Journal: Stem Cell Research & Therapy

Article Title: Efficient bone regeneration of BMP9-stimulated human periodontal ligament stem cells (hPDLSCs) in decellularized bone matrix (DBM) constructs to model maxillofacial intrabony defect repair

doi: 10.1186/s13287-022-03221-3

Figure Lengend Snippet: Isolation and characterization of primary human dental mesenchymal stem cells (hDMSCs). A Morphology of primary human DPSCs, SCAPs and PDLSCs. Primary human DPSCs, SCAPs and PDLSCs were isolated from different dental tissue sections and photographed at the indicated times (3, 5 and 7 days). Representative images are shown. B The expression of the mesenchymal stem cell surface markers CD90 and CD29 and the hematopoietic marker CD45 in isolated primary human DPSCs, SCAPs and PDLSCs was detected by flow cytometry

Article Snippet: The fluorescence-labeled antibodies were CD90-FITC (Sino Biological, China), CD29-FITC (Sino Biological, China) and CD45-FITC (Sino Biological, China) as previously described [ – ].

Techniques: Isolation, Expressing, Marker, Flow Cytometry

Journal: bioRxiv

Article Title: Calpain-2 regulates hypoxia/HIF-induced amoeboid reprogramming and metastasis

doi: 10.1101/2020.01.06.892497

Figure Lengend Snippet: a, In-silico modelling of cell elongation. Individual cell migration in dependence of friction and contractility using a two-dimensional phase field simulation. b, Active and total β1 integrin protein content (2D monolayer culture). D, densitometric analysis (representative Western blot, n=2-3). c, Schematic of cell isolation for harvesting attached (highly adhesive) and detached (weakly adhesive) cells in 2D culture. d, Active and total β1 integrin surface expression (MFI) in 4T1 subpopulations 48 h after treatment (left panel, representative flow cytometry histogram). Ratio of active/total β1 integrin surface expression normalized to vehicle control ratios (right panel). Columns show the median from independent experiments (data points). ** P=0.007, * P=0.03 (unpaired t-test, two-sided). e, Confocal micrographs of active (mAb 9EG7) and total β1 integrin (mAb CD29) expression of 4T1 tumoroids invading into 3D collagen (left panel) and the ratio of active/total β1 integrin per invading 4T1 single cell (right panel, 106 cells). Data show ratios of single cells; horizontal lines the median. Insets, single cell invasion phenotypes (arrowheads). Scale bars, 100 μm (overview), 10 µm (inset). **** P<0.0001 (Mann-Whitney test, two-sided). f, Brightfield micrographs of tumoroids after 72 h of 3D collagen invasion (left panel) and morphology-based single-cell subtypes (right panel) for indicated conditions in the presence or absence of β1 integrin-activating mAb 9EG7. Insets, morphologies of migrating single-cells. Data show the experimental means ± s.d. (5 tumoroids/condition each experiment, n=3). **** P<0.0001, *** P=0.0008, ** P<0.006, * P<0.03 (two-way ANOVA). Abbreviations: E, elongated; P pseudopodal-amoeboid; B, blebbing-amoeboid; N, normoxia; H, hypoxia; V, vehicle (DMSO); D, DMOG.

Article Snippet: The following antibodies and dyes were used: rabbit polyclonal anti-HIF1α (1:1000 WB, Novus Biologicals, 100-479), rabbit polyclonal anti–calpain-2 large subunit (1:1000 WB, Cell Signaling, 2539), chicken polyclonal anti-β-actin (1:1000 WB, Abcam, 13822), rabbit polyclonal anti-β-actin (1:2000 non-reducing WB in 4T1, Cell Signaling, 4967), rabbit polyclonal anti–talin-1 (clone 8d4; 1:200 WB, Sigma-Aldrich, T3287), mouse monoclonal anti-human CD29 (activated β1-integrin, clone HUTS-4; 1:500 WB, MerckMillipore, 2079Z), mouse monoclonal anti-human CD29 (clone 4B4; 1:1000 WB, 0.5-10 μg/ml functional studies, Beckman Coulter, 6603113), rat monoclonal anti-mouse CD29 (primed β1-integrin, clone 9EG7; 1:500 WB, 1:25 FC, 1:50 IF, BD Biosciences, 553715), rabbit monoclonal anti-mouse CD29 (clone EP1041Y; 1:1000 WB, Millipore, 04-1109), rat IgG2a isotype control (Clone R5-95; 1:25 FC, BD Biosciences, 553927), FITC-conjugated Armenian hamster anti-CD29 (clone HMβ1-1; 1:100 IF, 1:50 FC, Biolegend, 102206), FITC-conjugated Armenian hamster IgG (CloneHTK888; 1:20 FC, Biolegend, 400906), rabbit anti-cleaved caspase-3 (1:200 IF, Cell Signaling, 9664), mouse anti-human CD29 (clone TS2/16; 20 μg/ml, BioLegend, 303010), rabbit-on-rodent HRP-polymer (IHC, Biocare Medical, RMR622), rabbit monoclonal anti-cytokeratin 8 (clone EP1628Y; 1:250 IHC, Abcam, 53280), secondary goat anti-rabbit/mouse/chicken antibodies conjugated to horseradish peroxidase (1:10,000 WB, Jackson, 211-032-171/115-0450174/ 103-035-155), secondary goat anti-rat IgG AF488 (1:200 FC, Thermo Fisher Scientific, 11006), secondary goat anti-rat IgG AF647 (1:200 IF, Thermo Fisher Scientific, A21247), secondary goat anti-rabbit IgG AF633 (1:200 IF, Thermo Fisher Scientific, A21071), phalloidin AF488/546/568/633 (1:100 IF, Thermo Fisher Scientific, A12379/A22283/A12380/A22284), DAPI (1:500 IF, Thermo Fisher Scientific, D21490).

Techniques: In Silico, Migration, Western Blot, Cell Isolation, Expressing, Flow Cytometry, MANN-WHITNEY