A253 and Detroit 562 (D562) are human head and neck squamous cell carcinoma cell lines, cultured in S-MEM with 10% FBS and 2 mM L-glutamine . For all SiRNA experiments, cells were transfected by Lipofectamine® RNAiMAX Reagent (Invitrogen, Carlsbad, CA) using the manufacturer’s protocol. The following SiRNAs were purchased from Thermo Scientific (Lafayette, CO): ON-TARGETplus Non-targeting siRNA #1 (#D-001810-01-0005) for control siRNA, ON-TARGETplus PCSK7 siRNA (#J-005988-07-0005) for PC7, ON-TARGETplus FURIN siRNA-SMARTpool (#L-005882-00-0005) for Furin, and ON-TARGETplus PCSK6 siRNA (#J-005983-05-0002) for PACE4.
Cell lysates were harvested two days after siRNA transfection and subjected to Western blot analysis as described . For CoCl2 and PPP treatments, the transfected or non-transfected cells were incubated with drugs overnight. The treatment time with MG-132 was 30 min. For CHX treatment, the cells were incubated for different times as labeled in the corresponding figures. Regarding VEGF-A and VEGF-C analysis, the cells were first transfected with SiRNA for 2 days and serum-free media were collected after 1-day incubation. The conditioned media were concentrated with Microcon centrifugal filters (#42407) from Millipore (Billerica, MA) and used for Western blot analysis. The following antibodies were used: HIF-1α (#610958) from BD Biosciences (San Jose, CA), VEGF-A (#sc-152), VEGF-C (#sc-1881) and IGF-1Rβ (#sc-713) from Santa Cruz Biotechnology (Santa Cruz, CA), and GAPDH (#ab8245) from Abcam (Cambridge, MA). The quantification of Western blot results was performed by using ImageJ developed by the National Institutes of Health. Full paper
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