Figure 2.

Drug screening for cystine content and apoptosis. (A) High throughput screening for cystine level normalized for protein content in CTNS^−/− ciPTECs treated with 1200 small molecules of Prestwick chemical library and incubated for 24 h. Black dots: individual library compound at a final concentration of 10 µM; blue dots: DMSO (vehicle as negative control); green dots: cells treated with 100 µM of cysteamine (positive control). (B) High-content screening of small molecules that reduce apoptosis in CTNS^−/− ciPTECs. Relative caspase 3/7 positivity of each well was shown; percent values of each well were normalized with the average percent of apoptosis in untreated cells exposed to apoptosis stimuli (yellow line) of each plate. Each black dot represents the mean value obtained with each compound. Blue dots show the same results for non-induced cells that were exposed to vehicle. After plotting the results, an arbitrary threshold was selected, which made it possible to identify 27 compounds that reduced by at least of 40% the apoptosis rate (red dash line). All experiments were performed in triplicates in different plates. (C) HTS and HCS data sets for cystine content and apoptosis rate were combined in a single plot, allowing to identify 6 drugs that potentially corrected both phenotypes (red dots). (D) Dose-response curve of cystine-depleting lead compounds was generated using four-parameters logistic regression model to interpolate data.