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  • br Expression of YAP in mouse

    2019-10-08


    Expression of YAP1 in mouse cervical tissues showed in Figure 2B was detected by alkaline phosphatase-based detection systems using VECTASTAIN ABC-AP Staining Kit (Rabbit IgG, # AK-5001). The basic experimental steps of ABC-AP kit are similar with ELITE ABC system, except that the endogenous enzyme activity was blocked with BLOXALL Blocking Solution (#SP-6000, Vec-tor Laboratories) and the signal was visualized using Vector Red Substrate kit (#SK-5100) (Vector Laboratories, Burlingame, CA).
    All stained sections were scanned with an iSCAN Coreo Slide Scanner (Ventana Medical Systems, Inc. Oro Valley, AZ). The signal intensity and positivity (the ratio of positive cell number relative to the total cell number) of immunosignal on each section were quantified using an Aperio ImageScope software (Leica Biosystems Imaging, Inc. Vista, CA).
    Fluorescent Immunocytochemistry
    The expression and cellular location of YAP1, IRF3, IRF9, NFKB1, and STAT1 were analyzed by fluorescent immunocytochemistry as described previously (Lv et al., 2017). Cells were grown on coverslips in a 24-well plate. After treatment, cells were fixed with 4% formaldehyde (in 1X PBS) at 4 C for 20 minutes, rinsed with PBST for 3 times X 5 minutes, blocked cells with normal donkey serum (10%) for 1 hour before incubating with primary antibody ( 200x) at 4 C for 16 hours. Antigen was visualized with fluorochrome-conjugated secondary antibodies. Fluorescence-conjugated secondary antibodies for immunofluorescent analyses, including Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (#711-545-152) and Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (#715-545-150), were from the Jackson Immunoresearch Laboratories Inc. (West Grove, PA); Rhodamine Phalloidin (#R415) for visualizing Amiloride HCL was from Thermo Fisher Scientific (Rockford, IL). Nuclei were stained with DAPI. Images were captured using a
    ZEISS Xradia 810 Ultra Confocal Laser Scanning Microscope and analyzed with Zeiss Zen 2012 software (Carl Zeiss Microscopy, LLC, Thornwood, NY).
    Western Blot Analysis
    Western blot was used to determine the relative protein levels in cells and tissues with a protocol described previously with minor modification (Hua et al., 2016). Cells were harvested and lysed (150ml/106 cells) on ice. Protein concentration was measured using BCA assay kit. Thirty micrograms protein per sample was loaded onto the SDS-PAGE gel, fractioned using a Bio-Rad electrophoresis system, and then transferred from gel onto PVDF membranes. The protein containing membrane was blocked with 5% BSA for 60 minutes, incubated with primary antibodies at 4 C for 16 hours and then probed with a HRP-linked secondary antibody (#7074 for Rabbit IgG, #7076 for Mouse IgG, Cell Signaling Technology, Inc. Danvers, MA). The immunosignal was visualized with a SuperSignal West Femto Maximum Sensitivity Substrate Kit (#34096, Thermo Fisher Scientific, Waltham, MA). Images were captured and analyzed using a UVP gel documentation system (UVP, Upland, CA).
    Quantitative Real Time-PCR
    Total RNA from cultured human cells and mouse cervical tissues were extracted using TRIzol reagent (Invitrogen; Carlsbad, CA) and QIAGEN RNeasy mini kit (#74106QIAGEN, Carlsbad, CA) following instructions provided by the manufacture. cDNA was synthesize using an iScript Reverse Transcription Supermix for RT-qPCR Kit (Bio-Rad Laboratories, Inc.). qT-PCR was performed with the Bio-Rad CFX96 real-time PCR system using an iTaq Universal SYBR Green Supermix Kit (Bio-Rad Laboratories, Inc.). GAPDH and 18S were used as internal references. All primer sequences are shown in Table S1.
    YAP1 Overexpression
    Primary hCerECs were cultured following a protocol provided by the vendor (Catalog #7060, ScienCell Research Laboratories, Inc.). SiHa (a human cervical cancer cell line contained HPV16) and ECT1 (a human endocervical cell line immortalized by HPV16 E6/E7) cells were cultured with 2% Ultroser G serum substitute (Pall Corporation) in DMEM/F12 medium. hCerEC-MX and ECT1-MX cells was generated by transfecting hCerEC and ECT1 cells with retrovirus-based empty MXIV vector (MX) as control; hCerEC-YAP and