Fig 2: GRA18 promotes Ccl17, Ccl22 and Ccl24 chemokines expression in a β-catenin-dependent fashion.(A) Schematic diagram of gRNAs targeting the Ctnnb1 locus. The Protospacer Adjacent Motif (PAM) sequence is lined and highlighted in red; the targeting sequences is shown in green. Bi-directional arrow indicates the Cas9 cleavage site. DNA sequences from the wild-type and mutated RAW264.7-derived cell lines were analyzed by DNA sequencing; the identified deletions are indicated with (-). No mutations in control samples were observed. (B) Immunoblot analysis of β-catenin in parental RAW264.7 and the Ctnnb1-/- mutant confirmed the absence of β-catenin expression. As positive controls, cells were treated with 3 mM of BIO or 2 mM of CHIR GSK3 inhibitors for 12 hr. TBP was used as loading control. (C–E) RAW264.7 (WT) and β-catenin-deficient (Ctnnb1-/-) RAW264.7-derived cell lines were transfected with mCherry vector control (pcDNA-mCherry-HF) or the FH-GRA18II expression vector (pcDNA-FH-GRA18FL). At 18 hr after transfection, cells were either (C) fixed for IFA using anti-HA (red) and anti-β-catenin (green) antibodies or (D) cells were harvested and analyzed by immunoblot using the indicated antibodies. Anti-HA was used to detect FH-GRA18 and mCherry-HF. In (E) and (F) transcripts for Ccl17, Ccl22, Ccl24, and Ifnb1 were quantified by qPCR and normalized using Tbp. Data are mean value ± s.d. of three replicates. The P-values were calculated using two-tailed unpaired Student’s t-test or one-way ANOVA with Bonferroni posttests analysis of variance; *p<0.05 and P values greater than 0.05 were considered not significant (ns). In (F) RAW264.7 (WT) and β-catenin-deficient (Ctnnb1-/-) RAW264.7-derived cell lines were either left uninfected or infected with the indicated wild-type and Δgra18 mutant strains at a MOI of 1:6 for 18 hr. Data are representative of two independent experiments.

Fig 3: Effects of RFC3 on the regulation of proteins associated with epithelial-mesenchymal transition and the Wnt/β-catenin signaling pathway. (A) RFC3 overexpression plasmid and empty vector were transfected into the lung cancer A549 cell line. (B) RFC3-specific siRNA and non-specific control siRNA were transfected into the lung cancer H1299 cell line. Total protein was extracted and subjected to immunoblotting of Wnt1, p-GSK3-β (Ser9), GSK3-β, β-catenin, c-MYC, E-cadherin, N-cadherin and Vimentin. Actin was used as a loading control. (A and B) Representative image data of immunoblotting, semi-quantitative representation of protein expression and ratios of p-GSK3-β/GSK3-β from three separate experiments. *P<0.05, **P<0.01. GSK3, glycogen synthase kinase 3; p-, phosphorylated; RFC3, replication factor C subunit 3; siRNA, small interfering RNA.

Fig 4: KIF23 knockdown inhibits the Wnt/β-catenin signaling pathway. (A) KIF23 knockdown reduced the levels of p-GSK3-β, β-catenin and c-Myc. (B) KIF23 knockdown reduced β-catenin levels in the nucleus. (C,D) Immunofluorescence staining of β-catenin in 786-O and A498 cells.

Fig 5: Pharmacological inhibition of GSK-3 with 9-ING-41 potentiates the effect of anticancer therapeutics in RCC cells. Relative cell proliferation was measured by MTS assay in RCC cell lines (A and B) ACHN and (C and D) Caki-1 treated with 9-ING-41 in combination with (A and C) targeted therapeutics (sorafenib, sunitinib, pazopanib and cabozantinib) or (B and D) autophagy inhibitors (chloroquine and bafilomycin) for 72 h as indicated. RCC, renal cell carcinoma.