PMC

The impact of PTHLH/PTHrP on the cell cycle. (A) Cell cycle was analyzed in WSU-HN6, HN13 and CAL-27 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA. (B) The proteins of Cyclin D1, CDK4 and p21 which related to cell cycle were determined in WSU-HN6 and HN13 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA. (C) The fold change in western blot (Figure B) was quantified and analyzed. (D) Immunocytochemistry staining of cells transfected with PTHLH-siRNA was used to analyze the expression of PTHLH, Cyclin D1, CDK4 and p21.

The effect of PHTLH/PTHrP on cell proliferation and colony formation. (A) PTHrP protein expression was confirmed in WSU-HN6, HN13 and CAL-27 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA using western blotting. (B) Cell proliferation of WSU-HN6, HN13 and CAL-27 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA were analyzed using the CCK-8 kit, each point showed the mean values of triplicate wells (Mean ± SE). (C) Plate colony formation of WSU-HN6, HN13 and CAL-27 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA was analyzed. (D) Soft agar colony formation assay was analyzed between the cell lines transfected with anti-PTHLH P-si and cell lines transfected with scrambled siRNA.

The clinical significance of PTHrP overexpression was analyzed in 93 patients with HNSCCs. (A) PTHrP expression was detected in poorly differentiated, moderately differentiated and well differentiated specimens and in normal epithelial specimens, as shown by immunohistochemistry. (B) Kaplan-Meier survival curves illustrating the overall survival of 93 patients with HNSCC according to the PTHrP protein levels. (C) Two patients with base of tongue squamous cell carcinoma in this study were positive for HPV16/18 and p16 expression using ISH and IHC methods.

PTHLH mRNA and PTHrP protein expression in OSCC specimens and cell lines. (A) PTHLH mRNA expression was analyzed in 36 paired OSCC specimens including cancerous tissues and apparently normal surgical margins using real-time PCR (Mean ± SE: 2.618 ± 0.495, rang: 0.0817 ± 12.010 vs. Mean ± SE: 7.376 ± 1.040, rang: 0.988 ± 20.873; P = 0.0001; two-tailed Student’s t-test). (B) PTHrP protein expression was determined in 5 representative paired OSCC specimens by western blotting analysis (N, apparently normal surgical margin; C, cancerous tissue). (C) PTHLH mRNA levels were determined in 9 representative OSCC cell lines, primary normal epithelial cells and normal tissues showing in Figure 1A using real-time PCR (P < 0.0001; One-way ANOVA). (D) PTHrP protein levels were determined in 9 representative OSCC cell lines and primary normal epithelial cells using western blotting.