Rock1

The AML cell line (MV4-11 and OCI-AML3) cells were treated with increasing concentrations of DJ4 (0–1 μM) or DMSO and harvested at 24 h. Whole cell lysates were then collected in a RIPA buffer (Sigma) containing phosphatase and protease inhibitor cocktails (Sigma). Protein quantification was performed using a bicinchoninic acid (BCA) assay kit (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Denatured protein samples run on a NuPAGE 4–12% Bis-Tris gel (Life Technologies, Carlsbad, CA, USA) were subsequently probed with various primary antibodies as previously described [51 (link),52 (link)]. Bands were detected using the Bio-Rad ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA) and quantified via the ImageJ software [53 (link)]. The following antibodies were obtained: ROCK1 (611136; BD Biosciences), ROCK2 (610623; BD Biosciences), MYPT1 (07-672-I; Millipore Sigma), Phospho-MYPT1 (Thr696) (5163S; Cell Signaling Technology, Danvers, MA, USA), MLC2 (3672S; Cell Signaling Technology), Phospho-MLC2 (PA5-17726; Thermo Scientific), and GAPDH (sc-32233; Santa Cruz Biotechnology, Dallas, TX, USA); the goat anti-rabbit IgG–horseradish peroxidase (HRP) conjugates and the horse anti-mouse IgG-HRP conjugates were purchased from Cell Signaling Technology. All the whole western blot figures can be found in the Figure S9.

Cells were harvested in ice by scraping in ice cold 1X PBS and the pellets were lysed in RIPA buffer (50 mM TRIS pH 8.0, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, complete protease inhibitor cocktail (Roche), and phosphatase inhibitors 10 mM NaF, 1 mM Na₃VO₄, 1 mM sodium pyrophosphate, 10 mM beta-glycerophosphate). After sonication and centrifugation the protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad). Samples were loaded on 4-12% Bis-Tris polyacrylamide-SDS gels (NuPAGE) and transferred on to nitrocellulose membranes (Amersham). Membranes were blocked in 4% skimmed milk powder dissolved in 0,2% Tween-containing 1X PBS and incubated with primary antibodies followed by secondary antibodies (Invitrogen). Primary antibodies used were EGFR (sc-03, Santa Cruz), EGFR_Y2068 (ab5644, Abcam), ROCK1 (611137, BD), MYPT_Thr696 (ABS45, Millipore), ERK_{Thr202/Tyr204} (4370S, Cell Signaling), ERK (9102, Cell Signaling), Hsp90 (sc-7947, Santa Cruz), p27 (610241, BD), pRB_Ser807/811 (9308S, Cell Signaling), CDK2 (sc-163, Santa Cruz), Cyclin A (sc-596, Santa Cruz).

COS7, HeLa and MDA-MB-231 cells were grown in DMEM supplemented with 10% FCS, 1% pyruvate. PC3 cells were grown in RPMI supplemented with 10% FCS. All media contained 100 µg/ml streptomycin and 100 U/ml penicillin. The following antibodies were used: myc tag (A-14, sc-789, or 9E10, sc-40; Santa Cruz Biotechnology), GFP (FL) (sc-8334; Santa Cruz Biotechnology), Cullin3 (611848; BD), ROCK1 (611136; BD), ROCK2 (610623; BD), HA tag (3F10; Sigma–Aldrich), pMLC2 (Thr18/Ser19) (#3674, Cell Signaling), MLC2 (#3672, Cell Signaling), RhoA 67B9 (#2117, Cell Signaling), GAPDH (MAB374, Merck Millipore). Secondary horseradish peroxidase (HRP)-labelled antibodies were from GE Healthcare (anti-mouse, anti-rat and anti-rabbit). Complete protease inhibitor cocktail and PhosphoStop were from Roche. Active recombinant human GST-ROCK1 (17–535, # R10–11G) was from SignalChem. MLN4924 was from BostonBiochem (R&D Systems). H1152 Rho kinase inhibitor was from Calbiochem.