Fig. 2: OTULIN mediates genotoxic Wnt activation by inhibiting linear ubiquitination. | Nature Communications

Fig. 2: OTULIN mediates genotoxic Wnt activation by inhibiting linear ubiquitination.

From: ABL1-dependent OTULIN phosphorylation promotes genotoxic Wnt/β-catenin activation to enhance drug resistance in breast cancers

Fig. 2

a Immunoblotting analysis of β-catenin in parental and OTULIN knockout MDA-MB-231 cells treated with Dox (2 µg/ml) or Wnt3a (20 ng/ml) for 24 h. b TOPFlash assay of parental and OTULIN knockout MDA-MB-231 cells treated with Wnt3a (20 ng/ml), Dox (2 µg/ml), CPT-11 (10 µM) or IR (10 Gy) for 24 h. n = 3 independent experiments. p1 = 4.39E−06, p2 = 3.55E−05, p3 = 1.56E−05, p4 = 5.48E−05, p5 = 8.73E−06, p6 = 5.22E−05, p7 = 1.59E−05, p8 = 1.86E−05, p9 = 3.29E−05. c qPCR analysis of AXIN2 mRNA level in parental and OTULIN knockout MDA-MB-231 cells treated with Dox (2 µg/ml) or Wnt3a (20 ng/ml) for 24 h. n = 3 independent experiments. p1 = 0.000188, p2 = 0.000503, p3 = 0.000117, p4 = 0.00413. d Immunoblotting analysis of β-catenin expression in parental and LRP5/6-DKO HEK293A cells with or without HA-OTULIN overexpression. e Immunoblotting analysis of β-catenin expression in parental and DVL1/2/3-TKO HEK293T cells with or without HA-OTULIN overexpression. TOPFlash assay (f) and immunoblotting analysis of β-catenin (g) in MDA-MB-231 cells transfected with HA-OTULIN wildtype (WT) or catalytic activity defect C129S mutant (CS). n = 3 independent experiments in f. p1 = 0.000783, p2 = 0.000497. h TOPFlash assay of HEK293T cells transfected with LUBAC components HOIP and HOIL. n = 3 independent experiments. p1 = 0.00670. i Immunoblotting analysis of β-catenin in MDA-MB-231 cells transfected with HOIP and HOIL and treated with or without LUBAC inhibitor Gliotoxin (1 µM) for 24 h. j Immunoblotting analysis of β-catenin expression in parental and HOIP knockout HEK293T cells. k TOPFlash assay of parental, HOIP knockout, or HOIP-KO HEK293T cells reconstituted with HOIP WT or C885S mutant. n = 3 independent experiments. p1 = 4.06E−05, p2 = 9.49E−06. l TOPFlash assay of MDA-MB-231 cells transfected with HOIP and HOIL and treated with Dox (2 µg/ml) for 24 h. n = 3 independent experiments. p1 = 0.000297, p2 = 0.000717, p3 = 0.000462. The statistical analysis in b, c, f, h, k and l was performed by two-sided unpaired t-test and p values are indicated as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Data are presented as Mean±SD. Source data are provided as a Source Data file.

Back to article page