Cell treatments

Transfection. Cells were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction with following modifications. One million cells in 1 ml or 500 μl of growth media were plated in 1 well of a 6- or 12-well plate, Lipofectamine-DNA mix was immediately added on top of the cells, and media containing transfection mix were replaced with fresh growth media 12 hours after transfection. Cells were imaged 24 to 36 hours after transfection.

HCT 116 cell serum stimulation. For serum stimulation, cells were first starved by washing three times with 1×phosphate-buffered saline (PBS) and culturing for 40 hours in serum-free media, and then either left untreated or treated with serum for indicated time before harvesting.

1,6-Hexanediol (Sigma) treatment for live imaging. HCT 116 cells were grown on a glass-bottom culture plate with 1 ml of media, and cells were imaged every 30 s. After the second acquisition, 1 ml of 6% 1,6-hexanediol was added to the plate.

KL-1 treatment for live imaging. After transfection for 24 hours, HCT 116 cells were treated with 20 μM SEC inhibitor KL-1 for 6 hours (42).