Hoescht

Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester (TMRM, Thermo Fisher Scientific, Eugene, OR, USA). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich, Saint Louis, MO, USA) was used as a negative control and wells without TMRM or CCCP were included as blanks. Additional staining with Hoescht was used to normalize the values to the cell number. LRMSC were seeded in black 96-well plates with a clear bottom (SPL, 33,396) at 10,000 cells/well. The next day, the cells were washed once with PBS and incubated in 2% FCS, 1% P/S, and αMEM medium containing the drugs. At least 4 replicates were used per condition. After 30 min of incubation time, the cells were washed with PBS and fluorescence was measured with a Synergy H1 microplate reader at 548/574 nm. The cells were further incubated with Hoescht (Sigma-Aldrich, Saint Louis, MO, USA) at 5 µg/mL for an additional 30 min and the plate was read again at 355/546 nm.

Fluorescent in situ hybridization (FISH) experiments were performed on 4 mice per group to confirm the lack of Malat1 in cell nuclei of Malat^−/− mice and to qualitatively evaluate the effect of hypoxia on Malat1 expression in Malat^+/+ mice. Mice were anesthetized and then perfused through the heart with sterile saline solution until the liver was translucent. The lung was subsequently excised and fixed by perfusing formalin (10%, containing 4% paraformaldehyde) for 5 min. The fixed lung was embedded with OCT and cut in 10 μm-thick sections. Stellaris® FISH probes, consisting of a set of mixed oligonucleotides recognizing mouse Malat1 (NCBI gene ID:72289, NR_002847.2, from nucleotides 751 to 6982) and labelled with QASAR 570 Dye (SMF-3008-1, Biosearch technologies, Inc., Petaluma, CA, United States), were hybridized as recommended by the manufacturer onto at least two lung sections per mouse. Nuclei were stained with Hoescht (Sigma-Aldrich, ON, Canada) in Fluoromount G medium (Electron Microscopy Sciences, Hatfield, PA, United States). Imaging was performed on a Zeiss Observer Z1 (objective 63X with oil) coupled with LSM 800 lasers system (Zeiss, ON, Canada) and analyzed with Zen Blue software (Zeiss, ON, Canada). Fiji ImageJ software was used to quantify RNA FISH signals on a total of 3–5 pictures per animal.

Migration/invasion assays were performed using BD cell culture inserts (BD Biosciences, San Jose, CA, USA) with a pore size of 8 μm on 24-well culture plates. For invasion assays, the inserts were first coated with 10% Matrigel^TM diluted in serum-free media. Cells were seeded (1 × 10⁴ cells for migration assay and 2 × 10⁴ cells for invasion assay) with serum-free media in the upper chamber only, while 10% FBS containing media in the bottom well was used as a chemoattractant. After incubation for 24 h, the transwell inserts were washed with PBS and fixed with 4% PFA for 15 min. The non-migrated and non-invaded cells were removed using cotton buds. Migrated or invaded cells on the lower surface of the insert were stained with Hoescht (Sigma-Aldrich, St. Louis, MO, USA) solution (1% triton X 100 and 4 μg/mL Hoechst in PBS) and incubated in the dark at room temperature for 15 min. After rinsing with PBS, cells were counted under the fluorescence microscope.

For confocal microscopy studies, BMDCs were stained 30 min with Hoescht (3 nM, Sigma-Aldrich) and washed twice. Then, the cells were suspended in culture medium and incubated at 37 °C 5% CO₂ in 8-well glass chambers (Ibidi GmbH, Gräfelfing, Bayern, Germany) for 2 h in the presence, or not, of FITC-NanoMBGs. Stained cells were visualized using a confocal laser scanning microscope TCS SP5II confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). FITC fluorescence was excited at 488 nm and measured at 491–586 nm and Hoechst fluorescence was excited at 405 nm and measured at 420–480 nm. Signals from different fluorescent probes were taken in parallel. Immunofluorescence analysis was done using a 63 × 1.4 objective lens. A 0.772 µm thickness section was performed. Image processing was performed using the Fiji ImageJ 1.52p public software.

For indirect immunofluorescence, cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100/PBS for 5 min at room temperature (RT), followed by a blocking step with 2% bovine serum albumin (BSA) and 10% horse serum for 30 min to 1h at RT. Primary and secondary antibodies were diluted in blocking solution and incubated sequentially for 1h at RT. Samples were then mounted with fluorescent mounting medium (Prolongold, Thermofischer) with Hoescht (Sigma) and imaged by confocal microscopy using the Zeiss SP85 confocal microscope, with 40× or 63× 1.4 NA Plan Apochromat oil-immersion objectives.