Lsm 800 instrument

After the H9c2 cells were exposed to the indicated stimuli and incubated for 24 h, cell viability was measured using a cell viability assay kit (Abfrontier, Seoul, Korea) according to the manufacturer’s protocol. The TUNEL assay was performed using the DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer’s instructions. TUNEL-positive cells with green fluorescein were visualized with the LSM 800 instrument (Carl Zeiss, Oberkochen, Germany). To determine the percentage of apoptotic cells, the TUNEL-positive nuclei and TUNEL-negative cells were counted using the Image J software (National Institutes of Health).

The expression of the TIGAR protein and E6 viral oncoprotein within the HPV16-infected cervical cancer clinical samples by immunofluorescence staining, as well as the detection of cellular apoptosis using Annexin V-FITC/PI (or DAPI), were visualized confocal fluorescence-microscopy on a Zeiss LSM800 instrument using a Plan-Apochromat 20x/0.8 objective lens and ZEN OS software (Carl Zeiss Microscopy). The relative fluorescence-intensities of the Anti-TIGAR-specific (Rhodamine Red-X-positive) and Anti-HPV16 E6-specific (Alexa Fluor 488-positive) signals were graphically quantified using the Zen 2.5D analysis tool (Carl Zeiss Microscopy).

Approximately 1 million cells were pelleted and resuspended in 1-2 μL PBS and streaked onto glass slides to dry. Cells were fixed with 4% paraformaldehyde/PBS solution for 10 minutes and permeabilized with 0.5% (v/v) Triton X-100/PBS solution for 5 minutes, with PBS washing in between and after each of those steps. Cells were incubated with 20% normal goat serum (NGS) blocking reagent for 1 hour. EBNA1-specific OT1x antibody was diluted 1:100 in the blocking reagent and added to cells for 1 hour. Cells were washed and incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (diluted 1:1000 in blocking reagent) for 30 minutes. Cells were washed and incubated with a 10 μg/mL Hoechst 33258/PBS solution for 3-5 minutes. Cells were washed and successively dehydrated in 70% (1 minute), 90% (1 minute) and 100% ethanol (1 minute). ProLong Gold antifade reagent was added to each well and a No. 1.5 coverslip was attached. Data acquisition was performed using a Zeiss LSM 800 instrument. Processing and analysis were performed using ZEN Blue software (Zeiss). All steps were performed at room temperature.

After the H9c2 cells were exposed to the indicated stimuli and incubated for 24 h, cell viability was measured using a cell viability assay kit (Abfrontier, Seoul, Korea) according to the manufacturer’s protocol. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed using the DeadEnd Fluorometric TUNEL System (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. TUNEL-positive cells were visualized with the LSM 800 instrument (Carl Zeiss, Oberkochen, Germany).

Epifluorescence images of P_T4B8.5::gfp expression were obtained using an AxioImager Z2 upright microscope (Zeiss) and ZEN software (Zeiss). Confocal images of P_cyp-36A1::gfp expression were obtained using an LSM 800 instrument (Zeiss) and ZEN software. Fluorescence intensity for the P_T24B8.5::gfp reporter was quantified by measuring average intensity in a 300 μm section of the intestine centered on the vulva using FIJI software. P_T24B8.5::gfp reporter imaging conditions were optimized for observation of fluorescence in the midbody; fluorescence was not saturated in this region in quantified images.

Seeded cells were permeabilized with 0.5% Triton X-100/PBS for 5 mins, blocked with 1% BSA/PBS for 1h at room temperature and incubated with anti-mTOR (1:250) and anti-LAMP2 (1:150) primary antibodies for 1h at 37°C. Then, cells were washed three times with TBS and incubated with anti-mouse (1:250) and anti-rabbit (1:250) secondary antibodies for 1h at 37°C in the dark. Cells were washed three times with TBS and stained with DAPI (1:5000) for 10min. Image acquisition was performed by a Zeiss LSM 800 instrument. Image analysis was performed with Zeiss Zen Lite (Blue) software. Image J Coloc2 was used to score the colocalization of mTOR and lysosomes.