Cyclosporine h

A library of twenty-five 20-mer, 10-mer overlapping, peptides was designed based on the amino acid sequence of alpha-gliadin by solid phase synthesis (S1 Table). N-formyl-Methionine-Leucine-Phenylalanine (fMet-Leu-Phe) and cyclosporine H were purchased from Sigma (St. Louis, MO). Unlabeled and FITC-labeled gliadin peptide, TLPAMCNVYIPPYCTIVPFG, and FITC-labeled fMet-Leu-Phe were synthesized and conjugated in the Peptide/Protein Core Facility (Massachusetts General Hospital, Charlestown, MA. Director: A. Khatri).

For analysis of ΔΨm, cells were plated into 384- or 96-well plates (CellCarrier, PerkinElmer). After 24–48 h in culture, cells were rinsed with 10 mM HEPES buffered saline solution (HBSS—Hank’s Buffered Salt Saline, Invitrogen, pH 7.4 with 1 g/L of glucose) and subsequently incubated with 20 nM TMRM (Thermo-Fisher Scientific) in the presence of 1 μM cyclosporine H (Sigma) for 30 min at 37 °C; the same buffer was used during image acquisition. Alternate brightfield, Hoechst 33,342 (Ex:360–400/Em:410–480 nm), and TMRM fluorescence (Ex:520–550/Em:560–630 nm) images were acquired sequentially, by using different magnification air objective (10 × −20 × −40 ×) of the high-content screening (HCS) imaging system Operetta^® and Harmony^® 4.8 analysis software (PerkinElmer). After acquisition of basal fluorescence intensity, cells were treated with oligomycin and then with FCCP. The analysis was performed by means of Harmony^® software (PerkinElmer), as summarized in Supplementary Figures S1, S2, S4, and S5. Briefly, image segmentation was performed via detection of regions of interest (ROI; each ROI corresponds to a single cell) in the Hoechst 33,342 channel. Background-corrected TMRM fluorescence intensity was then measured per each ROI and averaged. In the next paragraphs, details for each model will be provided.