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. 2020 Mar 26;8(1):e000462. doi: 10.1136/jitc-2019-000462

Figure 4.

Figure 4

Validation of thiostrepton facilitated ATP release with mitoxantrone (MTX) and oxaliplatin (OXA). (A, B) Thiostrepton (THIOS; 0.5, 1, 2, 3, 4, 6 µM) decreased intracellular ATP content in human osteosarcoma U2OS wild-type cells within 6 hours in the presence of low doses of the ICD inducers mitoxantrone (MTX, 1 µM) and oxaliplatin (OXA; 200 µM) detected by quinacrine. High-dose MTX (5 µM) and OXA (400 µM) were used as positive controls. Representative images are shown in (A). Scale bar equals 10 µm. Data are means±SD of four replicates (compared with vehicle control (CTRL), *p<0.05, **p<0.01, ***p<0.001 compared with CTRL; ##p<0.01, ###p<0.001 compared with cells treated with low-dose MTX; &p<0.05, &&&p<0.01 compared with cells treated with low-dose OXA; unpaired Student’s t-test). (C–E) Thiostrepton increased extracellular ATP levels in murine fibrosarcoma MCA205 wild-type cells in the presence of low doses of the ICD inducers MTX (0.5 µM) and OXA (150 µM), but much less efficient in MCA205-TFE knockdown or ATG5 knockdown cells. Cells were treated with THIOS (1.56, 3.12, 6.25, 12.5 µM) alone or in combination with low-dose MTX or OXA for 6 hours as described above. Data are means±SD of at least three replicates (*p<0.05, **p<0.01, ***p<0.001 compared with thiostrepton single treatment; #p<0.05 compared with cells treated with low-dose MTX alone, &&&p<0.01 compared with cells treated with low-dose OXA alone; unpaired Student’s t-test).