Fig 1: Effects of CXCL14 on cell proliferation and migration of human colorectal cancer cells. CXCL14 was stably over-expressed in the HCT116 cells, as determined by RT-PCR assay (A) and western blot assay (B). (C) Quantitative results of CXCL14 expression levels. (D) The proliferative capacity of HCT116 cells stably expressing CXCL14 (HCT116/CXCL14) was stronger than that of HCT116/Control only transfected with empty vectors, as measured by colony formation assay. (E) The migratory capacity of HCT116/CXCL14 was stronger than that of HCT116/control cells, as measured by wound healing assay. (F) Quantitative results of cell migration numbers in modified Boyden chamber assays in HCT116/Control cells and HCT116/CXCL14 cells. (G) The expression levels of cell cycle-related and EMT-related proteins in HCT116/CXCL14 cells and HCT/Control cells were measured by western blot. (H) Quantitative results of cell cycle-related protein expression. (I) Quantitative results of EMT-related protein expression. CXCL14 was stably over-expressed in the SW620 cells, as determined by RT-PCR assay (J) and western blot assay (K). (L) Quantitative results of CXCL14 expression levels. M. The expression levels of CDK2, Cyclin A1, vimentin in HSW620/CXCL14 cells, and SW620/Control cells were measured by western blot. (H) Quantitative results of protein expression. The experiments were performed in triplicate (n = 3). Error bars represent mean ± S.D. p < 0.05 was considered to be statistically significant. * p < 0.05; ** p < 0.01, *** p < 0.001.
Fig 2: The regulation of AR in the cell cycle of resistant cells. A. Having been treated with 100 nM DHT for 24, 48 or 72 hours, Cell viability of MCF-7 and MCF-7pR was measured by the Cell Counting kit-8 assay. *P < 0.05, or **P < 0.01 or ***P < 0.001. B. The expression of AR and CCND1 was detected by Western blot following the treatment of vehicle or 100 nM DHT for 48 hours in MCF-7 and MCF-7pR cells. C. The lysates of MCF-7pR treated with vehicle or 100 nM DHT were subjected to immunoprecipitation with AR antibodies. The immunoprecipitates were then blotted with indicated antibodies. D. AR mRNA levels were determined by real-time PCR in MCF-7pR cells following transfection with shAR or shNC for 48 hours. Data shown were from triplicate experiments and reported as the mean ± SD, ***P < 0.001. E. The expression of AR was detected by Western blot following transfection with shAR or shNC for 48 hours. *P < 0.05, or **P < 0.01 or ***P < 0.001. F. MCF-7pR cells were treated with DHT and/or transfected with shAR for 48 hours and measured by a flow cytometer. Results are reported as mean percent cell cycle distribution with standard errors. G. The protein expression of AR, CCND1, CDK2 and CCNE1 was determined by Western blot in MCF-7pR following treatment DHT and/or transfection with AR for 48 hours.
Fig 3: MMPs and CDKs are the targets of WDR5. (A) Results of ChIP-seq and bioinformatics analysis on L02 with or without CCDC144NL-AS1 overexpression showed the involvement of cancer metastasis (MMP2 and MMP9) and the cell cycle (CDK1, CDK2, and CDK4) pathways in CCDC144NL-AS1-overexpressed cells. (B) Results of Western blotting showed that the expression of MMPs and CDKs are regulated by CCDC144NL-AS1/miR-940/WDR axis.
Fig 4: CCDC144NL-AS1 promotes WDR5 to be recruited to the promoter region of MMPs and CDKs. ChIP assay showed that (A) the recruitment of WDR5 and (B) the H3K4me3 level in the promoter region of MMP2, MMP9, CDK1, CDK2, and CDK4 was regulated by CCDC144NL-AS1/miR-940 axis. Data were expressed as mean ± SEM. *P<0.05, **P<0.001.
Fig 5: H2O2 regulated the expression levels of cell cycle-related and EMT-related proteins. (A) The HCT116 cells were treated with or without 15 μM H2O2 for different time. The expression levels of Cyclin A1, CDK2, Cyclin B1, CDK1 were measured by western blot. (B) Quantitative results of cell cycle-related protein expression levels. (C) The SW620 cells treated with or without 20 μM H2O2 or pretreated with 10 mM NAC. The expression levels of Cyclin A1, CDK2, surviving and vimentin were measured by western blot, which could be down-regulated by the antioxidant NAC. (D) Quantitative results of protein expression. (E) The expression of PCNA in SW620 cells treated with different concentration of H2O2 was measured by western blot. (F) Quantitative results of PCNA expression levels. (G) The HCT116 cells treated with or without 15 uM H2O2 for different time. The expression levels of E-cadherin, vimentin and N-cadherin were measured by western blot. (H) Quantitative results of EMT-related protein expression levels. All experiments were repeated three times or more. Error bars represent ±S.D. p < 0.05 was considered to be statistically significant. * p < 0.05; ** p < 0.01, *** p < 0.001.
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