Table 1.
Composition of normal and high phosphate-modified AIN-93G rodent diets.
Table 2.
ICP-MS and ICP-OES operational conditions.
Fig 1.
Tumor incidence in the lungs of K-rasLA1 mice provided with an ND or HPD.
Mice (n = 6 per group) were fed an ND (0.3% Pi) or HPD (1% Pi) for 1, 2, or 4 months. (A) Adenocarcinoma and adenoma in the lung tissue. (B) Total number of tumors, number of tumors with diameter >1.5 mm, and tumor volume in ND- and HPD-fed mice. The results are mean ± standard deviation (SD) of six independent measurements. Error bar represent SD. *p < 0.05; **p < 0.01; ***p < 0.001. (C) Tumors on the lungs of ND- and HPD-fed mice, as visualized by H&E staining. Scale bar: 100 μm.
Table 3.
Daily food and inorganic phosphate intake and body weight in K-rasLA1 mice fed a normal or high-phosphate diet for 1, 2, or 4 months.
Table 4.
Tumor incidence in the lungs of K-rasLA1 mice fed a normal or high-phosphate diet for 1, 2, or 4 months.
Fig 2.
Changes in ion levels in the liver and lungs of K-rasLA1 mice provided with an ND or HPD for 4 months.
LA-ICP-MS analyses represent mean values from three trials of solution ICP-MS/ICP-OES data (n = 6 per group). The vertical bar in graded color from blue to red represents the ion level from low to high, respectively.
Table 5.
Changes in ion levels in the livers and lungs of K-rasLA1 mice fed a normal or high-phosphate diet for 1, 2, or 4 months
Fig 3.
Western blot analysis of cellular metabolism-related proteins.
Protein expression of p-acetyl CoA, acetyl CoA, SDHA (complex II), cytochrome c (complex III), COX IV (complex IV), and Rieske (Fe-S complex) relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in liver tissue homogenates of K-rasLA1 mice fed an ND or HPD for 1, 2, or 4 months (n = 6 per group), as determined by western blotting and quantified by densitometry. The results are mean ± SD. Error bar represent SD. *p < 0.05; **p < 0.01.
Fig 4.
Western blot analysis of EMT- and autophagy-related proteins and qRT-PCR analysis of epithelial marker.
(A) Protein expression of the autophagy markers LC3 and ATG5 in lung tissue homogenates as determined by western blotting, using actin as a loading control. (B) LC3 expression (green) in lung tissue sections was assessed by immunocytochemistry. The highest immunoreactivity was observed in the 4-month HPD group relative to the corresponding control (ND) group. Scale bar: 10 μm. (C) Protein expression of the EMT marker N-cadherin in lung tissue homogenates of K-rasLA1 mice fed an ND or HPD for 1, 2, or 4 months (n = 6 per group), as determined by western blotting, using actin as a loading control. The results are mean ± standard deviation SD. Error bar represent SD. *p < 0.05; **p < 0.01; ***p < 0.001. (D) qRT-PCR analysis of epithelial markers, E-Cadherin. The results are mean ± standard deviation SD. Error bar represent SD. *p < 0.05; ***p < 0.001.
Fig 5.
Analysis of protein translation in the lungs of K-rasLA1 mice provided with an ND or HPD.
The expression of the translation-related proteins p70S6K, eIF4E, p-4E-BP-1/2, and 4E-BP-1/2 in lung tissue homogenates of K-rasLA1 mice fed an ND or HPD for 1, 2, or 4 months was determined by western blotting and quantified by densitometry (n = 6 per group) relative to the expression level of actin. The results are mean ± standard deviation SD. Error bar represent SD. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 6.
Analysis of proliferation and angiogenesis.
Proliferation and angiogenesis were evaluated in the lungs of K-rasLA1 mice fed an ND or HPD for 1, 2, or 4 months (n = 6 per group). (A) Expression of proteins associated with proliferation (PCNA) and angiogenesis (FGF-2) in lung tissue homogenates was evaluated by western blotting, using actin as a loading control. (B) PCNA-expressing cells in the tumor region (upper right corner of each panel) were visualized by immunohistochemistry. Scale bar: 20 μm. The results are mean ± standard deviation SD. Error bar represent SD. *p < 0.05; **p < 0.01.
Fig 7.
Apoptosis in the lungs of K-rasLA1 mice provided with an ND or HPD.
Apoptosis in the lungs was analyzed in K-rasLA1 mice fed an ND or HPD for 1, 2, or 4 months (n = 6 per group). (A) The expression of the mitochondrial apoptosis-related proteins Bad, Bax, and cytochrome c was evaluated by western blotting of lung tissue homogenates, with actin used as a loading control. (B) Immunohistochemical analysis of caspase-3 expression in the tumor region. Scale bar: 20 μm. The results are mean ± standard deviation SD. Error bar represent SD. *p < 0.05; **p < 0.01.