Figure 1. Establishment and characterization of an MCA205 cell line expressing tetracycline-inducible Rtn-1c. (A) Vectors used to stably transduce murine fibrosarcoma MCA205 cells to generate tetracycline (TET)-inducible iRTN-1c MCA 205 cells. (B) Western-blot analysis of the expression of reticulon-1 (Rtn-1C) in control conditions (CT) or upon TET treatment for 12 or 24 h. β-actin was used as loading control. A representative experiment is shown. (C‒F) Immunogenic cell death markers of iRTN-1c MCA 205 cells analyzed in response to 0.3 µM TET, 150 µM cisplatin (CDDP), CDDP + TET, 1 µM MTX, or no treatment. (C) Extracellular ATP as measured by luciferin-luciferase assay, (D) Immunofluorescence staining of calreticulin (CRT) exposure to the surface, (E) HMGB1 release from cells as detected by ELISA. (F) Apoptosis assayed by Annexin V detection of externalized phosphatidylserine and secondary necrosis as detected by staining with the vital dye propidium iodide. MTX treatment was used as a positive control. Tetracycline treated samples were compared with their untreated counterpart. Results are reported means ± SEM of triplicates. *P < 0.05, **P < 0.01 (unpaired Student’s t test).