Fig 2: DKK-1 regulates the CBX7-mediated Wnt signaling pathway(A) Effect of the inhibition of DKK-1 on CBX7-induced increased activity of the E-Cadherin and oppositively on other invasion related proteins. Cells treated with DKK-1 siRNA were subjected to immunoblotting with appropriate antibodies. (B) CBX7-induced reduction of phosphorylated AKT and GSK3-ß and ZEB1 were reversed by treatment of siDKK1. ß-actin was used as an endogenous normalizer in immunoblotting. (C-D) Wound-healing assay, (E-F) Transwell assay and (G) three-dimensional spheroid assays were performed in LN229 and T98G treated with siDKK1. All experiments were performed in triplicate. * P=0.05; ** P = 0.01 and *** P = 0.001.

Fig 3: CBX7 enhanced DKK1 expression by binding promoter(A) CBX7 impact the expression of DKK1, analyzed by immunoblotting with the indicated antibodies. ß-actin was used as control. (B-D) A luciferase reporter assay was performed to measure DKK-1 promoter activity in LN229 and T98G. TSS, transcription start site. (E) Schematic illustration of the promoter regions of the human DKK-1 gene and the regions containing the primers for ChIP assay (under). (F) The results of ChIP analysis show the CBX7 was recruited to the DKK-1 promoter regions in CBX7-overexpressing LN229 and T98G cells. IgG was used as an immunoprecipitation control. * P=0.05; ** P = 0.01 and *** P = 0.001.

Fig 4: CBX7 function was demonstrated in vivo(A) LN229/VEC and LN229/CBX7 stabled cells were counted and transplanted subcutaneously in nude mice respectively. After about 4 weeks, xenografted tumors were formed and difference in tumor volume was counted. Size in CBX7 overexpressing groups was smaller than vector groups. (B) Cells were counted and transplanted in orthotopic nude mice model. After about 20 days, HE assay was performed and showed that not only the volume became smaller, but the invasion ability became weak in CBX7 overexpressing groups. Arrow sign points to the invasion sites. (C) CBX7 enhanced survival ability of nude mice injected in orthotopic. (D) CBX7 promote the level of DKK1 in vivo tested by immunohistochemical analysis. (E-G) EMT-like processes were blocked and the expression of MMPs were reduced in vivo, which were analyzed via immunohistochemical analysis.

Fig 5: DKK1 inhibited proliferation and self-renewal of RAB6-/- AEC2 cells.a WT and RAB6-/- AEC2 cells were exposed to DKK1 protein (10 ng/ml) or PBS for 48 h and then cells were exposed to PM2.5 (100 µg/ml) for 48 h. The apoptosis of AEC2 cells was determined by FACS (Representative FACS images are shown, n = 3). b Statistical analysis of apoptosis rate. c The colony forming efficiency (CFE) of WT and RAB6-/- AEC2 cells was examined by plate colony formation assay (Representative images are shown, n = 3). d Statistical analysis of CFE (represented by colony numbers). e The relative protein expression of ß-catenin, and c-myc in AEC2 cell lysates was detected by western blot analysis. f Statistical analysis of relative expression levels of proteins. g The relative protein expression of ß-catenin in nuclear lysates of AEC2 cells was detected by western blot analysis. h Statistical analysis of relative expression levels of proteins. i–k The relative RNA expression of SOX2 (i), OCT4 (j), and NANOG (k) was measured by qRT-PCR. (n = 3; unpaired two-tailed t test. **P < 0.01; ***P < 0.001. Bar graphs represent the mean ± SEM for b, d, f, h–k).