Lsm780 uv

Immunofluorescence (IF) staining was used to detect EMT markers expression and localization in CSC-exposed HPV-16 Ect1/E6E7 cells. HPV-16 Ect1/E6E7 cells treated with CSC (3R4F (1 × 10⁻³, and 10 μg/mL)) for 72 h were fixed and incubated with primary antibodies to E-Cadherin rabbit monoclonal antibody (1:200, Cell Signaling, #31950) and Vimentin mouse monoclonal antibody (1:500, Santa Cruz, sc-6260) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-Mouse (red fluorescence) secondary antibody (1:5000, Molecular Probes^®, Eugene, OR, USA, A11001) and Alexa Fluor 555 goat anti-Rabbit (green fluorescence) secondary antibody (1:5000, Molecular Probes^®, A21428) at room temperature for 1 h. After counterstaining with DAPI (CAT# 1306, Molecular Probes) for 30 min, slides were examined under a Zeiss LSM780-UV and LSM880-UV meta confocal microscope (Carl Zeiss Inc.) using a Plan-Apochromat 40′/1.3 Oil DIC objective. Fluorescence intensities and percent of positive cells were measured by ImageJ/Fiji (NIHV 2.9.0/1.54b, National Institutes of Health, USA).

The PLA kit (DUO92101-1KT, Sigma-Aldrich, St. Louis, MO) was used to determine the association between FOXM1 and cyclin D1 in Cd-treated ht-UtLM cells by colocalization of these two proteins following the manufacturer’s protocol. Briefly, the cells were seeded on 3.5 cm glass bottom plate at density of 30,000 cells/plate. After ON culture, cells were treated with Cd at 0, 0.1 or 10 µM with or without PD for 24 h, fixed with 4% of paraformaldehyde for 20 min, and permeabilized with 0.1% triton X-100 for 30 min. The cells were incubated with a rabbit polyclonal anti-FOXM1 (#5436, Cell Signaling) at a 1:100 dilution and a mouse monoclonal anti-Cyclin D1 antibody (#MA1-10324, Thermo Fisher Scientific Inc.) at a 1:100 dilution, overnight at 4 °C (the cells were only incubated with FOXM1 antibody in the negative control plate). The PLUS and MINUS secondary PLA probes against rabbit and mouse IgG were added and incubated for 1 h followed by incubation with ligase for 30 min at 37 °C. Amplification was then applied for 120 min at 37 °C. The coverslips were mounted on plates with Duolink Mounting Medium with DAPI (DUO82040, Sigma-Aldrich). The cells were imaged on a Zeiss LSM780-UV (Carl Zeiss Inc, Oberkochen, Germany) using a Plan-Apochromat 40X/1.40 oil DIC M27 objective. The number of PLA dots (Bustos et al. 2017 (link)) was measured by Fiji.