Novoexpress software

FGF1E-AviTag was labeled with Alexa Fluor 488 C₅ maleimide (Thermo Fisher Scientific) according to manufacturer’s protocol and then biotinylated. U2OS-R1 cells were seeded onto 12-well plates (200,000 cells per well) in full medium and left to attach overnight. Then, medium was removed, cells were washed with PBS buffer and starved with serum-free medium for 4 h. Next, plates were cooled on ice, and labeled FGF1E-AviTag-Biot (500 ng/mL) or labeled FGF1E-AviTag-Biot (500 ng/mL) assembled with non-labeled SA-4A (500 ng/mL) were added to the cells in the presence of heparin, in a serum-free medium supplemented with 1% BSA. After 40 min of incubation on ice, the cells were moved to 37 °C for 15 min to allow for internalization. Then, the medium was removed and the cells were washed with serum-free medium supplemented with 0.2% BSA pH 3.5 (three times, 5 min) and then with PBS buffer (three times, 1 min). Cells were subsequently detached with 10 mM EDTA in PBS buffer, pH 8.0, harvested by centrifugation and resuspended in PBS supplemented with 1% BSA. Cells were analyzed using a NovoCyte 2060R Flow Cytometer and NovoExpress software (ACEA Biosciences, San Diego, CA).

The role of celastrol in cell cycle distribution was monitored by flow cytometry with PI/RNase staining buffer (BD Biosciences, San Jose, CA, USA). Briefly, cells were passaged in 6-well plates at a density of 3 × 10⁵ cells/well. After 24 h, the cells were exposed to various concentrations of celastrol (0, 0.3, 1, 3, and 10 μM) for 24 h. Then, the cells were harvested, fixed with 75% ethanol at -20 °C overnight, and stained with PI/RNase staining buffer for 15 mins. The cell cycle analyses were performed with a NovoCyte instrument (ACEA Biosciences, San Diego, CA, USA.), and the data were analyzed by using NovoExpress software (ACEA).

MSC surface markers were examined by flow cytometry analysis. Parental T-MSCs or clones were resuspended in PBS-based buffer containing 0.5% FBS and 0.1% (w/v) sodium azide and incubated with the following antibodies for 30 min on ice: fluorescein isothiocyanate-labeled anti-human CD11b (ICRF44, mouse IgG₁; BioLegend, San Diego, CA), Alexa Fluor 488 anti-human CD34 (561, mouse IgG_2a; BioLegend), peridinin chlorophyll protein-labeled anti-human CD45 (2D1, mouse IgG₁; BioLegend), allophycocyanin-labeled anti-human CD73 (AD2, mouse IgG₁; BioLegend), phycoerythrin-labeled anti-human CD90 (5E10, mouse IgG₁; BD Biosciences, San Jose, CA), and phycoerythrin-labeled anti-human CD105 (43A3, mouse IgG₁; BioLegend). Marker expression was measured using a NovoCyte flow cytometer and analyzed using NovoExpress software (ACEA Biosciences, San Diego, CA).

For determination of apoptosis, Annexin V/PI kit (BD bioscience) was used to stain cells. Cells were harvested by 0.25% trypsin, washed twice with chill PBS, followed by being resuspended in 250 μl of binding buffer. Staining solution containing Annexin V/FITC and propidium iodide was added in cell suspension. The stained cells were measured by ACEA Flow Cytometer (ACEA, USA). Data was analyzed using Novoexpress software (ACEA, USA).

At harvest, cells were counted on a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, cell culture medium was discarded, and cells were washed twice in PBS, detached from the plates in trypsin-EDTA, resuspended in complete medium, and washed twice in PBS. EGFP-positive cells were detected in the FITC channel. Data were acquired with BD FACSDiva software and analyzed with NovoExpress software (ACEA Biosciences, San Diego, CA, USA). Fluorescent and bright field images were taken on an Olympus IX71 (Olympus Life Science, Tokyo, Japan).

Intracellular Ca²⁺ levels were quantified using a Ca²⁺ quantification kit (Abcam, ab112115) following the standard manufacturer’s protocol. Fluorescence was determined using a microplate spectrophotometer at Ex/Em = 540/590 nm (Varioskan LUX, Thermo). Additionally, cytosolic Ca²⁺ levels were measured by flow cytometric estimation of Fluo-4 AM. The cells were collected and loaded with 5 μM Fluo-4 (Beyotime, ab145254) for 30 min at 37° C, and then resuspended in 500 μL of phosphate-buffered saline. Fluorescence signals were recorded with a flow cytometer at Ex/Em =488/525 nm and analyzed with NovoExpress software (NovoCyte, ACEA Biosciences, San Diego, CA, USA).

An annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was used to assess apoptotic cell death. MDA-MB-231 cells were seeded at a density of 20 × 10⁴ cells/well in a 6-well plate and transfected with the control or GLI1 siRNA at 20 nM. The culture medium was aspirated and replaced with fresh medium 6 h after transfection. At 48 h after transfection, the cells were harvested and fixed with 70% ethanol for 24 h at -20°C. After centrifugation, cells (1 × 10⁶ cells/mL) were resuspended in 1× binding buffer. Then, 100 μL of the solution was transferred to a 5-mL culture tube to which 5 μL of annexin V-FITC and 5 μL of PI were added, and the tubes were incubated for 30 min at 37°C. The stained cells were analyzed using a NovoCyte flow cytometer (ACEA Bioscience, Inc. San Diego, CA, USA). The percentage of cells in the G0/G1, S, and G2/M phases was determined using the NovoExpress software (ACEA Bioscience, Inc.).