Journal: Molecular Therapy Oncology
Article Title: Allele-specific depletion of GNAQ Q209L via siRNA or an rAAV2-shRNA vector induces selective toxicity in GNAQ Q209L uveal melanoma cells
doi: 10.1016/j.omton.2025.201020
Figure Lengend Snippet: P5 GNAQ Q209L -targeting siRNA preferentially reduces GNAQ Q209L transcripts and YAP transcriptional activity (A) Total GNAQ expression measured via reverse-transcription (RT) quantitative PCR between NTC and P5 siRNA-transfected samples, 24 h post-transfection. Data consist of three experimental replicates and three technical replicates each and are normalized to the housekeeping gene human GAPDH for each sample and to the NTC data. (B) The results of next-generation Amplicon-EZ sequencing (NGS) of a 263-base pair GNAQ cDNA amplicon from NTC and P5 GNAQ Q209L -targeting siRNA-transfected Mel202 cells are depicted as the percentage of total sequences. Each replicate is a single independent experiment, with a total of five independent experiments performed. Total sequence number varied among samples, with an average of 3.9×10 5 (±1.0×10 5 ) total sequences analyzed per sample. B also depicts a representative image of the Partek Flow software analysis, portraying the abundance of recovered GNAQ cDNA sequences from NTC and P5 GNAQ Q209L -targeting siRNA-transfected Mel202 cells. A black box is placed around the mutant nucleotide at position 626 on the GNAQ cDNA sequence. Blue = cytosine; yellow = guanine; red = thymine; green = adenine. (C) The functional impacts of the constitutively active Gα q protein in UVM were measured via the relative abundance of CYR61 and CTGF transcripts, which are both transcriptionally activated via the YAP protein, for both NTC- and P5 GNAQ Q209L -targeting transfected Mel202 cells. Gene expression was normalized to human GAPDH . The data shown consist of three independent experiments with three technical replicates each. Error bars represent ± the SD of the mean. Statistical significance was determined using an unpaired t test. Significance levels are indicated by the following: ∗ p < 0.05.
Article Snippet: B also depicts a representative image of the Partek Flow software analysis, portraying the abundance of recovered GNAQ cDNA sequences from NTC and P5 GNAQ Q209L -targeting siRNA-transfected Mel202 cells.
Techniques: Activity Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Amplification, Sequencing, Software, Mutagenesis, Functional Assay, Gene Expression
