Additional file 3 of N-acetyl cysteine induces quiescent-like pancreatic stellate cells from an active state and attenuates cancer-stroma interactions

Additional file 3: Supplementary Figure S1. Analyses of α-SMA and CK19 immunofluorescence staining in SUIT-2 or PSCs isolated from PDAC patients. PSCs presented a fibroblast-like appearance and expressed α-SMA, but not CK19 (original magnification: × 200). Pancreatic cancer cell (PCCs) line SUIT-2 expressed CK19, but not α-SMA. Supplementary Figure S2. Toxicity effects of NAC to PSCs and PCCs. (A-B) IC50 values were measured in PSC1–3 and PCCs by determined by CellTiter-Glo luminescent cell viability assay after 72 h treatment with indicated NAC concentrations. (C) Representative microscopy time-lapse images of PSC1 and PSC2 cells treated with 6 mM NAC in which concentration toxic to PSCs. (D) (Left) PSC1 and PSC2 cells were treated with 2.5 mM NAC for 48 h, stained with annexin V-FITC/PI, and then analyzed by flow cytometry. (Right) Quantification of apoptotic cells induced by NAC treatment. (E) the expression of cleaved caspase-3, a marker of apoptosis, was determined by western blotting in PSCs. PSCs were treated with PBS (as control), 2.5 mM NAC, or 20μM H2O2 (positive control). (F) the expression of α-SMA, an active marker of PSC, was determined by western blotting in PSC2, which treated with different concertation of NAC with or without supernatant from SUIT-2 (SUIT-SN). (G) Migration of PSC2 after 1 mM NAC treatment with or without SUIT-SN was analyzed. Migrated cell numbers were normalized by total cell numbers. H&E staining; original magnification, × 100. Scale bar = 100 μm. (H-I) Migration and invasion assays of PSCs and PCCs from Fig. 1c-d. Graphs show numbers of cells calculated from five fields. The exact number of migrated cells was counted. Scale bar = 100 μm. H&E staining; original magnification, × 100. (J) Effects of NAC were performed in PCCs and PSCs on mRNA expression of Nrf2, HMOX-1, NQO1, and GCLC. PBS was used as Control. *P