Fig 4. Secondary screening and ‘hit’ validation.

(A) Dose-dependent inhibition of the NDST1 promoter activity. HeLa cells stably expressing NDST1 promoter were treated with serially diluted potential NDST1 repressors. Dose-response curves were produced from 9 out of 10 drug candidates. Experiments were performed in singles. (B) Western blot densitometry analysis of NDST1 protein expression after treatment with selected potential NDST1 repressors. Normal human fibroblast cells were treated with potential NDST1 inhibitors at their calculated 1x IC₅₀ or 2x IC₅₀. The effect of the compound on the NDST1 expression was evaluated using densitometry analysis of NDST1 band normalized to its corresponding GAPDH band. Distinct decrease in NDST1 expression was observed for 2x IC₅₀ SAHA treatment. (C) ELISA assay of total NDST activity in normal human fibroblast cells. Lysates of cells treated with either SAHA or DMSO were assessed for total NDST activity. Treatment with 1.2 μM of SAHA resulted in a ~ 40% decrease in NDST enzyme activity. Experiments were conducted in triplicates. (D) The effect of SAHA on NDST1 mRNA expression in normal human fibroblast cells. Normal human fibroblast cells were treated with 2x IC₅₀ (2.4 μM) of SAHA for 5 days. The level of NDST1 mRNA was reduced by 60% after the treatment. Expression of NDST1 mRNA level was normalized to 18S rRNA level. Experiments were performed in duplicates. MEC–meclocycline, THIO–thiostrepton, ENTA–enthacapone, MIF–mifepristone, SAHA–SAHA, GRIS–griseofulvin, ANT–antralin, PENT–penthamidine, PYR–pyrimethamine. r.u.–relative units, l.u.–luminescence units.