Sodium thiocyanate

Dextromethorphan hydrobromide, chloroquine, denatonium benzoate, quinine hydrochloride, phenylthiocarbamide (PTC), 6-n-propylthiouracil (PROP), sodium thiocyanate, salicin, nicotine, yohimbine, colchicine, thiamine, caffeine, chloramphenicol and picrotoxinin were purchased from Sigma. Fluo-4 NW calcium assay kit was purchased from Invitrogen. hPASMCs and hASMCs were either purchased from ATCC or were a kind gift from Dr. Andrew Halayko, Dept. of Physiology, University of Manitoba. Cell culture media and hPASMC growth kit were purchased from Cedarlane, Canada. Nucleofection kit for smooth muscle cell transfection was purchased from Lonza. Polyclonal antibodies specific for T2R1, T2R38 and normal IgG specific rabbit antisera were obtained from Abcam (Cambridge, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA) respectively [6] (link), [7] (link). Phospho-MLC-Ser19 monoclonal antibody and MLC polyclonal antibody were purchased from Cell Signaling technology (Danvers, MA, USA). DyNazyme hot start was purchased from Thermo Fisher Scientific (Toronto, ON, Canada). The shRNA specific for T2R1 was purchased from Qiagen (Toronto, ON, Canada).

The paraffin sections on the glass slides were processed for FISH by the sodium thiocyanate (Sigma Aldrich, USA) according to the method mentioned elsewhere21 (link). For performing FISH on cells cultured from the TCV, the cells were harvested using 0.25% trypsin-EDTA and processed according to the manufacturer’s instructions (Abott Molecular Inc., USA). The dehydrated slides were air dried and FISH was carried out using CEP® X Spectrum Orange™ / Y Spectrum Green™ DNA Probe Kit (Abott Molecular Inc., USA) according to the manufactures instructions. For each TCV sections, a minimum of 500 cells with proper signals from the captured images of 30 different fields were evaluated for the enumeration of cells of maternal (XX, female) and fetal (XY, male) origin. For spheroids at each tested time points, sections from 5 different spheroids, each containing a minimum of 50 cells from the captured images of 20 different fields were evaluated.

In situ hybridisation for Y chromosome-specific sequences in combination with immunostaining for α-SMA was performed to detect BM-derived myofibroblasts as previously reported [11 (link)] with slight modification. Briefly, sections were cut at 4 µm and incubated in 1 M sodium thiocyanate (S7757; Sigma-Aldrich) to improve access of the probe to DNA. Following PBS washing, sections were digested in 0.4% w/v pepsin (P6887; Sigma-Aldrich) in 0.1 M hydrochloric acid to improve further access of the Y chromosome probe. The protease reaction was quenched in 0.2% w/v glycine (G4392; Sigma-Aldrich) in double concentration PBS and sections were then rinsed in PBS, postfixed in 4% w/v PFA (P6148; Sigma-Aldrich) in PBS, dehydrated through graded alcohols, and lastly air dried. A fluorescein isothiocyanate-labelled Y chromosome paint (1189-YMF-01; Cambio, Cambridge, UK) was used in the supplier's hybridisation mix. The probe mixture was added to the sections, sealed under glass with rubber cement, heated to 60°C for 10 minutes, and then incubated overnight at 37°C in a humidified chamber.
The next day, all slides were washed in 0.5x Standard Saline Citrate. The anti-FITC antibody (ab6656, Abcam) was applied in a dilution of 1 : 100 and slides further developed in DAB and counterstained lightly with haematoxylin, dehydrated and mounted in DPX-type mount.