Supplementary Figure S5 In vitro cytotoxicity assays. Luciferase-expressing BMF
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cells were added to 24-well plates at 5x104 per well. Three days later, cells were either
not treated or treated with 1 µg/mL mANT2 shRNA-1 alone, treated with combined 1
µg/mL mANT2 shRNA-1 and 1 x 106 MUC1-associated CTLs, or treated with 1 x 106
MUC1-associated CTLs alone. CTL-mediated killing of tumor cells reduced
luminescence activity, which was determined using a Series 200 IVIS Luminescence
Imaging System. D-Luciferin (potassium salt; Xenogen/Caliper Life Sciences, Alameda,
CA) at 150 µg/ml in medium was added to each well 7-8min before imaging.
Bioluminescence signals were acquired for 10 sec. (a) Representative luminescence
images of 24-well plates showing tumor cell lysis. (b) Bar graph depicting the
luminescence intensities of tumor cells treated with various treatments (notreatment,
MUC1-associated CTLs, mANT2 shRNA-1, and mANT2 shRNA-1 plus MUC1associated CTLs). Note that BMF tumor cells treated with mANT2 shRNA-1 plus
MUC1-associated CTLs showed a significant loss of luminescence intensity, indicating
enhanced BMF lysis by MUC1-associated CD8+ T cells. Columns contain mean values.
Bars represent SDs. RLU = relative luciferase unit. n = 8 mice per group. Data are
representative of three independent experiments.
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