Fig 1: Identification of adhesive molecules that were up-regulated in SSP6 cells.(A) Western blots showing the change in adhesive proteins in the M-SSP6 and A-SSP6 cells. Quantified data are presented as the means ± SD from three independent experiments. (B) Representative immunofluorescence (IF) staining images illustrating the distribution of DSC2, PKP1, vimentin (VIM), E-cadherin (CDH1), β-catenin, fibronectin (FN1), and integrin β1 (ITGB1) in the A549-C3 and MCF7-C3 cells before and after SS15 treatment for 12 hours. Scale bar, 10 μm. (C) qRT-PCR results showing the normalized mRNA levels of DSC2, PKP1, VIM, CDH1, FN1, and ZEB1 in the A549-C3 cells under SS15 treatment at various time points. As glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a housekeeping gene whose mRNA level in A549-C3 cells at 0 hours was normalized to 1 and used as an internal reference for calculating the mRNA levels of the other tested genes. The results represent the means ± SD from three independent experiments. Significant differences were determined by one-way analysis of variance (ANOVA) (C). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Fig 2: Signaling pathways of DSC2 and PKP1.(A) Western blots showing the higher expression of p-AKT, Bcl-2, and p-ERK1/2 in A-SSP6 cells. (B) Western blots showing the knockdown effects of DSC2, PKP1, DSC2 + PKP1, VIM, and ZEB1 on themselves and other proteins in A-SSP6 cells. (C) Western blots showing the effects of knockout ITGB1 on various indicated proteins in A-SSP6 cells. (D) DSC2 was overexpressed in A549-C3 cells and Western blots showing the up-regulation of VIM and ITGB1. (E) DSC2 was first overexpressed in A549-C3 cells, and then VIM was knocked down in these overexpressed cells, Western blots showing the down-regulation of VIM and ITGB1 after knockdown of VIM. (F) Western blot results indicating that shFN1 reduced the levels of ITGB1, FAK, and p-Src. (G) Western blotting showing that the levels of FAK and p-Src were decreased after VIM was knocked down. (H) The left side of the schematic illustration shows that CTCs with high levels of DSC2 can form clusters, survive in circulation, and subsequently form lung colonies by tail vein injection or metastasize to the liver, intestine, and brain in the lung orthotopic model. The right side of the schematics illustrates the proposed signaling pathways through which DSC2/PKP1 and VIM support cluster formation, cell survival in circulation, lung colony formation, and metastasis.
Fig 3: The dual expression of DSC2 and PKP1 is important for cluster formation and survival in the circulation of A-SSP6 cells.(A to C) Representative phase images and quantified percentages of clustered and viable cells upon knockdown of the DSC2, PKP1, DSC2 + PKP1, VIM, FN1, ZEB1, and CDH1 genes in A-SSP6 cells circulated under SS15 for 10 hours (the results were quantified from three independent experiments, cell numbers of ≥300 for each independent experiment). shCtrl, negative control shRNA. Scale bar, 100 μm. (D to F) Representative images and quantified percentages of clustered and apoptotic cells obtained from mouse blood 4 hours after tail vein injection of the A-SSP6 cells upon knockdown of the expression of the DSC2, PKP1, and DSC2 + PKP1 or injection of A549-C3 cells upon overexpression of the DSC2 (n = 5 mice per group). EV, empty vector; O-DSC2, overexpression of DSC2. Scale bar, 50 μm. The results represent the means ± SD. Significant differences were determined by one-way ANOVA (B, C, E, and F) except for the differences between empty vector and overexpression of DSC2 in which Student’s t test was used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Fig 4: Knockdown of DSC2, DSC2 + PKP1, VIM, FN1, or ZEB1 effectively reduced the metastatic potential of A-SSP6 cells.(A and B) Representative images and quantified results of the migrated cells with 20,000 cells seeded per insert for 24 hours after silencing DSC2, PKP1, DSC2 + PKP1, VIM, CDH1, FN1, and ZEB1 in the A-SSP6 cells. Two Transwell inserts were used in each migration assay, which was repeated two more times. Scale bar, 200 μm. (C) Quantified numbers of invading cells with 20,000 cells seeded on the Matrigel-coated membrane of the Transwell insert for 24 hours after silencing DSC2, PKP1, DSC2 + PKP1, VIM, CDH1, FN1, or ZEB1 in A-SSP6 cells. Two Transwell inserts were used in each invasion assay, which was conducted in triplicate. (D to F) Representative images and quantified results of the lung colony-forming ability upon DSC2, PKP1, DSC2 + PKP1, VIM, FN1, or ZEB1 knockdown in A-SSP6 cells for 21 days or overexpression of VIM or ZEB1 in A549-C3 cells for 30 days (n = 5 mice per group). O-VIM, overexpression of VIM; O-ZEB1, overexpression of ZEB1. Scale bars, 1 mm (D and E). The data are shown as the means ± SD. Significant differences were determined by one-way ANOVA (B, C, and F). *P < 0.05, ***P < 0.001, and ****P < 0.0001.
Fig 5: Clinical significance of DSC2 and PKP1 in lung and breast cancer.(A) Representative images of IHC staining for DSC2, PKP1, VIM, and ZEB1 between lung tumors and adjacent tissues from patients with lung cancer. Scale bar, 100 μm. (B) The positive correlation between the IHC score of DSC2 and that of PKP1 in patients with lung cancer (n = 30). (C to F) Plots showing the IHC scores of DSC2, PKP1, VIM, and ZEB1 between the adjacent tissues and human lung tumors (n = 30). Each dot represents one patient. (G to L) Representative images and quantified IHC scores of DSC2, PKP1, VIM, and ZEB1 among normal tissues, primary tumors, and metastatic tumors in patients with breast cancer. Plots showing the IHC scores for DSC2, PKP1, VIM, and ZEB1 in normal tissues (NT), primary tumors (PT), and metastatic tumors (MT). Scale bars, 100 μm. Significant differences were determined by Student’s t test (C to F) and one-way ANOVA (H, I, K, and L). **P < 0.01, ***P < 0.001, and ****P < 0.0001.
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