methylation of Thy1-CGI1 showed an increase in methylation in ACT cells compared to media alone, while treatment with the methylation inhibitor, 5-aza-dC resulted in a decrease of overall methylation in both groups. C) Individual CpG position sites were measured across Thy1-CGI1 and showed increases in methylation with ACT compared to media alone. D-E) Both media alone and ACT groups had decreases in methylation in all individual CpG sites when treated 5-aza-dC. * p<.05, ***p<.001

Article Snippet: The cells were then incubated with anti-mouse Thy1.2-PE conjugated antibody, 1:500, (BD Biosciences, San Jose, CA) for 1 h on ice.

Techniques: DNA Methylation Assay, Methylation

Journal: bioRxiv

Article Title: Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis

doi: 10.1101/365809

Figure Lengend Snippet: 3T3-L1 cells were pretreated for 24 h with either DMSO or 5-aza-dC, along with continuous daily treatment and then harvested corresponding to days with adipogenic cocktail (ACT) (Day 0= no ACT). A) Average percent DNA methylation of Thy1-CGI1 showed an increase in methylation in normal adipogenesis differentiation process, while treatment with the methylation inhibitor, 5-aza-dC resulted in a decrease in overall methylation. B-C) Individual CpG position sites were measured across Thy1-CGI1 and day 0 showed no relevant changes, while day 6 had a notable decrease in methylation when treated with 5-aza-dC. *= p<.05

Article Snippet: The cells were then incubated with anti-mouse Thy1.2-PE conjugated antibody, 1:500, (BD Biosciences, San Jose, CA) for 1 h on ice.

Techniques: DNA Methylation Assay, Methylation