Anti gef h1

Cells were scraped into RIPA buffer with protease/phosphatase inhibitor (5872S, Cell Signaling) then centrifuged at 13,000 rpm at 4°C for 20 min. Lysate was reduced in sample loading buffer and dithiothreitol (R0861, Thermo Fisher) and boiled for 10 min at 100°C. Samples were stored at –20°C until use. Ten µg of sample were run on a 10% stain-free polyacrylamide gel (161-0183, Bio-Rad) then transferred onto a PVDF membrane on ice for 1.5 hr. Membranes were blocked in OneBlock (20-313, Prometheus) for 1 hr at RT then washed 3× in PBST. Anti-GEF-H1 (1:1000, ab155785, Abcam), anti-VE-cadherin (1:14,000, 2500S, Cell Signaling), or anti-GAPDH (1:5000, 97166S, Cell Signaling) was added overnight at 4°C. Membranes were washed 3× in PBST then donkey anti-rabbit HRP secondary antibody (1:10,000, A16035, Thermo Fisher) was added for 1 hr at RT. Immobilon Forte HRP Substrate (WBLUF0100, MilliporeSigma) was added for 30 s. Blots were exposed for 8 s.

LM3 cells were seeded and grown 80% confluent until treatment with 80 µM hemin or vehicle. Immunofluorescence was performed as previously described^{23 (link)}. Rabbit anti-talin (1:100, Sigma), anti-tubulin-Cy3 (1:300, Sigma) and anti-GEF-H1 (1:500, Abcam) primary antibodies were used. For actin filaments, we used staining with Phalloidin-TRITC (1:300; Invitrogen) for 1 h at room temperature. Secondary Alexa 546 and 488 fluoro-conjugated antibodies (Molecular Probes) were used. DAPI was used to stain the nucleus. The coverslips were mounted on slides with mowiol mounting medium and we obtained superresolution and confocal z-stacks images in Zeiss LSM900 microscope with an Airyscan 2 module. Orthogonal x/y, x/z or y/z projection views were performed with Zeiss ZEN Software. Image intensity and protein distribution were analysed with Image J free software.

LM3 cells were seeded and grown sub-confluent until treatment with 80 µM hemin or vehicle. Immunoblotting was performed as previously described^{23 (link)}, incubating membranes with anti-HO-1 (1:1000; Enzo), anti-GEF-H1 (1:2000; Abcam), anti-actin (1:1000; Santa Cruz Biotechnology, Inc.), anti-tubulin (1:1000; Santa Cruz Biotechnology, Inc.), anti-PTEN (1:1000; Santa Cruz Biotechnology, Inc.), anti-Smad2/3 (1:1000; Santa Cruz Biotechnology, Inc.), anti-p53 (1:500; Santa Cruz Biotechnology, Inc.), anti-vimentin (1:1000; Santa Cruz Biotechnology, Inc.), anti-GAPDH (1:5000; MERK) and anti-transferrin receptor 1 (1:500, Santa Cruz Biotechnology, Inc.). To differentiate the HO-1 cleaved band from the native HO-1, we performed a 10% gel and ran it until the running front fell off. For talin we performed an 8% gel. For the rest of the proteins we performed a 12% gel. Western blot bands relative density was calculated with Image J free software and expressed in relative units (RU) (https://imagej.nih.gov/ij/). Uncropped western blots are shown in supplementary Fig. S4.