(A–C) RT-qPCR analysis to monitor (A) efficacy of CRISPR/Cas9-mediated Slfn5 disruption, (B) expression of Slfn5 in response to IFN-β treatment (5,000 IU for 6 hours) in Slfn5 WT cells, and (C) IFN-β–mediated (5,000 IU for 6 hours) induction of indicated murine ISGs in Slfn5-WT and Slfn5-KO cells. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over the corresponding controls, and the graphs represent mean ± SEM of 3 independent experiments. *P < 0.05, ***P < 0.001 by 2-tailed unpaired t test with Welch’s correction (A and B) or 1-tailed unpaired t test with Mann-Whitney test (C). (D) Slfn5-WT and Slfn5-KO KPC1199 cells were plated in 6-well plates and counted on days 1, 2, and 3 after seeding. Data are mean of number of cells ± SEM of 3 independent experiments, each done in duplicate. Two-way repeated-measures ANOVA with Šidák’s multiple-comparison test; **P < 0.01, ****P < 0.0001. (E) Slfn5-WT and Slfn5-KO KPC1199 cells were plated into round-bottom 96-well plates under stem cell–permissive conditions to form 3D spheroids. After 7 days, spheres were imaged using a Cytation 3 cell imaging multi-mode reader to determine cross-sectional area. Data are expressed as percentages of WT parental spheres and represent mean ± SEM of 3 independent experiments, each done in triplicate. Two-tailed unpaired t test with Welch’s correction; *P ≤ 0.05.