Plan apochromat 100x 1.4 na

Cells were incubated with 100–500 HPV16 PsVs particles per cell, washed in PBS and fixed with 2% paraformaldehyde (PFA) prepared in PBS for 10 min at room temperature (RT). For EGFR co- staining, the cells were fixed with 2% PFA and treated with 0.2% Triton X-100 in PBS for 2 min at RT. Fixed cells were stained using primary, Alexa-conjugated secondary antibodies, and Hoechst33342 (Invitrogen). Fluorescence imaging was performed using a Zeiss Axiovert 200 M microscope equipped with a Plan-Apochromat 100x (1.4 NA) and Axiovision deconvolution and colocalization software 4.7 (Carl Zeiss, Jena, Germany). For determining amount of L1 (viruses) colocalizing either with CD151 or EGFR at 5 hr after PsVs exposure, we used randomly chosen rectangular areas of ~130 µm² and normalized assessed L1 pixels colocalizing with CD151 to the total L1 pixels.

HeLa cells were transfected with siRNAs targeting CD9. Two days later, the cells were infected with HPV16 PsVs (with ≈ 100 particles per cell) and incubated at 37 °C for 7 h. Subsequently, the cells were fixed with 100% methanol and processed for staining with mAb L1-7 as described previously [13 (link)]. This mAb recognizes a specific epitope located in the interior of the pseudovirion capsid and is not accessible in intact virions. The samples were analysed by fluorescence microscopy using a Zeiss Axiovert 200 M inverted microscope equipped with a Plan-Apochromat 100x (1.4 NA) (Carl Zeiss, Jena, Germany) and quantified by ImageJ software (https://imagej.nih.gov/ij/). For quantification, the internalized particles were determined based on the L1-7 positive pixels relative to the cell nucleus signal (DNA/Hoechst 33342-positive pixels). Quantification was performed by analysis of at least 20 images (3–5 cells per image).

NHEK or HeLa cells were grown on coverslips. After transfection and/or infection (with approximately 200 particles per cell), cells were treated with paraformaldehyde/Triton X-100 or methanol. For LBPA staining, cells were fixed with 4% paraformaldehyde, and afterwards incubated with 50mM NH₄Cl. Fixed cells were stained with the indicated primary antibodies (LBPA in the presence of 0,5% saponin51 (link)) and with Alexa-conjugated species-specific secondary antibodies (Invitrogen, Carlsbad, CA, USA). DNA was stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA). Coverslips were mounted onto slides using Fluoprep mounting medium (bioMérieux). Images were acquired using the LSM 710 (Carl Zeiss, Jena, Germany) and the LSM software ZEN 2008 or using a Zeiss Axiovert 200 M microscope equipped with a Plan-Apochromat 100x (1.4 NA) and Axiovision deconvolution and colocalisation software 4.7 (Carl Zeiss, Jena, Germany). Image files were assembled into figures using InDesign software (Adobe). For colocalisation analysis, cells were grown on coverslips and/or transfected with siRNA. Cells were infected with pseudoviruses for 7 or 8 (EdU-viruses) hours and stained with antibodies as indicated. Colocalising pixels of at least 10 images (3–5 cells per image) were analysed using Colocalisation Software 4.7 (Carl Zeiss, Jena, Germany).