Fig. 5

Nelfinavir and bortezomib prevent tumor spheroid growth in Tsc2−/− MEFs. a Tsc2–/– MEFs were plated in soft agar and treated over 11 days with either 20 µM nelfinavir (NFV), 20 nM bortezomib (BTZ), as single agents or in combination. Images of the colonies were taken and the diameters measured using ImageJ. Scale bar is 20 µm (n = 4, with >100 spheroids measured per condition, per replicate). b Tsc2−/− MEF spheroids were treated with DMSO vehicle control, 10 μM nelfinavir combined with 10 nM bortezomib (NFV BTZ), or 25 nM rapamycin (RAP), for 96 h. DRAQ7 was added for the final 36 h to monitor cell death before images were taken and quantified (n ≥ 3, average of 12 spheroids per condition per replicate). c Spheroid diameter was determined from phase contrast images of (b) after 96 h drug treatment and plotted against DRAQ7 staining intensity. d Spheroids were re-plated onto standard tissue culture plates and grown under drug-free conditions. Images were taken every 24 h and the area of outgrowth calculated using ImageJ. Scale bar is 200 µm. Relative outgrowth areas are graphed in (e). Statistics compare the 72 h timepoint. Graphs in (a), (b), and (e) shown mean ± S.E.M. f Treated spheroids were stained using phalloidin (actin—green in merged images) and DRAQ7 (nuclei–white) and imaged using confocal microscopy. A representative slice (×63 oil lens) through the spheroid is shown with a scale bar of 30 µm, alongside the maximum projection (×20 lens) with a scale bar of 75 µm