Nhdf ad

We used a collection of 9 stem cell models of glioblastoma (HSR-GBM1 [23 (link)]; JHH520 [24 (link)]; NCH421k, NCH644 [25 (link),26 (link)]; BTSC-23, BTSC-233, BTSC-268, BTSC-349, BTSC-407 [27 (link)]), and exposed them to a library of clinical drugs (231 substances, each in 9 different concentrations; 4.33 nM to 25 $\mathsf{\mu }$ M) using a semi-automated screening platform. For each cell model, repetitive drug resistance tests were performed: two biological (except for BTSC-23) and three technical replications. For each biological replication, the mean of the technical replications was used. Undifferentiated and immortalized human neural progenitor cells (ReNcell^®CX, Sigma-Aldrich, St Louis, MO, USA) and neural stem cells (H9-Derived, Gibco) were used as healthy controls to evaluate the toxicity of the drugs. Additionally, we also tested the inhibition efficiency using three different normal adult human dermal fibroblasts (NHDF-Ad, Lonza, Basel, Switzerland). Effects on cell growth were assessed 72 h after substance exposure using the CellTiterGlow^® assay (Promega, Madison, WI, USA). All procedures were in consent with the local ethical commission oversights.

Normal Human Dermal Fibroblasts derived from an adult donor (NHDF-Ad, Lonza, CC-2511) were cultured using Dulbecco’s modified Eagle medium (DMEM, Thermofisher Scientific, 41965-039) supplemented with 10% FBS (Thermofisher Scientific, 10270-106), 1% Insulin-Transferrin-Selenium (Thermofisher Scientific, 41400045) and 1% penicillin-streptomycin (Thermofischer Scientific, 10378-016). Cell cultures were routinely checked for mycoplasma. CO₂-independent media was prepared by using CO₂-independent DMEM (Thermofischer Scientific, 18045-054) supplemented with 10% FBS, 1% penicillin-streptomycin, 1.5% HEPES 1 M, and 2% L-Glutamine (Thermofischer Scientific, 25030-024). One day before experiments, cells were co-transfected with the membrane-targeting plasmid peGFP-mem and the Amph1-pmCherryN1. Transfection was performed using the Neon transfection device according to the manufacturer’s instructions (Invitrogen). peGFP-mem was a kind gift from Pr. F. Tebar. and contained the N‐terminal amino acids of GAP‐43^{51 (link)}, which has a signal for post‐translational palmitoylation of cysteines 3 and 4 that targets fusion protein to cellular membrane, coupled to a monomeric eGFP fluorescent protein. Amph1-pmCherryN1 was a kind gift of Pr. De Camilli and contained the full-length Amphiphysin 1 coupled to a mCherry fluorophore.

The cultured NHDF-Ad (CC-2511, Lonza, Basel, Switzerland) in confluent phase of growth and at a final number of 2 × 10³ cells per 1 well were used for the test. The cells were seeded in 96-well plates to which CsA was added at a concentration range from 1.0 to 10,000 ng per 1 mL of the medium (Sandimmun, Novartis, Basel, Switzerland) and cultured for 8, 24, 48 h in standard conditions. Each procedure included a two blank sample with a fresh medium with and without cells. After incubation time the viability assay was performed using 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) according to the standard producer’s protocol (M2128; Sigma-Aldrich). After 4 h of incubation with dye and liberation of the incorporated dye, absorbance was measured at λ = 570 nm and λ = 690 nm (reference wavelength) [38 (link)] using the MRX Revelation plate reader (Dynex Technologies, Chantilly, VA, USA). The viability test was repeated three times and there were eight replicates for each concentration of CSA.