Cells (5 × 103) were seeded in a 96-well plate (in either RPMI-1640 (Gibco #10566) or DMEM medium (Gibco #61870), supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) 100 U/mL penicillin-streptomycin) and allowed to adhere overnight under standard tissue culture conditions (37 °C, 5% CO2 and 95% humidity). The next day, they were treated with threefold serially diluted concentrations of MMAF (unmodified), BG-linker-AURIF, alphaCSPG4 (scFv)-SNAP (unconjugated) or alphaCSPG4 (scFv)-SNAP-AURIF and incubated for 72 h in a 5% CO2 incubator with 95% humidity at 37 °C. Untreated cells served as negative controls (100% cell viability), while Zeocin-treated (100 ug/mL) cells were used as positive controls (0% cell viability). The Cell Proliferation Kit II (XTT) (product number: 11465015001, Roche, Switzerland) was used (according to the manufacturer's protocol) to assess cytotoxicity. In this assay, cleavage of the tetrazolium salt XTT occurs in the presence of metabolically active cells and results in the formation of orange formazan crystals, which absorb light at 450 nm. Thus, at 68 h post-treatment, cells were treated with the XTT reagent and at 72 h, absorbance readings (at 450 nm as the measurement filter and 650 nm as the reference filter) were taken using a spectrophotometer (iMark^TM Microplate Absorbance Reader, Bio-Rad, USA). All experiments were carried out in triplicate (n = 3), with 3 technical repeats. The absorbance values were normalized with respect to the untreated and Zeocin controls, and the results were presented as a percentage of cell viability. The concentration required to achieve a 50% reduction in cell viability (IC50 value) was calculated using GraphPad Prism v5 software.

Cells (5 × 103) were seeded in a 96-well plate (in either RPMI-1640 (Gibco #10566) or DMEM medium (Gibco #61870), supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) 100 U/mL penicillin-streptomycin) and allowed to adhere overnight under standard tissue culture conditions (37 °C, 5% CO2 and 95% humidity). The next day, they were treated with threefold serially diluted concentrations of MMAF (unmodified), BG-linker-AURIF, alphaCSPG4 (scFv)-SNAP (unconjugated) or alphaCSPG4 (scFv)-SNAP-AURIF and incubated for 72 h in a 5% CO2 incubator with 95% humidity at 37 °C. Untreated cells served as negative controls (100% cell viability), while Zeocin-treated (100 ug/mL) cells were used as positive controls (0% cell viability). The Cell Proliferation Kit II (XTT) (product number: 11465015001, Roche, Switzerland) was used (according to the manufacturer's protocol) to assess cytotoxicity. In this assay, cleavage of the tetrazolium salt XTT occurs in the presence of metabolically active cells and results in the formation of orange formazan crystals, which absorb light at 450 nm. Thus, at 68 h post-treatment, cells were treated with the XTT reagent and at 72 h, absorbance readings (at 450 nm as the measurement filter and 650 nm as the reference filter) were taken using a spectrophotometer (iMark^TM Microplate Absorbance Reader, Bio-Rad, USA). All experiments were carried out in triplicate (n = 3), with 3 technical repeats. The absorbance values were normalized with respect to the untreated and Zeocin controls, and the results were presented as a percentage of cell viability. The concentration required to achieve a 50% reduction in cell viability (IC50 value) was calculated using GraphPad Prism v5 software.

Cells (5 × 103) were seeded in a 96-well plate (in either RPMI-1640 (Gibco #10566) or DMEM medium (Gibco #61870), supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) 100 U/mL penicillin-streptomycin) and allowed to adhere overnight under standard tissue culture conditions (37 °C, 5% CO2 and 95% humidity). The next day, they were treated with threefold serially diluted concentrations of MMAF (unmodified), BG-linker-AURIF, alphaCSPG4 (scFv)-SNAP (unconjugated) or alphaCSPG4 (scFv)-SNAP-AURIF and incubated for 72 h in a 5% CO2 incubator with 95% humidity at 37 °C. Untreated cells served as negative controls (100% cell viability), while Zeocin-treated (100 ug/mL) cells were used as positive controls (0% cell viability). The Cell Proliferation Kit II (XTT) (product number: 11465015001, Roche, Switzerland) was used (according to the manufacturer's protocol) to assess cytotoxicity. In this assay, cleavage of the tetrazolium salt XTT occurs in the presence of metabolically active cells and results in the formation of orange formazan crystals, which absorb light at 450 nm. Thus, at 68 h post-treatment, cells were treated with the XTT reagent and at 72 h, absorbance readings (at 450 nm as the measurement filter and 650 nm as the reference filter) were taken using a spectrophotometer (iMark^TM Microplate Absorbance Reader, Bio-Rad, USA). All experiments were carried out in triplicate (n = 3), with 3 technical repeats. The absorbance values were normalized with respect to the untreated and Zeocin controls, and the results were presented as a percentage of cell viability. The concentration required to achieve a 50% reduction in cell viability (IC50 value) was calculated using GraphPad Prism v5 software.