Pre collagen coated 8 well chamber slides

To assess the influence of HDAC inhibitor induced EMT on ROS production, SW13 cells were seeded into pre-collagen coated 8-well chamber slides (Ibidi, Fitchburg, WI, USA) at 4000 cells/well and treated with 2 nM FK228 for 48 h. Cells were washed once with Hank's Balanced Salt Solution (HBSS) (Cellgro, Lincoln, NE, USA) prior to incubation with 5 μM CellRox™ Deep Red Reagent (ThermoFisher, Waltham, MA, USA) for 30 min at 37 ᵒC. Cells were then washed twice with HBSS, fixed with 4% paraformaldehyde, and slides were mounted ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA). Oxidized probe was visualized at 40X using a BZ-X700 Fluorescent Microscope (Keyence, Osaka, Japan) with fluorescence 640 nm_excitation/665 nm_emission. Low photobleach settings and exposure were held consistent through imaging process.

To examine the effects of iron availability on the morphologic changes that occur with HDAC inhibitor-induced EMT, SW13 cells were seeded at 4000 cells/well into pre-collagen coated 8-well chamber slides (Ibidi, Fitchburg, WI, USA) and treated as described above. Following their respective treatments, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton-X, and blocked with 1% BSA before incubation with phalloidin conjugated to Alexafluor 488 (A12379, Invitrogen, Waltham, MA, USA) at 1:1000 or Vimentin (Cell Signaling, #5741) at 1:100 in 1% BSA for 1 h. Cells were washed three times with PBS and slides were mounted ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA). Images were obtained using a BZ-X700 Fluorescent Microscope (Keyence, Osaka, Japan) at 495 nm_excitation/518 nm_emission and 360 nm_excitation/460 nm_emission for actin filaments and DAPI, respectively, with uniform exposure.