Journal: Molecular Therapy. Nucleic Acids

Article Title: A genome-wide CRISPR screen unveils the endosomal maturation protein WDR91 as a promoter of productive ASO activity in melanoma

doi: 10.1016/j.omtn.2025.102577

Figure Lengend Snippet: Functional validation of tcT3 and lnaT3 activity enhancer candidates in 501Mel cells and confocal microscopy experiment (A) Workflow of the experimental design. (B) Setup of the 501Mel cell density assay with ANXA2 positive control. Cell density measurement was performed by crystal violet assay after tcT3 transfection on normal, control or ANXA2 knockdown cells, NT, non-treated. The relative cell density in each condition is normalized to the NT cells. (C) 501Mel cell density assay after tcT3 transfection on normal, control, or candidate’s knockdown cells. Data are presented with a cell density normalized to 100% for each treatment with the siRNA alone, to discard any proliferation bias induced by the siRNA’s effects. (D) 501Mel cell density assay after LNA T3 transfection on normal, control, WDR91 or ANXA2 knockdown cells. Data are presented with a cell density normalized to 100% for each treatment with the siRNA alone, to discard any proliferation bias induced by the siRNA’s effects. (E) Confocal microscopy of 501Mel cells, transfected with either a control or a WDR91 siRNA and then with 50 nM of a FAM-labelled LNA TSB T3 ASO (Blue: Hoechst 33258 - nucleus; Green: FAM-labelled LNA TSB T3 ASO). Scale bar, 10 μm. Experiments were performed in independent biological replicates. Data are presented as mean (SD). Unpaired t tests were performed. The statistical significance is (C): ∗ p = 0.0468 (TADA2B, n = 3); ∗∗ p = 0.0011 (WDR91, n = 4); ∗∗∗∗ p ≤ 0.0001 (ANXA2, n = 5); NS = non-significant ( p = 0.0588); (D): ∗ p = 0.0154 (WDR91, n = 5); ∗∗∗ p = 0.0004 (ANXA2, n = 5).

Article Snippet: siRNAs pools were purchased from Santa Cruz (negative control A sc-37007; WDR91 sc-89398; WBSCR16 sc-89899; FH sc-105377; ELMOD1 sc-77261; TADA2B sc-156169; and ANXA2 sc-270151).

Techniques: Functional Assay, Biomarker Discovery, Activity Assay, Confocal Microscopy, Positive Control, Crystal Violet Assay, Transfection, Control, Knockdown

Journal: Molecular Therapy. Nucleic Acids

Article Title: A genome-wide CRISPR screen unveils the endosomal maturation protein WDR91 as a promoter of productive ASO activity in melanoma

doi: 10.1016/j.omtn.2025.102577

Figure Lengend Snippet: Functional validation of WDR91 in an immortalized DMD muscle cell line KM571 (A) Experimental setup for the double transfection protocol of siRNAs and SSOs in KM571 (B) WDR91 and ANXA2 mRNAs silencing efficiency. (C) WDR91 and ANXA2 mRNAs silencing effects on exon 51 skipping efficiency of the SSOEx51. (D) Experimental setup for the double transfection protocol of siRNAs and a GM against ING2 (GM ING2) in KM571. (E) WDR91 and ANXA2 mRNAs silencing efficiency. (F) WDR91 and ANXA2 mRNAs silencing effects on ING2 knockdown induced by the GM ING2. Results are shown as the average of at least three independent biological replicates. Data are presented as mean (SD). The relative mRNA FC was normalized to the appropriate control condition (Mock or siCTL). Unpaired t test were performed. The statistical significance is ∗∗ p < 0.005; ∗∗∗∗ p < 0.00005; NS = non-significant.

Article Snippet: siRNAs pools were purchased from Santa Cruz (negative control A sc-37007; WDR91 sc-89398; WBSCR16 sc-89899; FH sc-105377; ELMOD1 sc-77261; TADA2B sc-156169; and ANXA2 sc-270151).

Techniques: Functional Assay, Biomarker Discovery, Transfection, Knockdown, Control