Journal: STAR protocols

Article Title: Enzyme-free isolation of mesenchymal stem cells from decidua basalis of the human placenta.

doi: 10.1016/j.xpro.2023.102498

Figure Lengend Snippet: Figure 2. Immunophenotypic characterization of isolated PL-MSCs The antibodies used were (A) human CD73, (B) human CD90 (C) human CD105 (D) human CD166 (E) human CD34 (F) human HLA-DR and their respective isotype controls. Open histogram indicates background signal, while, shaded histogram represents positive reactivity with the indicated antibodies. Table shows percentage of cells expressing the respective surface markers (n = 3) in mean G SEM.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies PE Mouse Anti-Human CD73 (1:33) BD Pharmingen Catalog No 550257 PE Mouse Anti-Human CD90 (1:33) BD Pharmingen Catalog No 555596 CD105 PE-conjugated Antibody (1:50) R&D Systems Catalog No FAB10971P PE Mouse Anti-Human CD34 (1:50) BD Pharmingen Catalog No 550761 PE Mouse Anti-Human CD166 (1:50) BD Pharmingen Catalog No 559263 FITC Mouse Anti-Human HLA-DR (1:33) BD Pharmingen Catalog No 555811 PE Mouse IgG, k Isotype Control (1:33 as isotype for CD90, 1:800 as isotype for CD34/ CD73/CD166) BD Pharmingen Catalog No 550617 Mouse IgG1 PE-conjugated Antibody (1:50) R&D Systems Catalog No IC002P FITC Mouse IgG2a, k Isotype Control (1:33) BD Pharmingen Catalog No 555573 Vimentin (D21H3) XP Rabbit mAb Antibody (1:150) Cell Signaling Technology Catalog No 5741S Human STRO-1 antibody (1:100) R&D Systems Catalog No MAB1038 Oct-4 (D705Z) Mouse mAb (1:400) Cell Signaling Technology Catalog No 75463S Alexa Fluor 488 goat anti-mouse (1:500) Invitrogen Catalog No A11001 Alexa Fluor 568 goat anti-rabbit (1:500) Invitrogen Catalog No A11011 Alexa Fluor 488 anti-rabbit (1:500) Cell Signaling Technology Catalog No 4412S Biological samples Human placenta N/A N/A Chemicals, peptides, and recombinant proteins 0.9% w/v Saline Kunal Remedies Pvt.

Techniques: Isolation, Expressing

Journal: The Journal of Cell Biology

Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

doi: 10.1083/jcb.200608093

Figure Lengend Snippet: MSCs expressed no VEGFRs. The expression of VEGFR mRNA transcripts was examined by semiquantitative RT-PCR analysis and cell surface protein expression by single-color flow cytometry, using human MSCs, HUVECs as a VEGFR-positive control cell, and HDF as a VEGFR-negative control cell. (A) RNA isolated from MSCs, HUVECs, and HDFs were used to amplify VEGFR1-3, VEGF-A, NP-1, and NP-2 transcripts, with GAPDH as a control, and then resolved by agarose gel. Lane 1, VEGFR1 (99 bp); lane 2, VEGFR2 (81 bp); lane 3, VEGFR3 (87 bp); lane 4, VEGF-A (98 bp); lane 5, GAPDH (71 bp); lane 6, NP-1 (77 bp); lane 7, NP-2 (83 bp). Data are representative of six independent experiments, with two different pairs of primers for VEGFR1-3 used. (B) Flow cytometry using PE-conjugated antibodies. Analysis of VEGFR1-3 is represented by VEGFR1-, VEGFR2-, and VEGFR3-PE expression, respectively, with IgG 1 -PE expression as a control. A representative example of three independent experiments is shown.

Article Snippet: For single-color flow cytometry, MSCs, HUVECs, or HDFs (4×10 6 cells/ml) were incubated with either PE-conjugated anti–human VEGFR1-PE (FAB321P), VEGFR2-PE (FAB357P), or VEGFR3-PE (FAB3492P) antibodies, or control anti–IgG 1 -PE antibody (IC002P; R&D Systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Positive Control, Negative Control, Isolation, Control, Agarose Gel Electrophoresis

Journal: The Journal of Cell Biology

Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

doi: 10.1083/jcb.200608093

Figure Lengend Snippet: VEGF-A stimulated both PDGFRα and PDGFRβ tyrosine phosphorylation. Human phospho-RTK arrays were used to examine VEGF-A–induced RTK phosphorylation levels in MSC lysate samples. Arrays contain phosphotyrosine-positive control spots in each corner, having coordinates (A1, A2), (A23, A24), (F1, F2), (F23, F24), which were assigned a pixel density value of 100, which was used to normalize positive RTK spot pixel density values. Relevant RTK duplicate spot coordinates: PDGFRα = (C7, C8), PDGFRβ = (C9, C10), VEGFR1 = (D9, D10), VEGFR2 = (D11, D12), VEGFR3 = (D13, D14), EGFR = (B1, B2), FGFR3 = (B13, B14), Axl = (B21, B22), EphA7 = (E3, E4). (A) RTK array analysis of control MSC lysate, not stimulated with exogenous growth factor (basal). (B) RTK array analysis of lysates from MSCs transfected with 3 μg scrambled siRNA as a control, siRNA PDGFRα or siRNA PDGFRβ, stimulated using 20 ng/ml VEGF-A 165 in serum-free conditions for 10 min at 37°C. Each array was identically exposed to detection reagents and film. (C) Bar graph representing data from arrays (A and B). Mean pixel density ± the SD of duplicate spots, normalized against duplicate phosphotyrosine-positive control spots = 100. A representative example of two independent experiments is shown for each array analysis. *, P < 0.001 compared with the respective VEGF-A 165 –stimulated scrambled siRNA control.

Article Snippet: For single-color flow cytometry, MSCs, HUVECs, or HDFs (4×10 6 cells/ml) were incubated with either PE-conjugated anti–human VEGFR1-PE (FAB321P), VEGFR2-PE (FAB357P), or VEGFR3-PE (FAB3492P) antibodies, or control anti–IgG 1 -PE antibody (IC002P; R&D Systems).

Techniques: Phospho-proteomics, Positive Control, Control, Transfection

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation

Journal: bioRxiv

Article Title: YAP1 status defines two intrinsic subtypes of LCNEC with distinct molecular features and therapeutic vulnerabilities

doi: 10.1101/2023.12.19.572449

Figure Lengend Snippet: YAP1 status does not predict sensitivity to cisplatin in LCNEC cell lines (A) or cell line/PDX xenograft models (B). C, Treatment of LCNEC cell lines with DMSO, cisplatin, MYF-01-37, XAV-939 and decitabine did not change YAP1, but verteporfin reduced YAP1 levels. D, YAP1 status does not predict response to verteporfin. E, Waterfall plot of drug sensitivity in LCNEC cell lines. A comparison of high YAP1 levels and IC50 values identified several drugs with similar targets, including MEK1/2, CDK4/6, and Src family kinase inhibitors. F, Treatment of YAP1-high LCNEC and SCLC (SW1271) cell lines with trametinib did not change YAP1 levels. G, Tumor growth curves from YAP1-high cell line xenografts H1915 (LCNEC) and SW1271 (SCLC) demonstrate a delay in tumor growth with trametinib treatment.

Article Snippet: One million cells each for H810, H1155, H1755, H1833, H2106, MKL1, HCC2374, HCC4017, HOP92, H1359, H2066, H1770, H2106, H1570, H661, HCC3051, H460, HCC4017, H1299, and H1915 in triplicate were surfaced stained with DLL3 (FAB4315P; R&D Systems), AXL (386202; Biolegends), CD56 (362534), EPCAM (324222) or IgG control and then fixed in 2% PFA.

Techniques: Comparison