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. 2010 Oct;12(10):789–796. doi: 10.1593/neo.10396

Figure 1.

Figure 1

CIN85 upregulates the growth of HNSCCs. (A) CIN85 mRNA and protein expression in representative clinical samples. Total RNA and proteins were extracted from the tissues of untreated patients with oral squamous cell carcinoma. CIN85 mRNA and protein expression and phospho-ERK1/2 expression were evaluated by quantitative RT-PCR and Western blot analysis. CIN85 mRNA expression in no. 155 sample was taken as 1.0. In Western blots, β-actin served as a loading control. The intensity of bands was quantified and normalized with respect to those of corresponding β-actin. The resulting values were expressed as the percentage in reference to that of no. 212. (B) CIN85 mRNA and protein expressions and EGFR protein expression in three HNSCC cell lines (KB, YCU-H891, and SCC9) by quantitative RT-PCR and Western blot assays. Total RNA and proteins were extracted from exponentially growing cells and subjected to quantitative RT-PCR analysis and Western blots. In quantitative RT-PCR analysis (left panel), the level of CIN85 mRNA expression in YCU-H891 was taken as 1.0. In Western blot analysis (right panel), the resulting values were expressed as the percentage in reference to that of KB cells. (C) Establishment of stable CIN85-overexpressing and CIN85 knockdown KB cells. Either an shRNA construct for CIN85 or a full-length CIN85 construct was transfected with KB cells. Independent clonal lines were designated as KB/KD1, KB/KD2, KB/OE1, and KB/OE2. Expression levels of CIN85 mRNA (upper panel) and proteins (lower panel) in parental, vector ,control and transfected cells were measured by quantitative RT-PCR or Western blots. In quantitative RT-PCR analysis, the level of CIN85 mRNA expression in parental KB cells was taken as 1.0. In Western blot analysis, the resulting values were expressed as the percentage in reference to that of KB/OE2 cells. Note that a full-length CIN85 transgene is V5-tagged, and thus, its product migrates slower than endogenous CIN85 on a gel (arrow). (D) Growth curves of control and transfected cells. Two thousands cells of the indicated cell lines were cultured for 96 hours and counted every other day. The results are representative of three independent experiments. The values at each point indicate the average of triplicate dishes; bars, SD.