Dapi mounting medium

HUVECs were pressurised to 0, 120 mmHg alone or 120 mmHg with inhibitors apocynin (3 µM), BEC 100 µM), or SN50 (20 µM) for 30 min. and then fixed in 4% formaldehyde and stained as previously described^{68 (link)}. Briefly, cells were blocked for 1 h in PBS (0.3% TritonX100, 5% normal rabbit serum) and then incubated overnight at 4 °C with primary antibody (1:50; NFĸB; Cell Signaling Technology, Danvers, MA, USA) in PBS (0.1% tween20, 0.1% BSA). Cells were washed with PBS and incubated for 1 h at room temperature with the secondary antibody alexa fluor 546 (1:500; Thermo Fisher Scientific, Waltham, MA, USA) in PBS (0.1% tween 20, 0.1% BSA). Cells were then washed, counterstained and mounted in DAPI mounting medium (5 µl; Thermo Fisher Scientific, Waltham, MA, USA) and z-stack imaged using a Nikon AR1 confocal microscope (Monash Micro Imaging, Clayton, VIC, Australia).

Mid-sagittal sections (4 μm thick) of paraffin-embedded clinical synovial samples were deparaffinized and rehydrated. Antigen retrieval was conducted by soaking slides in Tris–EDTA pH9.0 in a microwave oven for 10 min. After soaking three times in PBS, slides were administered with 3% hydrogen peroxide for 10 min at room temperature. Next, slides were blocked with 10% bovine serum (Solarbio, Beijing, China) for 1 h at room temperature and incubated with primary antibodies at 4 °C for 16 h. Incubation of fluorescent dye lasted for 1 h at room temperature, then slides were mounted with DAPI mounting medium (Thermo Fisher Scientific). Agents applied for IF assay in this study include: rabbit anti-IGF2BP3 (Proteintech, 1:200, 14642-1-AP), mouse anti-CD68 (Proteintech, 1:200, 66231-2-Ig), species-matched Alexa-488 or -594-labeled secondary antibody (Life Technologies, Carlsbad, CA, USA).

Cells were cultured for 24 h after seeded on glass coverslips and then 2.5% bovine serum albumin was used as blocking reagent for 1 h. After that the cells were conjugated with PE anti-mouse CD11c antibody (Biolegend, San Diego, CA, USA) or PE anti-mouse CD206 antibody (Biolegend) at 25 °C for 1 h. The samples on the coverslips were gently mounted with 10 μL DAPI mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). The coverslips were sealed with the nail oil and images captured with Leica TCS SP5 confocal microscope.

The chromosomes spread assays to determine the aneuploidy induction were conducted after 5–7 days selection with puromycin following the infection with virus containing the ORP3-targeting shRNA vectors to eliminate the non-infected cells. The Y235T cells were then seeded on coverslips in 6-well plates. After 48 h, cells were treated with 10 µg/ml KaryoMAX^® Colcemid (ThermoFisher Scientific, Waltham, USA) for 4–6 h and washed three times with 1xPBS and incubated 20 min in 2 ml of 0.075 M KCl at 37 °C. Cells were then fixed with cold metaphase fixation solution (acetic acid: methanol 1: 3) for 15 min. After adding a drop of DAPI mounting medium (ThermoFisher Scientific, Waltham, USA) mounting medium on observation slides, the cover slides were reversed and placed on the drop of DAPI. The slides were dried under dark room overnight and analyzed under the Zeiss TCS SP5 confocal microscope with a 100 × objective.

Lymphoma cells were seeded at 3·10⁵ cells/mL (6 mL) in a cell culture dish and incubated at different times (15, 24 and 48 h) with 5 nM T22-PE24-H6 nanoparticle or buffer. Afterwards, cells were centrifuged at 400 g for 5 min, washed with PBS, centrifuged again, resuspended with 1 mL of 3.7% paraformaldehyde and fixed for 10 min at -20 ºC. Then, cells were washed, resuspended with ~10 µL of PBS and placed on a slide. Finally, slides were stained with DAPI mounting medium (Thermo Fisher Scientific) and visualized using a fluorescence microscope (Olympus BX53, Olympus). Representative pictures were taken using an Olympus DP73 digital camera and processed with the cellSens Dimension 1.9 software (Olympus) at 1000 magnifications.

Hematoxylin-eosin sections were prepared as in (22 (link)). For immunofluorescence staining, the samples were flash-frozen in liquid nitrogen and embedded into anOptimal cutting temperature compound (OCT) medium. Sections of 14 μm were prepared using the Frigocut 2800E (Reichert Jung/Leica) and were hydrated in PBS at RT for 10 min. The samples were fixed with acetone at 4°C for 15 min and washed in PBS. Staining (listed in Supplementary Table 2) was performed overnight at 4°C. Counterstaining was performed with DAPI mounting medium (ThermoFisher). Images were taken in AxioImager (Zeiss). Quantification of the epidermal thickness was performed in ImageJ (U.S. National Institutes of Health) as described in (25 (link)). Quantification of the immune populations infiltrating the ear was performed by measuring the percentage of the positive area and normalizing it to DAPI with ImageJ.