Fluoromount with dapi

Coverslips preseeded with HFF cells were infected with ΔKu80:mNeonGreen and incubated at 37 °C for 1 h to allow invasion. Following this, drug (5 μM) was added, and the plates placed back in the incubator for 24 hours. Cells were washed with 1 mL of sterile PBS, which was then again removed and 500 μL of 4% paraformaldehyde (PFA) was added and incubated at room temperature for 20 minutes. The PFA was removed, cell washed once with PBS. Cells were blocked and permeabilisated with incubation 500 μL of 2% bovine serum albubin (BSA), 0.5% Triton X-100 in PBD overnight at 4 °C. The coverslips were then removed and submerged in distilled water three times and mounted using Fluoromount with DAPI (Southern Biotech). The slides were then visualized using a DeltaVision fluorescent microscope (Applied Precision) and processed using SoftWoRx and FIJI software.

Immunocytochemistry (ICC) and immunohistochemistry (IHC) were used to confirm cell surface expression of TRPV1. Live cells were incubated with rabbit anti-TRPV1 (extracellular) polyclonal antibody (Thermo Fisher Scientific cat#PA5–77361) (1:50) in culture medium for 10 min at 37 °C followed by 5 washes with RT culture medium. Cells were then incubated with goat anti-rabbit Alexa Fluor 568 or 633 (1:1000, ) for 10 min at 32 °C followed by a further 5 washes with RT culture medium. Cells were then fixed in 3.7% paraformaldehyde in Hank’s balanced salt solution (HBSS) for 30 min at room temperature and washed 5 times in HBSS before mounting using Fluoromount with DAPI (Southern Biotech, Birmingham, AL). Images were acquired using a Zeiss LSM 880 inverted confocal microscope (Carl Zeiss, Oberkochen GER).