Withaferin a

HEK293T, HeLa, A549, MDA-MB-231 and MDCK cells were cultivated according to standard protocols. Briefly, growth media contained DMEM (high glucose, pyruvate) for HEK293T, HeLa, MDA-MB-231 and MDCK cells or DMEM/F-12 (high glucose, pyruvate) for A549 cells supplemented with L-glutamine and antibiotics if not stated differently. Cells were trypsinized for passaging and cultivated at 37 °C in a humidified chamber with a 5% CO₂ atmosphere. Stable A549_VB6-CB cells were generated as described below. For transient transfection of HEK293T of HeLa cells in p100 dishes, 24 μg DNA were mixed with 140 μL polyethylenimine (PEI, Sigma Aldrich) pre-diluted in 600 μL DMEM to generate DNA/PEI complexes, incubated for 15 min and added to the cells. Transient transfection of A549 cells using Lipofectamine LTX and reverse transfection using RNAiMAX was carried out according to manufacturer’s protocols (Life Technologies). Compound treatment with 5 ng/ml TGF-β (Peprotech) or 50–500 nM Withaferin A (WFA, Merck Millipore) was performed up to 72 h.

At 50% confluency, cell growth medium containing chemotherapeutic agents were added to the cells for a 72 h incubation. DMSO was used as a solvent. The concentrations were as follows: Temozolomide (TMZ) (cat#T2577, Sigma Aldrich) 250 μM, Withaferin A (WA) (cat#W4394, Sigma Aldrich) 1.5 μM, Sulforaphane (SFN) (cat#574215, EMD Millipore) 10 μM. Cells were also incubated in growth medium containing 0.2% DMSO, or in growth medium. Cells were photographed and apoptosis assays were performed as described above.

Withaferin A (WA) was commercially purchased from Sigma Aldrich (Cat# W4394 SIGMA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT; Cat# M2003) and paraformaldehyde was purchased from Sigma Aldrich. HIV-1 clade B recombinant Tat protein (86-amino acid) was obtained from NIH AIDS research and reference reagent program (Cat# 2222).

Healthy vimentin-transfected and well attached cells were chosen and imaged for 1 h prior drug treatment. Subsequently, Withaferin A (Sigma, Poole, UK) with concentrations of 1 µM, 2.5 µM, and 5 µM, or acrylamide (Bio-Rad Laboratories, Watford, UK) with concentrations of 2 mM, 4 mM, and 6 mM was added and cells were imaged for 6 additional hours. Images were captured every 10 min. Cell velocity was calculated as described above on the same cells before and after drug treatment.

Reference standards for withanoside IV (USP grade, Lot no:1719532), withaferin A (analytical standard, Lot no:89910), and withanolide A (USP grade, Lot no:1719500) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The internal standard, Tianeptine Cas No. 66981-73-5, was procured from Sigma-Aldrich (St. Louis, MO, USA). Methanol, Acetonitrile, o-phosphoric acid, and formic acid (LCMS grade) were purchased from Merck, India and water was collected from a Milli-Q organic-free water system (Millipore Corporation, Bedford, MA, USA). The OasisR HLB solid-phase extraction cartridge was obtained from Waters (Milford, MA).