AFM Measurements of Withaferin-A Effects

Cells were plated in collagen (0.3 mg/mL)-coated petri dishes for 24 h and placed in a petri dish heater on the AFM stage in 25 mM HEPES-containing media, for a maximum of one hour. Cells incubated with Withaferin-A were measured between 2.75 and 3.25 h of incubation with the drug. The AFM used was a JPK NanoWizard 4. Bruker MSNL-10 cantilevers with a nominal spring constant of 0.03 N/m were used with a setpoint of 3 nN. Measurements were taken in QI mode with a 300 µm/s cantilever speed over a grid of 32 by 32 pixels that covered the whole area of the cell. Data was processed using an in-house MATLAB pipeline which uses a contact point detection method optimised for adherent cells [33 (link),34 (link)]. Since we found a large variability between cell mechanical properties between experiments on different days, results were from a single experiment designed to maximise cell numbers captured in one day.