retinal pigment epithelia rpe low passage cells  (ATCC)

Journal: bioRxiv

Article Title: Dynamic modelling of EWS::FLI1 fluctuations reveals molecular determinants of phenotypic tumor plasticity and prognosis in Ewing sarcoma

doi: 10.1101/2025.04.03.647002

Figure Lengend Snippet: Generation and establishment of EF thresholds. A , Schematic representation of the A673 cell line editing strategy and EF titration using the dTAG system. sgRNA, single-guide RNA; RNP, ribonucleoprotein; mNG, mNeonGreen. B , Western blot analysis of FLI1 protein levels in EF-dTAG clone A2.2 and parental A673 cells treated with the indicated concentrations of dTAG V -1 for 24 hours (left). Tubulin was used as a loading control. Bar graphs depict EF protein levels normalized to Tubulin, presented as mean ± SEM (right; n = 3 independent experiments). P -values were determined using one-way ANOVA with post-hoc Dunnett’s multiple comparisons test. C , D Flow cytometry analysis of mNG fluorescence intensity in EF-dTAG clone A2.2 treated with increasing concentrations of dTAG V -1 for 24 hours ( c ) or following 16 or 24 hours of washout after treatment with 150 nM dTAG V -1 ( D ). Data shown are representative of at least three independent experiments.

Article Snippet: For nucleofection, 1 million low-passage A673 cells were resuspended in nucleofector solution (Cell Line NucleofectorTM Kit V, Cat# VCA-1003) together with 2 µg of either sense or anti-sense ssDNA and 2 µL of 10.8 µM Alt-R Cas9 Electroporation Enhancer (IDT, Cat# 1075916).

Techniques: Titration, Western Blot, Control, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Dynamic modelling of EWS::FLI1 fluctuations reveals molecular determinants of phenotypic tumor plasticity and prognosis in Ewing sarcoma

doi: 10.1101/2025.04.03.647002

Figure Lengend Snippet: Profile of EF-bound chromatin regions and its association with immediate response genes. A , UMAP plots summarizing the results of a genome-wide analysis of EF-bound chromatin regions with CUT&RUN. Each point indicates one peak. A total of 12,370 consensus EF binding peaks were identified, supported by replicates in either parental A673 or A2.2 cells. JASPAR2020 45 human core transcription factor binding motif matches were counted for every peak and the Manhattan metric was used to define peak-to-peak distances. Clustering was done with the Louvain method 75 and eight distinct EF-binding peak clusters were identified. B , For each peak cluster, the total number of peaks, their overlap with promoters or intergenic regions, number of matches to the EF binding motif (matrix profile MA0149.1), width, and CUT&RUN signal intensity (MACS2 reported peak signal rescaled to [0, 100] per sample, replicates averaged were summarized (median and interquartile range shown as points and ranges). C , Transcription factor binding motif match enrichments were calculated for every peak cluster, displaying the top five motifs. Motifs were filtered (one-sided Fisher’s exact test; FDR < 0.01, lower bound of 95% confidence interval of log 2 odds ratio > 2) and ranked by the log 2 odds ratio. D , Density contour plots overlaid on the UMAP plot from panel ( A ), indicating association of peaks with genes responsive to EF degradation. Every peak was linked to its closest response gene (out of the 810 differentially expressed SLAM-seq genes, cp. ) within 150kb distance. E , Cluster-specific enrichments were calculated for each EF-dependent gene cluster (G1-G5) and each EF-binding peak cluster (P1-P8) using two-sided Fisher’s exact test. Circle size and color indicate the FDR and odds ratio, respectively. Significant associations (FDR < 0.05) are highlighted with a solid border.

Article Snippet: For nucleofection, 1 million low-passage A673 cells were resuspended in nucleofector solution (Cell Line NucleofectorTM Kit V, Cat# VCA-1003) together with 2 µg of either sense or anti-sense ssDNA and 2 µL of 10.8 µM Alt-R Cas9 Electroporation Enhancer (IDT, Cat# 1075916).

Techniques: Genome Wide, Binding Assay