18 well μ slides

Cells were seeded in 18-well μ-slides (ibidi GmbH, Gräfelfing, Germany) and incubated with PT and defensin for 4 h at 37 °C. Afterwards, cells were washed with PBS, fixed with 4% PFA, permeabilized and blocked with 10% NGS and 1% BSA in PBS-T. Then, cells were incubated with rabbit anti-PT (Abcam) and mouse anti-HNP (= α-defensin-1) (Santa Cruz) or mouse anti-α-defensin-5 (Abcam) primary antibodies for 1 h at 37 °C. PLA was performed according to the manufacturer’s protocol (Duolink using PLA technology, Sigma-Aldrich, Merck). In brief, cells were incubated with PLA secondary antibodies (anti-rabbit for detecting anti-PT and anti-mouse for detecting anti-defensin) having attached specific oligonucleotide sequences to each. If they get in close proximity, they can form a ring structure. By addition of ligase and polymerase, rolling circle amplification can occur. Samples were probed with fluorescence labeled oligonucleotides, complementary to the amplification product for detection of the protein interaction. PLA signals were counted from fluorescence images using ImageJ software v.1.52a (NIH).

Cells were cultured in 18-well μ-slides (ibidi GmbH, Gräfelfing, Germany), pre-incubated with respective inhibitors, and exposed to PT for a 4 h incubation at 37 °C. Subsequently, the cells underwent a series of steps: they were rinsed with PBS, fixed with 4% PFA, permeabilized, and blocked with a solution containing 10% NGS and 1% BSA in PBS-T. Following this, the cells were treated with primary antibodies, namely rabbit anti-PT (Abcam, Cambridge, UK) and mouse anti-CCT5 (Abcam) or mouse anti-α-defensin-5 (Abcam). This antibody incubation took place for 1 h at 37 °C. PLA was executed in accordance with the manufacturer’s guidelines, utilizing Duolink’s PLA technology (Sigma-Aldrich Merck, Darmstadt, Germany). PLA secondary antibodies were employed, with anti-rabbit antibodies for detecting PTS1 and anti-mouse antibodies for detecting CCT5. These secondary antibodies featured specific oligonucleotide sequences. When these oligonucleotides came into close proximity they could assemble into a ring structure. The addition of ligase and polymerase facilitated rolling-circle amplification. Samples were probed with oligonucleotides labeled for fluorescence, which were complementary to the amplification product, thus enabling the detection of protein interactions. The resulting PLA signals were quantified by counting them from fluorescence images using the Image J find maxima tool.

After bPHA and staining, cells were transferred to 18-well μ-slides (ibidi) and rested for 15 min to allow the attachment of cells to the slides. Samples were then imaged using a Zeiss 780 Meta confocal microscope (Carl Zeiss) equipped with a Zeiss Plan-Apochromat 63× oil immersion objective lens. Images were processed using ImageJ.