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. 2018 Oct 29;38(22):e00188-18. doi: 10.1128/MCB.00188-18

FIG 8.

FIG 8

SP1 is required for Wnt signaling-dependent β-catenin stabilization and regulation of Wnt-responsive genes. (A) Immunofluorescence analysis for expression of β-catenin and SP1 in siControl and siSP1-transfected HCT116 cells (bars, 20 μm). (B) Quantification of nuclear intensities of SP1 and β-catenin (Mann-Whitney test, two tailed, P < 0.0001). (C) Immunoblots for SP1, β-catenin, and TCF7 upon Wnt3A treatment in control and SP1-depleted HEK293 cells. HEK293 cells were transfected with siGFP and siSP1 and treated with Wnt3A after 42 h for 6 h. (D) Immunoblots for SP1 and β-catenin upon CHIR treatment in control and SP1-depleted CRL-1790 cells. CRL-1790 cells were transfected with siGFP and siSP1 and treated with CHIR for 48 h. (E) Immunoblots for FLAG and for SP1 in FLAG–β-catenin-expressing siLuciferase-transfected HEK293 cells and FLAG–β-catenin-expressing siSP1-transfected cells. Data representative of three independent experiments. (F) Immunoblots for MYC and FLAG using FLAG in MYC–β-catenin control and MYC–β-catenin FLAG-SP1 mutant HEK293 cells. RFP was used for transfection control. (G) Immunoblots for HA, axin1, GSK3β, β-TrCP, and β-catenin in control HA-ubiquitin and HA-ubiquitin-FLAG–mutant SP1 HEK293 upon coimmunoprecipitation of β-catenin with ubiquitin, axin1, GSK3β, and β-TrCP. Mouse IgG was used as a negative control. The asterisk denotes IgG heavy chain. (H) HEK293 cells were transfected with FLAG and FLAG mutant SP1. Lysates were subjected to IP using anti-axin1 antibody and rabbit IgG. Coimmunoprecipitated proteins were analyzed by Western blotting. Overexpression of mutant SP1 impedes the recruitment of GSK3β to the destruction complex. (I) Immunoblots for FLAG, axin2, TCF7, and SP1. Stable FLAG–β-catenin HEK293 cells were treated with CHIR in siControl, siSP1, and siSP1 MG132 HEK293 cells. RFP was used as a transfection control. (J) Graphical representation of TOP/FOP reporter activity under CHIR treatment in siControl and siSP1 HEK293 cells. The experiment was performed in triplicates and repeated twice. The data show a significant increase in TOP/FOP reporter activity upon CHIR treatment (P < 0.0001). Knockdown of SP1 significantly reduces TOP/FOP reporter activity (two-way ANOVA, P < 0.0001). Immunoblots of SP1 for the reporter assay are presented below the graph. The actin blot serves as endogenous control. (K) Immunoblots for HA, SP1, axin2, and TCF7 in control and in SP1-depleted HEK293 cells upon S37A β-catenin overexpression in a dose-dependent manner. Tubulin was used as an endogenous control for all immunoblots.