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. 2022 Apr 27;298(6):101986. doi: 10.1016/j.jbc.2022.101986

Figure 2.

Figure 2

Low-throughput reporter screen identifies CK1γ3 as an activator of WNT signaling.A, HEK293T cells were transiently transfected for 30 h with 100 ng of indicated positive control (β-catenin, NRF2, and NICD) construct or pGUS negative control or the indicated CK1γ construct. About 40 ng TK-Renilla and 40 ng of the indicated Response Element–Luciferase construct was included. Where indicated, cells were treated with (TGFβ 10 ng/ml, TNFα 10 ng/ml, and retinoic acid [RA] 10 ng/ml) for 6 h (TGFβ and TNFα) or 16 h (RA). Inset W. blot demonstrates protein overexpression for the BAR luciferase assay. Each condition was normalized to pGUS control. Error bars represent standard deviation, and statistics are compared to pGUS control. B, BAR luciferase assay from HEK293T B/R (stable BAR-firefly luciferase, TK-Renilla–expressing cell line) cells transfected for 24 h with indicated DNA mass. Statistics are compared to pGUS control. Inset blot is confirmation of overexpression for luciferase assay in B. Dashed line represents cropped blot for removal of redundant untreated sample. C, HEK293T B/R cells were transfected with the indicated construct for 14 h and then treated with Lcell or WNT3A CM for 16 h. All statistics are compared to FLAG-Control Lcell sample. D, HEK293T B/R cells were transfected with the indicated construct for 24 h before luciferase quantitation. Statistics are compared to pGUS control. E, HEK293T B/R cells were transfected with the indicated construct for 14 h and then treated with 10 μM C59 for 18 h. F, WT, LRP5/6 DKO, DVL TKO HEK293T cells transfected with 20 ng BAR-luciferase, 10 ng TK-Renilla, and 70 ng of indicated constructs for 14 h, and then treated with either Lcell CM or WNT3A CM for 18 h. Statistical significance presented is compared with Lcell-treated FLAG-Control cells for each cell type. For all panels: ∗∗∗p < 0.0005, ∗∗p < 0.005, and ∗p < 0.05. All data are plotted as box-and-whisker plots, with the boxes representing the median and the interquartile range. All biological replicates are averaged across three technical replicates. Statistical significance was evaluated by Student’s t test. CK1γ3, casein kinase 1γ3; CM, conditioned media; DKO, double KO; DVL, disheveled; HEK293T, human embryonic kidney 293T cell line; LRP, low-density lipoprotein receptor–related protein; NICD, Notch intracellular domain; NRF2, nuclear factor erythroid 2–related factor 2; TGFβ, transforming growth factor beta; TKO, triple KO; TNFα, tumor necrosis factor alpha; W. blot, Western blot.