Journal: STAR Protocols

Article Title: Generation of transgene-free human induced pluripotent stem cells from erythroblasts in feeder-free conditions

doi: 10.1016/j.xpro.2022.101620

Figure Lengend Snippet: Timeline and representative pictures of hiPSC generation The timeline represents the general procedure to obtain hiPSCs from erythroblasts as well as the timing to obtain these cells. (A–C) Flow cytometry assessment of erythroblast culture before electroporation showing high purity (more than 98%) in CD71+ cells (A), CD36+ cells (B) with combined analysis of double positive cells (C). Red histograms and plot represent the stained cells while grey histograms and plot represent the unstained control. (D–G) Evolution of the morphology during cellular reprogramming showing erythroblasts culture (D – day 11), early reprogramming events leading to the generation of hiPSCs (E – day 18), an emerging hiPSC clone (F – day 25) and finally an established hiPSC clone (or colony) (G – day 32). Scale bars: 100 μm.

Article Snippet: This procedure is performed using CD71 microbeads (Miltenyi) as described in the Manufacturer’s instructions: link .

Techniques: Flow Cytometry, Electroporation, Staining, Control

Journal: STAR Protocols

Article Title: Generation of transgene-free human induced pluripotent stem cells from erythroblasts in feeder-free conditions

doi: 10.1016/j.xpro.2022.101620

Figure Lengend Snippet:

Article Snippet: This procedure is performed using CD71 microbeads (Miltenyi) as described in the Manufacturer’s instructions: link .

Techniques: Recombinant, Electron Microscopy

Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: Design and characterization of cell type specific Wnt signaling enhancing mimetic molecules. ( A ) Scheme of designed molecules. Fragments of RSPO2 spanning the Fu1 and Fu2 domains (action module) were fused the C-terminus of cell type specific or control scFv (targeting module). Specific mutations used are indicated. ( B ) In vitro STF activity of four RSPO proteins. Furin domains of human RSPO1-4 were tested as fusions to an anti-GFP scFv. RSPO2 furin domain alone (N37-E143) was used as a control. Luciferase activity unit is arbitrary (RLU). ( C ) STF activity of RSPO mimetics on HEK293 ( top ) or HuH-7 ( bottom ) in the presence ( left ) or absence ( right ) of an exogenous Wnt source (30% Wnt3a conditioned media). ( D ) Quantitative PCR analysis of ASGR1 , ASGR2 and TFRC gene expression in HEK293, HuH-7 and A431 cells. The signals were relative to ACTB . ( E ) Western Blot analysis on LRP6 receptor and DVL2 phosphorylation in HuH-7 cells. Asterisk indicates a non-specific band that is detected in HuH-7 by the antibody used. The lower bands indicated by the arrow correspond to DVL2 with and without phosphorylation. Tubulin (TUB) is used as the loading control. 10% Wnt3a conditioned media was used as the exogenous Wnt source. Images were cropped for clarity. The uncropped images are presented in Fig. .

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: In Vitro, Activity Assay, Luciferase, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: Confirmation of receptor-specific activation of Wnt signaling. ( A ) STF activity in HEK293 cells transiently transfected with TFR1 (control, top ), ASGR1 ( middle ), and ASGR1 together with ASGR 2 cDNAs ( bottom ), either in the presence ( left ) or absence ( right ) of exogenous Wnt source (30% Wnt3a conditioned media). ( B ) STF activity in A431 cells with TFR1 or ASGR1 overexpression as specified. ( C ) Flow cytometry analysis of cell surface level of FZD proteins. HEK293 cells were transiently transfected with either ZNRF3 alone ( top ) or ASGR1 and ZNRF3 cDNAs ( bottom ), treated with RSPO mimetics as specified at 10 nM, then stained by the pan-FZD antibody 18R5.

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: Activation Assay, Activity Assay, Transfection, Over Expression, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Tissue-targeted R-spondin mimetics for liver regeneration

doi: 10.1038/s41598-020-70912-3

Figure Lengend Snippet: In vivo tissue-specific enhancement of Wnt signaling. ( A ) STF activity of the RSPO mimetic molecules in the appended-IgG format. ( B ) BLI analysis of the ASGR1 antibody (expressed as a Fab) on human and mouse ASGR1. The affinity (Kd) determined by steady-state fitting is indicated. ( C ) Axin2 and ( D ) Ki67 expression in liver ( upper panel ) and small intestine ( lower panel ) were analyzed by quantitative PCR, 48 h after i.p. injection of proteins as specified (n = 8 mice per group). Statistical analysis was performed using 1-way ANOVA: (ns) not significant, ** p < 0.01, **** p < 0.0001. ( E ) Immunofluorescence staining of liver samples for Ki67 and HNF4α from 10 mg/kg treatment groups. White arrows denote some double-positive cells as examples. DAPI was used to stain nuclei.

Article Snippet: For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters ( ASGR1 was clone OHU03658D from GenScript, ASGR2 was in-house cloned isoform d/NP_001188281.1, and TFRC was clone HG11020-UT from SinoBiologicals), then split into 96-well plates (20,000 cells per well) for STF assay 24 h post transfection.

Techniques: In Vivo, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Injection, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence

doi: 10.1038/s41467-020-16337-y

Figure Lengend Snippet: a BCA (protein concentration) of circulating EVs from healthy donors ( h EVs) and P. vivax patients ( Pv EVs) ( n = 10, individual samples). Data show mean ± SD. Two-sided, Mann–Whitney test, * p = 0.0232 (GraphPad). b P. vivax proteins identified by different unique peptides (sequences below the description of corresponding proteins). UniProtKB accession numbers and gene name corresponding to the P. vivax PvP01 strain are shown. c Western blot analysis of Pv EVs obtained from different individual patients (Pv1-7 and Pv10) and human donors (H1 and H3) using anti P. vivax MSP3.1 (upper membrane) and PHISTc (bottom membrane) antibodies. MSP3.1 and PHISTc recombinant truncated-proteins fused to GST, were used as positive controls. Molecular weight in kDaltons (kDa) is shown to the left. M: molecular size marker. Image representative of three independent experiments. d Nanoparticle tracking analysis (NTA) profile (size [nm] versus concentration [particles/mL]) of pooled h EVs and Pv EVs was analysed using NanoSight LM10-12. Table shows mean of three measurement of pooled h EVs and Pv EVs quantified by NTA (particles concentration) and BCA (protein concentration). e Beads-based flow cytometry analysis of pooled h EVs and Pv EVs using six EV markers (CD9, CD63, CD81, GAL3, CD5L and CD71). Data show median fluorescence intensity (MFI) of each antibody and control antibodies (rabbit and mouse-isotype) ± SD (technical replicates, n = 3). Unpaired and two-sided, t -test ** p = 0.0032 (CD63), *** p = 0.0006 (CD81, CD71), **** p < 0.0001 (CD9) (GraphPad). Source data are provided as a Source data file and Supplementary Data , and .

Article Snippet: Further, cells were stained using the following antibodies: CD90-PE/Cy7 [5E10] (Biolegend, Cat#328124) 1/100; CD44-FITC [KM201] (Abcam Cat#ab25340) 1/100; CD54-Alexa Fluor® 488 [HCD54] (Biolegend, Cat#322714) 1/400; CD71-PE [AC102] (Miltenyi Biotec, Cat#130-091-728) 1/400; CD45-PerCP [2D1] (BD Biosciences, Cat#345809) 1/50.

Techniques: Protein Concentration, MANN-WHITNEY, Western Blot, Membrane, Recombinant, Molecular Weight, Marker, Concentration Assay, Flow Cytometry, Fluorescence, Control

Journal: PLoS Genetics

Article Title: MYO5B mutations in pheochromocytoma/paraganglioma promote cancer progression

doi: 10.1371/journal.pgen.1008803

Figure Lengend Snippet: Proliferation with CyQuant NF assay in SK-N-AS clones (A) shown as fold change at 24h, 48h, and 72h compared to mean MYO5B wild type (WT). Graphs show three independent experiments, each run in sextuplicates. Grey = empty vector, black = MYO5B WT , red = p.L587P, blue = p.G1611S, green = p.R1641C. Mean fold change (FC) and significance ( p -value) compared to MYO5B WT within each time point are presented (lower panel). * p <0.05, ** p <0.01, paired t-test. Migration (Oris) in HEK293 clones (B) shown as representable photos from light microscope (5X) of cell area remaining in MYO5B-mutants (p.L587P, p.G1611S, and p.R1641C), MYO5B WT , and empty vector at 24 h compared to the starting wound area. Scale bar shown is 100μm. Graph show percent wound area left after 24 h (mean±SEM, n = 2) in each construct. Transferrin uptake assay in SK-N-AS clones (C), with transferrin stained in red and nuclei stained in blue (DAPI). Pictures are taken with 40X Zeiss Axioscope 2 Plus fluorescence microscope, and small inserts show representative pictures taken with 63X, LSM700 confocal microscope. Scale bars shown are 50 μm and 100μm, respectively. Graphs plot fold change of corrected total cell fluorescence (CTCF) of transferrin (Tf) uptake (from four fields of view taken with 40X), and Western Blot analysis of the Transferrin receptor (TfR) expression (from two independent experiments) in mutants and empty vector compared to MYO5B WT (n = 2). GAPDH was included as a loading control.

Article Snippet: Western blot was performed using antibodies with ECL-detection (Supersignal West Maximum Fempto, Thermo Fisher Scientific) as follows: FLAG-DDK M2 mouse mAb (1:750, #F3165, Sigma Aldrich), GAPDH rabbit Ab (1:500, sc-25778, Santa Cruz Biotechnology), MYO5B Rabbit mAb (1:250, HPA040902, Atlas Antibodies), transferrin receptor (CD71) rabbit mAb (1:500, #13208, Cell Signaling Technology).

Techniques: CyQUANT Assay, Clone Assay, Plasmid Preparation, Migration, Light Microscopy, Construct, Staining, Fluorescence, Microscopy, Western Blot, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD71 , REA902 , 50 , 130-115-028 , FITC (PE) , Miltenyi Biotec.

Techniques: Imaging