Rements prior to tissue harvest. Panels B, D, and F) The overall change in tumor size after no treatment, or Eated with recombinant TCTP/GST for one h before harvest.ImmunohistochemistryTissue treatment with DMSO (control) or PQ7 (25 mg/kg) via 7 IPs. * P-value < 0.05 compared to controls.doi: 10.1371/journal.pone.0067174.gwere 52232-67-4 site identified as adenosquamous carcinomas. Histological examination of the lung tissue from all Late stage mice showed no significant difference in the presence of metastatic foci between treatment groups.Effect of PQ7 on connexin expression in neoplastic tissuePQ7 has been shown to enhance GJIC and increase Cx43 expression in breast cancer cells [4], thereforeThe effect of PQ7 on mammary carcinomaFigure 4. 15481974 Number of developed tumors in PyVT female mice during development. Tumors identified grossly during the A) Pre, B) Early, and C) Late stages of tumor development after a 14 day period with either no treatment, or treatment with DMSO (control) or PQ7 (25 mg/kg) via 7 IPs. * P-value < 0.05 compared to controls.doi: 10.1371/journal.pone.0067174.gthe differential pattern of connexin proteins in PQ7treated tumors was determined. Though most connexins are tumor suppressors, Cx46 has been shown to be upregulated in breast cancer cell lines and tumors to provide protection in hypoxic conditions [10]. Immunoblot analysis of connexin expression indicates that PQ7 treatment increased Cx43 expression (Figure 5B) and decreased Cx46 expression (Figure 5C) during the early stages of carcinogenesis. During the control PyVT tumor development there was an increase in Cx43 and Cx46 expression from Pre to Late stage. Data suggests that the gap junction protein connexin 43 was expressed at higher levels in PQ7-treated animals compared to controls and the contrary for connexin 46 early in tumor formation. Cx46 expression in PyVT tumors treated with PQ7 from the Pre and Late stages of development had significantly lower levels than that of the controls (P-valuePre = 0.016, P-valueLate = 0.0007). The Pre stage tumors treated with PQ7 had a significantly greater level of Cx43 expression compared to controls (P-valuePre = 0.040), while during the Late stage they had significantly less Cx43 compared to controls (P-valueLATE = 0.034). This may be explained by the overall increase in both connexin 43 and connexin 46 during tumor development and metastasis of the PyVT mice. Histopathology of the harvested PyVT tumors showed no significant difference in morphology. Immunohistochemistry of PQ7 treatment at Pre and Early stages of tumor formation showed stronger positive cytoplasmic staining in Cx43, while during the Late stage there was stronger positive staining in the control tissue versus the PQ7 treated tissue (Figure 6A). The Cx46 immunohistochemistry indicated a weaker positive staining compared to controls. This supports the molecular analysis previously mentioned. Gap junction proteins are phosphoproteins that are targeted by kinases for efficient trafficking, assembly and disassembly, degradation, and gating of hemichannels [11?3]. Phosphorylation regulates GJIC in both a kinase and connexin specific manner [11,14]. Since PQ7 altered connexin expression, we explored the role of PKC in the PQ7 treated PyVT tumors at each stage of development by western blot analysis (Figure 5D) and immunohistochemistry (Figure 6C). No significant change in PKC expression was determined due to PQ7 treatment. Interestingly in the control tumors there appeared to be a stronger positive staining in the Pre stage compared to the Late stage.Rements prior to tissue harvest. Panels B, D, and F) The overall change in tumor size after no treatment, or treatment with DMSO (control) or PQ7 (25 mg/kg) via 7 IPs. * P-value < 0.05 compared to controls.doi: 10.1371/journal.pone.0067174.gwere identified as adenosquamous carcinomas. Histological examination of the lung tissue from all Late stage mice showed no significant difference in the presence of metastatic foci between treatment groups.Effect of PQ7 on connexin expression in neoplastic tissuePQ7 has been shown to enhance GJIC and increase Cx43 expression in breast cancer cells [4], thereforeThe effect of PQ7 on mammary carcinomaFigure 4. 15481974 Number of developed tumors in PyVT female mice during development. Tumors identified grossly during the A) Pre, B) Early, and C) Late stages of tumor development after a 14 day period with either no treatment, or treatment with DMSO (control) or PQ7 (25 mg/kg) via 7 IPs. * P-value < 0.05 compared to controls.doi: 10.1371/journal.pone.0067174.gthe differential pattern of connexin proteins in PQ7treated tumors was determined. Though most connexins are tumor suppressors, Cx46 has been shown to be upregulated in breast cancer cell lines and tumors to provide protection in hypoxic conditions [10]. Immunoblot analysis of connexin expression indicates that PQ7 treatment increased Cx43 expression (Figure 5B) and decreased Cx46 expression (Figure 5C) during the early stages of carcinogenesis. During the control PyVT tumor development there was an increase in Cx43 and Cx46 expression from Pre to Late stage. Data suggests that the gap junction protein connexin 43 was expressed at higher levels in PQ7-treated animals compared to controls and the contrary for connexin 46 early in tumor formation. Cx46 expression in PyVT tumors treated with PQ7 from the Pre and Late stages of development had significantly lower levels than that of the controls (P-valuePre = 0.016, P-valueLate = 0.0007). The Pre stage tumors treated with PQ7 had a significantly greater level of Cx43 expression compared to controls (P-valuePre = 0.040), while during the Late stage they had significantly less Cx43 compared to controls (P-valueLATE = 0.034). This may be explained by the overall increase in both connexin 43 and connexin 46 during tumor development and metastasis of the PyVT mice. Histopathology of the harvested PyVT tumors showed no significant difference in morphology. Immunohistochemistry of PQ7 treatment at Pre and Early stages of tumor formation showed stronger positive cytoplasmic staining in Cx43, while during the Late stage there was stronger positive staining in the control tissue versus the PQ7 treated tissue (Figure 6A). The Cx46 immunohistochemistry indicated a weaker positive staining compared to controls. This supports the molecular analysis previously mentioned. Gap junction proteins are phosphoproteins that are targeted by kinases for efficient trafficking, assembly and disassembly, degradation, and gating of hemichannels [11?3]. Phosphorylation regulates GJIC in both a kinase and connexin specific manner [11,14]. Since PQ7 altered connexin expression, we explored the role of PKC in the PQ7 treated PyVT tumors at each stage of development by western blot analysis (Figure 5D) and immunohistochemistry (Figure 6C). No significant change in PKC expression was determined due to PQ7 treatment. Interestingly in the control tumors there appeared to be a stronger positive staining in the Pre stage compared to the Late stage.