Culture flasks

RAW 264.7 mouse monocyte macrophage cell lines used as an osteoclast precursor were seeded (5 × 10⁶ cells/well) in culture flasks (75 mL BD Falcon, Franklin Lakes, NJ, USA) as previously described [28 (link)]. The cells were maintained in DMEM containing 10% Fetal Bovine Serum (FBS), 100 U/L penicillin G, and 100 mg/L streptomycin at 37°C with 5% CO₂. The medium was changed every 2 days.

In the presence of βGal, DDAOG substrate is hydrolysed to DDAO, which generates a far-red fluorescent signal. The staining protocol was adopted from Gong and colleagues with slight modifications (Gong et al. 2009 (link)). Briefly, cells were seeded into culture flasks (BD FALCON) cultured in DMEM containing 10% FBS for 24 h and subsequently treated with 0.1 µM Bafilomycin A1 to induce lysosomal alkalinisation and 0.02 mM DDAOG for 2 h at 37 °C. Cells were washed twice in PBS, detached by TryplE (Gibco, Life Technologies, USA) at 37 °C for 5 min for harvesting. The cells were stained for viability with a fixable viability dye (FV450; 1:1000) in PBS for 15 min at room temperature. They were then washed with 1× BD Wash buffer, followed by a 5-min centrifugation at 300×g before surface marker staining. Cells were then permeabilised and fixed with 200 µl BD Cytofix/Cytoperm solution for 30 min at room temperature. After incubation, the cells were washed twice in 1× BD Perm/Wash buffer before intracellular staining with antibodies. A 5 µl/sample of PerCP-Cy™5.5 Mouse Anti-H2AX (pS139 BD Biosciences) and 1:500 Anti-CDKN2A/p16^INK4a (EPR1473, Abcam, ab209579) antibody was added and incubated at room temperature for 30 min in the dark. Cells were washed twice in 1× BD Perm/Wash buffer prior to analysis on BD FACSCanto II flow cytometer (Beckton Dickinson, BD Bioscience).

The hTert-RPE1 and U87-MG cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) or Eagle’s minimal essential medium (E-MEM), respectively, supplemented with 10% (v/v) fetal bovine serum and 0.1% (w/v) penicillin and 0.1% (w/v) streptomycin. Cells were maintained in culture flasks (Falcon or Corning) at 37 °C and 5% CO₂ in a humid atmosphere in an incubator and subcultured (passaged) every 2 to 3 days after obtaining 80% to 90% confluence. Cells were seeded on 18-well glass-bottom dishes (ibidi) one day before transfection and then transfected using Lipofectamine 3000 (Thermo) or JetOptimus (Polyplus) according to manufacturer’s instructions. Cells were incubated in the incubator and visualized 24 h to 48 h post transfection.
Transfected cells were imaged using a Leica SP8 confocal microscope enclosed in an environmental chamber with constant temperature (36.9 °C), humidity (95%) and 5% CO2 levels. Images were acquired using a 63x/1.40 N.A. oil objective. Filter and detector settings were optimised to minimise any possible bleed-through between fluorescence channels.
Fluorescence images were adjusted in Fiji^{74 (link)}. All acquired z-stack were processed using Maximum intensity projection and Brightness/Contrast function. Only the Tub1C1 was additionally processed using Substract Background function with 150 pixels rolling ball radius.

Femurs and tibiae were removed from the mice and the BM cells were flushed out. The washed BM cell suspensions were seeded in culture flasks (BD Falcon, Bedford, MA) in DMEM F12 medium (Thermo Fisher Scientific, Bremen, Germany) supplemented with 10% FBS and 5% horse serum (Invitrogen). After two days, the non-adherent cells were removed, and the adherent cells were further propagated for characterization of the BM-MSC phenotypes (as previously described and demonstrated in^{13 (link)}). Osteogenic differentiation was induced using DMEM supplemented with 10% FCS, 10 mM β-glycerophosphate (Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma) and 0.01 μM hydrocortisone (Sigma-Aldrich). The cultures were incubated for 21 days and the medium was changed every 3 days.

For the temperature treatment, 500 μl of an exponentially growing QPX (isolate NY0313808BC7) culture was inoculated into twelve 25-ml culture flasks (Falcon) filled with 5 ml of Minimum Essential Medium (MEM, Sigma) supplemented with 10% of fetal bovine serum (FBS, Sigma). The flasks were incubated at four different temperatures: 27°C, 23°C, 13°C and 10°C, in triplicates (except 10°C which was done in duplicate) for each treatment. After seven days of incubation, the cultures were diluted with equal volume of filter sterilized artificial seawater and passed several times through a syringe to facilitate liquefaction of QPX mucus secretion. The mixtures were then transferred into 15-ml conical tubes and centrifuged at 3000 g for 40 minutes at 4°C. The supernatant was discarded and cell pellets collected and kept on ice for immediate RNA extraction. Trizol reagent (Molecular Research Center, Inc.) was used to isolate RNA from all samples following manufacturer’s protocol. RNA quality and quantity were estimated spectophotometrically using a Nandrop spectrophotometer.