Fungizone amphotericin b

The primary sarcoma cell lines (AA, AX, AW, BC, BD, BG, CE, and CP0024) used in this study were previously characterized.^{29 (link),41 (link),61 (link),73 (link)} A673, Saos-2, SK-UT-1, 93T449, HT-1080, and SW872 were commercial sarcoma cell lines. AA, AW, BC, BD, BG, and CE cells were maintained as a subconfluent monolayer in F-10 medium (Sigma). AX, Saos-2, SK-UT-1, 93T449, HT-1080, and SW872 were cultured using DMEM (Sigma), while A673 and CP0024 were maintained in RPMI (Sigma). Other characteristics of each cell line appear in Supplementary Data Table S5. All media were supplemented with 10% FBS, penicillin–streptomycin antibiotics (Sigma) and Fungizone (Amphotericin B, Sigma) as previously described.^{29 (link),41 (link),61 (link),73 (link)} All cell lines were authenticated and regularly tested for mycoplasma. AA cell line, transfected to overexpress MAP17, and AX cell line, transfected with a shRNA against MAP17, were previously described^{50 (link)} and maintained in complete DMEM media supplemented with puromycin 0.5 μg ml⁻¹.

The mouse corticotrope tumor cell line AtT-20, secreting adrenocorticotropic hormone [30 (link),31 (link)], was kindly provided by Dr. Ulrich Renner and Prof. Dr. Günter K. Stalla from the Clinical Neuroendocrinology Group, Max Planck Institute of Psychiatry, Germany. The rat Wistar-Furth pituitary tumor cell line GH3, secreting growth hormone and prolactin [32 (link)], was obtained from the Cell Bank of Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. The cell lines were maintained in Dulbecco’s modified culture medium that was supplemented with 10% fetal bovine serum, antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, LGC Biotechnology, São Paulo, Brazil), and 5 mg/mL fungizone (amphotericin B) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO₂.

The sarcoma cell lines used in this study were previously characterized [11 (link), 31 (link), 32 (link)]. The cells were maintained as a subconfluent monolayer in F-10 medium (Sigma) supplemented with 10% FBS, penicillin-streptomycin antibiotics (Sigma) and Fungizone (Amphotericin B, Sigma). Each cell line was cultured at 37°C and 95% humidity in 5% CO₂ under conditions of O₂ levels, culture medium and supplements indicated in the provider's instructions.

Primary normal human trabecular meshwork (HTM) cells were harvested from donor eyes (n = 4) obtained from the Liverpool Research Eye Bank and approved by the local ethics review board (RETH000833). Samples were handled in accordance with the Declaration of Helsinki. Eyes were obtained from the Royal Liverpool University Hospital Mortuary and medical history was unknown; although no donor had previous ocular surgery or a known diagnosis of glaucoma. Donor information is listed in Supplemental Figure S1. Donor eyes were excluded if the maximum post-mortem time exceeded 48 h and HTM cells were isolated using the blunt dissection method as reported previously [24 (link)]. Cells were maintained in Dulbecco’s Modified Eagle Media (DMEM)-low glucose (Sigma, Gillingham, UK) supplemented with 10% fetal calf serum (Biosera, Heathfield, UK), 2 mM L-glutamine (Sigma, Gillingham, UK), Pen/Step (Sigma, Gillingham, UK), and 2.5 µg/mL Fungizone (amphotericin B, Sigma, Gillingham, UK). Samples were incubated at 37 °C (5% CO₂ and 95% humidity). HTM characterisation was carried out as previously described [24 (link)] and included upregulated myocilin protein expression in response to dexamethasone treatment (polyclonal rabbit anti-myocilin primary antibody was a kind gift from Dr. W. Daniel Stamer) as previously described by our group [25 (link)].

The sarcoma cell lines used in this study were previously characterized [53 , 54 ]. The cells were maintained as a subconfluent monolayer in F-10 medium (Sigma) supplemented with 10% FBS, penicillin-streptomycin antibiotics (Sigma) and Fungizone (Amphotericin B, Sigma). Each cell line was cultured at 37°C and 95% humidity in 5% CO2 under conditions of O2 levels, culture medium and supplements indicated in the provider's instructions.

3−aminopropyltriethoxysilane, 4-α-Phorbol 12,13-didecanoate (4αPDD), capsaicin, capsazepine, EDTA, eugenol, fungizone (Amphotericin B), L-glutamine-penicillin, paraplast^®, 3−aminopropyltriethoxysilane, streptomycin solution, ruthenium red, and RPMI culture medium were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Melatonin standard was from Acros Organics™ (Fisher Scientifics, Villebon- sur-Yvette, France). Acetonitrile and hydrogen peroxide solution-HPLC grade were from Fisher Scientifics (Villebon- sur-Yvette, France). RNA later was from Life technologies SAS (Saint Aubin, France). The DIG labeling kit was from Roche Diagnostics (Meylan, France). The polyclonal rabbit anti-zebrafish antibodies (anti-TRPV1, Ab68969; anti-TRPV4, Ab69094) were from Abcam (Cambridge, England). The IHC revelation kit (IHC Select^® HRP/DAB) was from Merck-Millipore (Molsheim, Alsace, France).