J61899

At day 0, cells were plated at $3000\mathrm{cells}/{\mathrm{cm}}^2$ ( ${\displaystyle 2.25\times {10}^5}$ cells seeded in a T-75 flask). After 24 hr of incubation at 37°C, 5% CO₂, expression of exogen desmin was induced by supplementing the culture medium with 10 µg/mL doxycycline (D9891, Sigma-Aldrich) for 2 days. The medium was replaced with fresh medium every 24 hr. Iron oxide nanoparticles were incorporated into the cells on day 3. After 2 hr of recovery, a heat shock (HS) with a water bath at 42°C for 0, 30, or 120 min was applied to the cells. 2 hr later, cells were detached and used to form spheroids by magnetic moulding or seeded in 24-well plates for desmin aggregation evaluation. After an overnight incubation at 37°C, 5% CO₂, spheroids were magnetically flattened to measure their mechanical macroscopic properties, then fixed for 1 hr at room temperature in 4% paraformaldehyde (PFA, J61899, Alfa Aesar). The corresponding cells grown in 2D in the 24-well plates were fixed for 15–20 min at RT in 4% PFA to assess the percentage of cells with Myc aggregation for each condition.

The cell culture medium was aspirated and 75 μL of 4% paraformaldehyde (PFA) in phosphate-buffered saline (J61899, Alfa Aesar, USA) was added to all the perfusion inlet and outlet wells [10 (link)]. The device was placed on a rocker to induce flow and incubated overnight at 4°C. After fixation, the PFA was aspirated from the wells and the microfluidic chips were washed three times with 0.3% Triton X-100 in PBS in all the perfusion inlets and outlets, followed by a permeabilization step of 1% Triton-X100 (VWR 28817295). Nuclei were stained using 1:2000 Hoechst 33258 (H3569, Life Technologies, USA). Tissue construct was incubated with primary antibodies for overnight; 10 μg/mL αβ3-tubulin, AF488 (Invitrogen, 53-4510-82), 0.36 μg/mL αCD31, Rabbit (Invitrogen, PA5-16301), 5 μg/mL αPECAM1 (Millipore Sigma, WH0005175M1), 10 μg/mL αVE-Cadherin, Rabbit (Sigma-Aldrich, V1514), and 5 μg/mL αZO- 1, Mouse, AF488 (Invitrogen, 339188). Secondary antibodies with blocking solution are further incubated; 2 μg/mL αMouse, AF568 (Invitrogen, A-11004), 4 μg/mL αRabbit, AF568 (Invitrogen, A-11036), and 2 μg/mL, αRabbit AF488 (Invitrogen, A-11008). Image data was acquired and processed using confocal microscope (ZEISS Multiphoton LSM 710) and ZEN software. Imaging depth was set at 16 bits, binning at 1, and imaging resolution at 2048 × 2048. Autofocus was set at a 120 μm offset from channel bottom.

Cells were seeded in 35 mm glass bottom dishes (MatTek P35G-1.5-14-C) and treated as needed. At the end of treatment, cells were washed with PBS, and pre-extracted for 1 min with extraction buffer (50 mM PIPES pH 6.9, 25 mM KCl, 3 mM EGTA, 1 mM MgSO₄, 0.5% Triton X-100), followed by fixation in 4% paraformaldehyde in PBS (Alfa Aesar J61899) for 20 min at 4°C. Cells were washed 2× with PBS, permeabilized with 0.5% Triton-X100 in PBS for 10 min at RT, washed 2× with PBS and blocked for 1 h at RT (1% BSA, 10% goat serum in PBS). Primary antibodies were incubated overnight at 4°C in 1% BSA in PBS. Cells were washed 3x/5min with PBS, and incubated with secondary antibodies (1:2000, Invitrogen goat anti-mouse or anti-rabbit IgG (H+L) Alexa Fluor 488) in 1% BSA in PBS for 1 h at RT. After washing with PBS and drying, coverslips were mounted with Prolong Gold Antifade with DAPI (Invitrogen P36935). At least 50 cells were analyzed per condition. Images were taken with a Nikon Eclipse Ti-E epifluorescence microscope equipped with a 100x objective (Plan Apo, 100× 1.45, oil), a Photometrics CoolSnapHQ2 camera and appropriate filters (DAPI, GFP, Texas Red and Cy5). The numbers of foci per cell were quantified using Nikon Elements Software. Experiments were repeated in triplicate.