Six well dish

The cells were harvested 48 h after transfection, and an equal number of viable cells were plated onto soft agar after respective treatments for determination of anchorage-independent growth. For analysis of growth in soft agar, 5 × 10³cells were seeded in triplicate onto a six well dish (Falcon) in 3 ml of complete medium containing 0.33 % agar solution at 37 °C. Cells were fed with 500 μl of medium every 2 days. From each well randomly 10 field images were taken using Phase contrast Inverted microscope (Zeiss axiovert 200 m) and colonies were counted manually.
Growth curve analysis - 25,000 cells/well were seeded in 24 well plates and growth was assessed post day 2, 4 and 6 by counting the cells using a haemocytometer. Percent survival were plotted at day 4 relative to day 2 and later normalized against scrambled or empty vector control.

100 or 400–800 live (Trypan Blue-negative) NHK or SCC13 cells, respectively, were seeded per condition into duplicate or triplicate wells (containing a 3T3-J2 feeder layer) of a six-well dish (Falcon). After 12 days, feeder cells were completely removed by rinsing with PBS and colonies were either fixed in 4% (w/v) paraformaldehyde for 10 min and then stained with 1% Rhodanile Blue (1:1 mixture of Rhodamine B and Nile Blue A (Acros Organics))12 (link), or simultaneously fixed and stained with Crystal Violet solution (0.4% (w/v) crystal violet, 20% (v/v) methanol). Colonies were imaged and counted using an automated cell colony counter (Gelcount, Oxford Optronix, UK), and CFE was calculated as the average percentage of seeded cells that formed colonies. Colony area was measured using the Fiji image processing software package and the ‘Analyse Particles' tools, with a minimum particle size of 0.01 mm².

For analysis of growth in soft agar, 5 × 103 cells were seeded in triplicate onto a six-well dish (Falcon) in 4 ml of complete medium containing 0.33% agar solution along with respective treatments of GSI-XXI at 37°C in CO2 incubator. Ten images per well were photographed after 21 days using inverted phase contrast microscope and colonies were counted manually.