Journal: Oncogene

Article Title: Cell-to-cell heterogeneity of EWSR1-FLI1 activity determines proliferation/migration choices in Ewing sarcoma cells

doi: 10.1038/onc.2016.498

Figure Lengend Snippet: EWSR1-FLI1 expression is heterogeneous in Ewing cell lines. ( a ) Gene expression profiling at the single cell level by RT-ddPCR of EWSR1-FLI1 and RPLP0 mRNA. Representation of EWSR1-FLI1/RPLP0 ratio in A673, SK-N-MC and TC71 Ewing cell lines; and in shA673-1c, shSK-E17T, shA673-1c+DOX, and shSK-E17T+DOX clones (inducible systems), each dot represents one cell. Bars represent median with interquartile range. The EWSR1-FLI1 low threshold is defined by the upper limit of the interquartile range of DOX-treated cells. DOX-treated/untreated cells were compared using Wilcoxon test (*** P <0.001). See also . ( b ) Protein quantification by immunofluorescence using three-dimensional deconvolution microscopy completed with ImageJ quantification at the single cell level of EWSR1-FLI1 (FLI1) normalized to DNA content (DAPI). Three-dimensional microscopy was performed on Eclipse 90i upright microscope on Nikon Imaging centre (Institut Curie, Paris, France) with a 0.2 μm step for 3D stack and a X100 NA 1.4 oil-immersion objective. The deconvolution of each image stacks was performed automatically using an iterative 7 and algorithm Meinel by PICT-IBiSA imaging facility of the Curie Institut, Paris. Scale bar represents 10 μm ( c ) Representation of EWSR1-FLI1/DAPI ratio in A673 and TC71 Ewing cell lines; and in shA673-1c and shA673-1c+DOX clones (inducible systems), each dot represents one cell. Quantification experiments of the shA673 clone in the −/+ DOX conditions were repeated three times with fully consistent results showing highly significant differences in the quantification of the specific EWSR1-FLI1 staining between both conditions. Bars represent median with interquartile range. The EWSR1-FLI1 low threshold is defined by the upper limit of the interquartile range of shA673-1c+DOX cells. DOX-treated/untreated cells were compared using Wilcoxon test (*** P <0.001). ( d ) Characterization of ICAM1 − and ICAM1 + subpopulations by FACS analysis of A673 cell line stained with APC anti-ICAM1 (1:500, Biolegend, APC anti-CD54 HA58) and ( e ) mRNA expression analysis by RT-qPCR of direct EWSR1-FLI1-targets, data are represented as mean +/−s.e.m. ( f ) Characterization of ICAM1 − and ICAM1 + subpopulations at day 0, 20 and 38 days post-sorting.

Article Snippet: Droplet digital PCR amplification of EWSR1-FLI1 (FAM) and RPLP0 (VIC) was performed in 25 μl reactions by adding the ddPCR Supermix for Probes No UDP (Bio-Rad), the FAM probes and the VIC probes to the cDNA.

Techniques: Expressing, Gene Expression, Clone Assay, Immunofluorescence, Microscopy, Imaging, Staining, Quantitative RT-PCR

Journal: Communications biology

Article Title: Androgen receptor variant shows heterogeneous expression in prostate cancer according to differentiation stage.

doi: 10.1038/s42003-021-02321-9

Figure Lengend Snippet: Fig. 1 Expression of AR-FL, AR-V7, and AR-v567es in CTCs from patients with mCRPC. CTCs were enriched from 29 patients with mCRPC by CD45 negative depletion and processed for quantification of each AR transcript by ddPCR. Heatmap of AR-FL, AR-V7, and AR-v567es expression per patient (not detected; white).

Article Snippet: AR-FL, AR-V7, and AR-v567es transcript quantifications were carried out on a QX200 Droplet Digital PCR (ddPCR) system with automated droplet generation (Bio-Rad Laboratories).

Techniques: Expressing

Journal: Communications biology

Article Title: Androgen receptor variant shows heterogeneous expression in prostate cancer according to differentiation stage.

doi: 10.1038/s42003-021-02321-9

Figure Lengend Snippet: Fig. 3 Expression of AR-FL, AR-V7, and AR-v567es in EpCAMpos and EpCAMneg CTC pools in patients with mCRPC. a Experimental outline: CTCs were enriched from mCRPC patients by CD45 negative depletion followed by single-cell isolation using the CellCelectorTM micromanipulator. b Pools of EpCAMpos/CD45neg or EpCAMneg/CD45neg CTCs were isolated from 10 patients with mCRPC and processed for quantification of each AR transcript by ddPCR. Data were normalized by the number of CTCs in each pool. Heatmap of AR-FL, AR-V7, and AR-v567es expression per patient and per EpCAM status (AR-FL, red; AR-V7, green; AR-v567es, blue; not detected, white).

Article Snippet: AR-FL, AR-V7, and AR-v567es transcript quantifications were carried out on a QX200 Droplet Digital PCR (ddPCR) system with automated droplet generation (Bio-Rad Laboratories).

Techniques: Expressing, Single-cell Isolation, Isolation

Journal: Communications biology

Article Title: Androgen receptor variant shows heterogeneous expression in prostate cancer according to differentiation stage.

doi: 10.1038/s42003-021-02321-9

Figure Lengend Snippet: Fig. 4 Expression of AR-FL, AR-V7, and AR-v567es in single CTCs and organoids from patients with mCRPC and NEPC. a Single CTCs were isolated from three patients with mCRPC (A, B, C) and b two patients with NEPC (D, E) and processed for quantification of each AR transcript by ddPCR. Big circles represent individual patients and small circles represent single CTCs. (For patient A: we have single 81 CTCs, for patient B we have 33 CTCs, for patient C we have 45 CTCs, for patient D we have 18 CTCs and for patient E we have 33 CTCs). The total CTCs isolated from each patient were divided into three equally sized groups and each group was tested for only one of the three transcripts (For example, for patient A, we analyzed 27 single CTC for the expression of AR-FL, 27 single CTCs for the expression of AR-V7 and 27 single CTCs for the expression of AR-v567es). Colored small circles for each transcript represent positive expression, while white circles represent CTCs negative for the respective transcript. The CTCs positive for AR-FL shown in red circles; AR-V7, green circles; and AR-v567es, blue circles. c Prevalence of each transcript across in single CTCs from patients with CRPC vs. NEPC. Statistical significance was determined using two-tailed Fisher Exact test, n.s.; not significant for AR-FL+ and AR-V7+; p = 0.0002 in AR-v567es+ CTCs. d Table shows expression levels for each transcript in four patient-derived NEPC organoids. Data displayed in a doughnut format for three of the four organoids offer a visual display of relative transcript abundance. Transcript color coding as indicated. Data shown are copies per sample normalized to input RNA and internal loading control.

Article Snippet: AR-FL, AR-V7, and AR-v567es transcript quantifications were carried out on a QX200 Droplet Digital PCR (ddPCR) system with automated droplet generation (Bio-Rad Laboratories).

Techniques: Expressing, Isolation, Two Tailed Test, Derivative Assay, Control

Journal: Stem cell research & therapy

Article Title: New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies.

doi: 10.1186/s13287-023-03586-z

Figure Lengend Snippet: Fig. 5 Representative flow cytometry and ddPCR analysis of LSCs and 3T3 cells prepared as the feeder layer. A and B Gating strategy of single-cell LSCs based on forward scatter (FSC) and side scatter (SSC) parameters. Unspecific staining was set to 1% of the acquired cells according to isotype antibody staining. C The majority of LSCs expressed p63α, D but few were positive for the ABCG2 stem cell-associated marker. E Residual 3T3 feeder cells in LSC expanded culture were below the acceptance limit. F 1D plot for two sets of analyses on ddPCR analysis of sample 3 (A1 – D1) and sample 4 (E1 – H1) with mouse assay. G4 is analysis of mouse positive control (DNA from pure 3T3 culture), H11 is negative control (NTC) and H12 is test for possible cross-reactivity with human DNA (human positive control). G Analysis of sample 3 (A3) and sample 4 (B3) with the human assay. H4 is analysis of human positive control (DNA from human whole blood), A11 is NTC and A12 is test for possible cross-reactivity with mouse DNA (mouse positive control)

Article Snippet: Each 20 μL droplet digital polymerase chain reaction (ddPCR) consisted of 10 μL of ddPCR Supermix for Probes (Bio-Rad, USA), 1 μL of Hind III (New England Biolabs, USA) and 4 μL of DNA.

Techniques: Flow Cytometry, Staining, Marker, Mouse Assay, Positive Control, Negative Control