Fig 2: Hypoxia increases the expression of HIF-1α and ZEB1 in T24-P cells, and promotes the process of epithelial-mesenchymal transition. Induced hypoxia increased the expression of (A) HIF-1α and (B) ZEB1 in T24-P cells. (C) Western blotting revealed that HIF-1α, ZEB1, N-cadherin and vimentin were upregulated when T24-P cells were grown in hypoxic conditions, but that CK-18 was downregulated. ***P<0.001. HIF-1α, hypoxia inducible factor-1α; ZEB1, zinc-finger E-box-binding homeobox 1; T24-P, T24 parental cells; CK-18, cytokeratin-18.

Fig 3: CSNK2A1 promotes Wnt/β-catenin signalling in KRAS(G12C) mutant lung cancer cells.(a) GSEA analysis showed enrichment for Wnt pathway activation in CSNK2A1-high expressing lung cancer cell lines (CCLE dataset, n = 87, p = 0.008, Permutation test) and tumours from patients with LUAD (TCGA dataset, n = 517, p = 0.014, Permutation test). (b) Schematic depiction of the Wnt signalling pathway which can be influenced by casein kinase 2 (CK2) at different levels. Cytoplasmic-nuclear shuttling of active β-catenin is a hallmark of this pathway and crucial for Wnt-dependent target gene expression such as c-myc and Cyclin D1. (c) Transiently transfected 8xTOPFlash reporter (RLU – relative luminescence units) indicates reduction of basal and MEK inhibitor-induced (100 nM trametinib for 24 hrs) Wnt pathway activity in Calu1 KRAS(G12C) but not in A549 KRAS(G12S) cells upon siRNA-induced CSNK2A1 knockdown. A representative experiment of three independent experiments is shown (n = 3 biological replicates each). d) Protein fractionation indicates reduced levels of Wnt target proteins cMyc, cyclin D1 and Zeb1 as well as reduced nuclear translocation of total and transcriptionally active full length (92 kDa) and low molecular weight (LMW, 56 kDa) phospho-Ser552-β-catenin upon CSNK2A1 knockdown and 24 hrs of trametinib treatment (100 nM) in Calu1 but not A549 cells. “**” and “****” indicate p-values of < 0.01 and 0.0001, respectively (Student t-test).

Fig 4: CHFR is a ZEB1 E3 ligase.a Transiently transfected SFB-ZEB1 and different concentrations of MYC-CHFR in HEK293T cells and immunoblotted with the MYC and FLAG antibodies. b In SUM159 cells, different concentrations of MYC-CHFR were transfected, followed by immunoblotting with the antibodies as indicated. c, d SFB-ZEB1 and MYC-CHFR were transiently transfected into HEK293T cells, and 50 μg ml−1 CHX was added, and samples were collected at the indicated time points and analyzed by western blotting with FLAG and MYC antibodies. e, f In SUM159 stably expressed mock or CHFR cells, 50 μg ml−1 CHX was added, and samples were collected at the indicated time points, followed by immunoblotting with antibodies against ZEB1, MYC, and β-actin. g LM2 was transduced by scramble or sh-CHFR cells, and samples were collected and immunoblotted by ZEB1, CHFR, and β-actin antibodies. h Transiently transferred SFB-ZEB1, MYC-CHFR, or MYC-CHFR∆RING into HEK293T cells. Immunoblotting of MYC and FLAG. i Transiently transfected MYC-CHFR or MYC-CHFR∆RING into SUM159 cells, followed by immunoblotting of ZEB1, MYC, and β-actin. j HEK293T cells were transiently transfected with SFB-ZEB1, MYC-CHFR, or MYC-CHFR ∆RING and added MG132 for 6 h before collecting samples, followed by pulling down with s-S protein beads and immunoblotting of MYC, HA, and FLAG.

Fig 5: CHFR interacts and negatively correlates with ZEB1.a Mass spectrometry identified a partial list of ZEB1 associated proteins. b, c Transiently transfected SFB-ZEB1, MYC-CHFR, and MYC-GFP into HEK293T cells, followed by pulldown with Sepharose beads (s-S beads) (b) or MYC beads (c) and immunoblotting with the antibodies indicated. d The schematic diagram of FHA, RING, and CRD domains of CHFR. Full-length CHFR (FL), CHFR∆FHA, CHFR∆RING, and CHFR∆CRD were transiently transfected into HEK293T cells, followed by pulldown with Sepharose beads (s-S beads) or MYC beads and immunoblotting with the antibodies indicated. e Immunoblotting of ZEB1, CHFR, and β-actin in non-TNBC cell MCF7 and TNBC cells LM2, SUM159, BT549, and MDA-MB-231. f SUM159 cells were treated with 0.5 µg ml−1 nocodazole overnight, the mitotic cells were “shaken off,” and then released into normal medium. Samples were collected at 0 and 5 h after releasing and analyzed by western blotting with ZEB1, CHFR, and β-actin. Cell cycle distribution was marked by Cyclin A and p-H3 (S10).