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  • br ECT YAP cells were generated

    2019-10-08


    ECT1-YAP cells were generated by transfecting hCerEC and ECT1 cells with vectors expressing wild-type of YAP1 protein; hCerEC-YAPS127A and ECT1-YAPS127A cells were generated by transfecting hCerEC and ECT1 cells with vectors expressing constitutively active YAP1 (YAPS127A, Serine at residue 127 is replaced by an Alanine resulting in the constitutive activation of YAP1). Since hCerEC are primary cells, all experiments using hCerEC-MX, hCerEC-YAP and hCerEC-YAPS127A cells were performed within 4 passages after transfection. Expression of YAP1 gene was confirmed by both RT-PCR and western blot assay.
    Gene Silencing by siRNA
    For gene silencing experiments, hCerEC, ECT1, or SiHa cells were transfected with scrambled control siRNA (siGENOME Non-Targeting siRNA #1, D-001210-01-20) or YAP1 specific siRNAs (siYAP), or ITGA6 specific siRNAs (siITGA6) for 6 hours using Lipo-fectamine 2000 protocol (Invitrogen, Carlsbad, CA) after cells reaching 60% confluent. siRNAs targeting YAP1 and ITGA6 genes were from Dharmacon (Lafayette, CO). At least two different siRNAs for each gene were used to validate the knockdown efficiency of ITGA6 specific siRNAs. Gene knockdown was detected by RT-PCR and western blot. The results for validation of YAP1 siRNAs were presented in supplemental Figures S12A–S12C. The results for validation of ITGA6 siRNAs were presented in supplemental Figures S12D–S12E.
    Preparation of HPV PsVs
    HPV PsVs (HPV16- PsV) is a HPV16 virion-like particle in which HPV16 L1 and L2 proteins encapsulate a GFP plasmid (reporter gene). Infection efficiency of HPV16 is indicated by the signal intensity of GFP and the ratio of GFP-positive cells in HPV16-PsV treated cells. Previous studies have demonstrated that this PsV could well mimic HPV infection process (Conway and Meyers, 2009; Roberts et al., 2007). HPV16-PsV were generated as described previously13,59. Briefly, plasmids expressing HPV GSK1838705A proteins L1 and L2 (p16L1L2, #45291, Addgene. Donated by Dr. John T. Schiller’s Lab) and GFP reporter (pCIneoEGFP, #46949, Addgene. Donated by Dr. John T. Schiller’s Lab) were co-transfected into 293TT cells using a Lipofectamine 2000 (#11668027, Invitrogen) protocol. After 48 hours, the transfected cells were trypsinized, washed with DPBS-Mg solution for three times, and re-suspend with a cell concentration of 1 3 108 cells/ml in DPBS-Mg solution (with 0.5% Brij58, 0.2% Benzonase, and 0.2% Plasmid Safe). Cell lysate was incubated at 37 C for 24 hours to ensure capsid maturation and then chilled on ice for 10 minutes. After chilling on ice, the salt concentration of the cell lysate was adjusted to 850 mM NaCl and incubated on ice for 10 more minutes. The lysate was then clarified by centrifugation, and the supernatant was layered onto an Optiprep gradient. The gradient was spun for 4.5 hours at 16 C at 40,000 RPM in a SW40 rotor (Beckman Coulter, Inc., Brea, CA). The concentration of HPV PsVs was evaluated by analysis of GFP transduction efficiency in 293 cells.
    HPV16 PsV Infection of Cells
    hCerEC, ECT1 or SiHa Cells (2x104 cells / well) were seeded in a 12-well plate and cultured overnight. HPV16 PsVs (1.0 or 2.0 MOI) were added into culture medium and incubated for 72 hours. GFP signal was captured using a Nikon-ECLIPSE NI with X-CITE system. The images were analyzed using Zeiss Zen 2010 software (Carl Zeiss Microscopy, Thornwood, NY).
    QUANTIFICATION AND STATISTICAL ANALYSIS
    All experiments were repeated at least four times unless otherwise noted. Data are presented as mean ± SEM. Statistical analysis was conducted, and graphs were made with GraphPad Prism 7.04 (GraphPad Software, Inc. La Jolla, CA). Data were analyzed for significance using Student’s t test (two groups) or one-way ANOVA with Tukey’s post hoc tests (multiple groups). A value of p < 0.05 was considered statistically significant.