Multiscan go reader

RNA was extracted from tumor tissue using the AS1480 Maxwell RSC Simply RNA tissue kits (Promega, Madison, WI, USA) according to the manufacturer’s instructions. RNA concentrations were calculated using Multiscan GO reader, V.1.01.10 (ThermoFisher Scientific, Saclay, France). One μg RNA was reverse-transcribed in cDNA using the high-capacity RNA-cDNA kit (ThermoFisher Scientific, Saclay, France) according to the manufacturer’s instructions. cDNA amplification was performed using the TaqMan Universal PCR Master Mix, No AmpErase UNG (ThermoFisher Scientific, Saclay, France) according to the manufacturer’s instructions. TaqMan PCR primers and probes specific for Gli1 target genes and controls β-actin were purchased commercially and used as per the manufacturer’s instructions (ThermoFisher Scientific, catalog number 4331182) (Table S4). All values were normalized to the control gene β-actin using the ΔΔ Ct method. Two samples not expressing Gli1 as assessed by IHC were taken as reference samples or calibrators.

The study was carried out under standard conditions. Cells were obtained from culture bottles by enzyme digestion (trypsin/EDTA), and the cell suspension obtained was centrifuged (200× g, 3 min). The cells were then counted. The density of the cell suspension was 1 × 105 cells/mL. Using a multichannel pipette, cells were dispensed at 100 µL into 96-well plates (1 × 10⁴ cells/well). Before starting the experiment, cells were incubated for 24 h (5% CO₂, 37 °C, 90% relative humidity). This incubation time ensured cell regeneration, adhesion, and transition to the logarithmic growth phase. After this time, 100 µL of medium containing the appropriate sample extracts, control, or blank only, was added to each well. The test plates were incubated for another 24 h (5% CO₂, 37 °C, 90% humidity). The culture medium was then removed from the plates and 50 µL of 1 mg/mL MTT solution was added to each well. The discs were incubated for another 2 h at 37 °C. After this time, the MTT solution was removed, and 100 µL of isopropyl alcohol was added to each well. Five samples of each material were used to make the extract. The reference wavelength of 570 nm was used to read absorbance on a MultiscanGo reader (Thermo Fisher Scientific, Waltham, MA, USA).

The test was performed according to the guidelines of the standard for testing the cytotoxicity of biomaterials. Cells were obtained from culture NHDF. Tests were performed in 96-well plates at 1 × 10⁴ cells/well. Cells were incubated for 24 h (5% CO₂, 37 °C, 90% humidity) so that the cells formed a monolayer on the plate surface. Before the experiment, each plate was checked under a phase-contrast microscope to ensure that cell growth was relatively uniform across the test plate. After 24 h of incubation, the medium was removed from above the cells. Then, 100 µL of medium containing the appropriate sample extracts, control, or blank only was added to each well. The test plates were incubated for a further 24 h (5% CO₂, 37 °C, 90% humidity). After 24 h of incubation, each plate was viewed under a phase-contrast microscope to assess the growth of control and extract-treated cells. The observed changes in cell morphology may have been due to the cytotoxic effect of the test sample extract. The culture medium was then carefully removed from the plates and 50 µL of 1 mg/mL MTT solution was added to each well. The plates were incubated for a further 2 h in an incubator at 37 °C. After this time, the MTT solution was removed and 100 µL of isopropanol was added to each well. Absorbance was read on a MultiscanGo reader (Thermo Scientific, Waltham, MA, USA) at 570 nm.