Figure 3 from Uncovering Novel lncRNAs Linked to Melanoma Growth and Migration with CRISPR Inhibition Screening
Year: 2025
Type: other
Abstract: CRISPRi screen lncRNA hit functional characterization. A, Flow cytometry–based dotmplot showing GFP and mCherry signal as proxy for sgRNA and dCas9 expression (left). 7-AAD fluorescence–dependent cell cycle analysis in 501mel-dCas9-KRAB (top) and WM136a-dCas9-KRAB (bottom) cell lines upon sgBDNF-AS, sgXLOC_030781, and sgGMDS-AS1 CRISPRi knockdown (rigth). Note that the top panel is plotted linear and bottom logarithmic because of better cell cycle–gating parameters. B, Cell cycle phase quantification based on A in percent of cells (n = 2). C, PARP cleavage apoptosis assay performed by Western blotting upon sgBDNF-AS, sgXLOC_030781, and sgGMDS-AS1 knockdown in 501meld-Cas9-KRAB and WM1361a-dCas9-KRAB Representative Western blots (top). GAPDH detection was used as a loading control; densitometric quantification of apoptosis as cleaved PARP/GAPDH protein ratio (bottom; average of n = 2). sgROSA served as negative control and etoposide apoptosis inducer as positive control. D, Re-analysis of 7-AAD cell cycle assay staining using exact same gating parameters for sub-G1 cellular fraction indicative of apoptosis. E, Transwell cell migration assay. Fluorescence photomicrographs show calcein-stained migrated cells of sgBDNF-AS, sgGMDS-AS1, and sgXLOC_030781 knocked-down 501mel-dCas9-KRAB cells vs. sgROSA negative control and quantification of average sum count cell numbers (n = 2).
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4412112356