Fig. 8

PKMYT1 activates Wnt signaling. a Wnt signaling pathway was enriched by GSEA analysis. b The correlations between PKMYT1 and Wnt signaling factors, including β-catenin, Axin2, c-Myc and Cyclin D1 in TCGA-LUAD , were examined using pearson’s correlation analysis. c Wnt signaling pathway activity was examined using TOPFlash reporter assay in indicated cells after Wnt3a or LiCl (100 μM) treatment, respectively. d Relative expression of indicated transcripts were examined by Real-time RT-PCR upon PKMYT1 knockdown. e Membrane-tethered form of cancer stem cell marker CD133 was examined in indicated cells by flow cytometry assay. Quantification result was indicated (right). f Total and phosphorylated forms of β-catenin proteins were examined by immunoblot with indicated antibodies in A549 cells. β-catenin Phosphorylation level was detected after normalization of endogenous β-catenin proteins, but not β-actin. g Total β-catenin proteins were reduced after PKMYT1 knockdown in A549 cells, which can be reversed by MG132 (20 μM) treatment. h PKMYT1 knockdown inhibited nuclear accumulation of β-catenin proteins in A549 cells by immunoblot. Lamin B: nuclear fraction; GAPDH: cytosol fraction. i-j Lysates of indicated cells treated by cycloheximide (CHX: 100 μg/mL) were examined by immunoblot with indicated antibodies. (j) is the quantification data for (i). k-l Examining the ubiquitination levels of β-catenin proteins upon PKMYT1 knockdown (k) or over-expression (l) by immunoblot using indicated antibodies under different co-transfection conditions. m Co-IP assay was used to detect the association among PKMYT1, β-TrCP1 and β-catenin in HEK-293T cells. Myc-tagged and HA-tagged PKMYT1 were used for different assays. * P < 0.05, ** P < 0.01, *** P < 0.001