Fig. 2: Arrdc3 was validated as a TRP53 regulated gene and loss of Arrdc3 provided a competitive advantage to Eμ-Myc lymphoma cells after TRP53 activation.
From: Genome-wide CRISPR screening identifies a role for ARRDC3 in TRP53-mediated responses
A qRT-PCR analysis to assess the levels of Arrdc3 expression in AF47A Eμ-Myc lymphoma cells after treatment for 6 or 24 h with nutlin-3a (10 μM) or etoposide (40 ng/mL), relative to expression in DMSO-treated control cells. Additionally, expression of Pmaip1, Bbc3, and Cdkn1a were assessed as controls, and the expression of each gene was also examined in Trp53KO cells to assess the level of reliance of Arrdc3 expression on TRP53 activity. Gapdh was used as a housekeeping gene. Each treatment was performed three times, and the qRT-PCR was undertaken with 3 technical replicates. Error bars represent standard error of the mean. Statistical tests were one-way ANOVAs with Šídák’s multiple comparisons tests, performed to compare the indicated samples. Note that due to variation between biological replicates, some differences are not statistically significant despite obvious trends. B Cell cycle assay using the AF47A Eμ-Myc lymphoma cell line with CRISPR/Cas9-mediated knockout of Arrdc3 compared to the NTsgRNA transduced control lymphoma line, treated with 5 μM nutlin-3a for 6 h. Each treatment was performed 3 times. Data are presented as means +/− standard deviation. Statistical analyses are given in Table S1. C Cell death assays using the AF47A Eμ-Myc lymphoma cell line edited using CRISPR/Cas9 to ablate Arrdc3, treated for 24 h with one of three apoptosis-inducing drugs: nutlin-3a and etoposide that act via TP53/TRP53, or thapsigargin that functions in a TP53/TRP53-independent manner. Each cell line treatment was performed 3 times, with 2 technical replicates each time. Data are presented as means +/− standard deviation. D Cell competition assays using the 560 Eμ-Myc lymphoma cell line with CRISPR/Cas9-mediated knockout derivative lines of either Arrdc3 or Trp53, or a non-targeting sgRNA (NTsgRNA). Mixed cell populations (1:1) were treated with sub-optimal doses of either nutlin-3a (1.5 µM, ~IC20) or thapsigargin (1 nM, ~IC90) over a period of 14 days. Each competition assay was performed twice for each cell line, with 2 technical replicates each time, with a representative example being shown. Data are presented as means +/− standard deviation.
