Fig 2: C-Abl kinase is activated by mechanical forces and its actin-binding domain is mechanosensitive.a The kinase activity of endogenous c-Abl is elevated after hypo-osmotic treatment. Endogenous c-Abl was immunoprecipitated from cells treated with iso-osmotic or hypo-osmotic medium (60 mOsm, 10 min) and in vitro kinase assay was performed using GST-CrkII as substrate. Phosphorylation (radiolabeled) and the total protein (Coomassie stained) signals are shown. N = 3 (control) and n = 5 (hypo-osmotic) biologically independent samples from 3 independent experiments. Statistical analysis with a two-tailed unpaired t test. *P < 0.05. b Kinetics of c-Abl activation upon osmotic swelling (30 mOsm) and after shock release is shown. Endogenous CrkII was immunoprecipitated from cells and pY221CrkII and CrkII was detected, normalized to iso-osmotic. From left to right, n = 5, 3, 5, 9, 2, 2, from biologically independent samples from the corresponding independent experiments. Statistical analysis with a two-tailed unpaired t test. *P < 0.05. c c-Abl kinase mechanosenses substrate rigidity. Endogenous CrkII was immunoprecipitated from cells grown in soft or stiff hydrogels and pY221CrkII and CrkII was detected. N = 4 biologically independent samples from 4 independent experiments. Statistical analysis with a two-tailed unpaired t test. *P < 0.05. d c-Abl kinase is activated by mechanical strain. Endogenous CrkII was immunoprecipitated from human fibroblasts unstretched and stretched for 10 min and pY221CrkII and CrkII was detected. #P = 0.08, n = 9 from biologically independent samples from 5 independent experiments. e Cartoon representing the different tensional states of a cell in culture and the activity of c-Abl depending on those tensional states. f Schematic representation of the heteropolyprotein (I91 ?Cys)2-ABD-(I91 ?Cys)2 used to characterize the mechanical properties of ABD. g Typical force-extension trace showing four unfolding peaks of the fingerprinting domain I91, which are preceded by a featureless extension which suggests that ABD unfolds at low forces. Solid lines show worm-like chain plots used to estimate contour lengths. h Final contour lengths of fingerprinted single-molecule force-extension traces. Dashed lines indicate theoretical final contour length values if ABD unfolds during the experiment (black) or remains folded (gray) are indicated. Data represent mean ± S.E.M.

Fig 3: AgFL transiently suppresses the phosphorylation of ERK1/2 in primary human myoblasts. (A–E) Myoblasts were treated with 1 nM AgFL for 5–240 min and 10 µM DPH for 240 min in the serum-free medium. Activity of c-Abl, ERK1/2, JAK/STAT, and FAK pathways was estimated by measuring the phosphorylation of (A) c-Abl (at Tyr412), (B) Crk-II (Tyr221), (C) ERK1/2 (Thr202/Tyr204), (D) STAT3 (Tyr705), and (E) FAK (at Tyr397). Results are means ± SD, n = 5 donors (7Y, 7Y, 8Y, 15Y, 65Y) except for ERK1/2, where n = 9 donors (7Y, 7Y, 8Y, 12Y, 15Y, 17Y, 65Y, 67Y, and 77Y). (F) The phosphorylation of ERK1/2 (at Thr202/Tyr204) in myoblasts treated with 1 nM AgFL for 5–240 min and 10 µM DPH for 240 min in the presence of 10% FBS. Results are means ± SD, n = 4 donors (15Y, 17Y, 22Y, and 65 Y). (G) The phosphorylation of ERK1/2 (Thr202/Tyr204) in myoblasts treated with 1 nM AgC90 for 5–240 min or 1 mM H2O2 for 15 min. Results are means ± SD, n = 4 wells (2 donors: 5Y and 80Y). * p < 0.05 vs. Basal.

Fig 4: Stabilization of geminin protein by c-Abl phosphorylation and the expression of geminin and c-Abl in breast cancer cell lines.(A) The expression level of geminin in naïve HME, uninduced and induced Gem9 cells following c-Abl silencing or inactivation using imatinib or transfection of the dominant negative c-Abl (K290R). (B) The expression of c-Abl, geminin, p-CrkII in induced Gem9, MCF7 or MDA-MB-231 cells silenced from c-Abl or treated with imatinib. (C) The expression level of c-Abl, p-CrkII and geminin in induced Gem9 or MDA-MB-231 cells silenced of c-Abl or treated with nilotinib. (D) RT/PCR analysis of geminin mRNA in uninduced, induced Gem9 or MDA-MB-231 cells in the presence of vehicle or imatinib. (E) The expression of geminin in naïve HME or induced Gem9 cells in the presence of vehicle, imatinib or imatinib + MG132. (F) The expression of c-Abl and geminin mRNAs and proteins (inset) in several breast cancer cell lines. Note that Hs578T cells express high level of geminin mRNA, but no protein. (G) The re-expression of geminin in Hs578T cells reconstituted with WT and not kinase dead (KD) c-Abl (left). The re-expression of geminin in Hs578T cells reconstituted with WT or constitutively active (CA) c-Abl was blocked by imatinib (right). (H) The expression of endogenous (left) or overexpressed (right) geminin in MDA-MB-231 cells following treatment with the translational inhibitor cycloheximide (CHX) for 0-10 h with cells collected at 2 h intervals. (I) The expression of exogenous Myc tagged WT- (left), Y150A- (middle) or Y150E- (right)-geminin following no treatment (1st lanes), 10 h of CHX (2nd lanes), 10 h CHX followed by 24 h of complete serum (3rd lanes) or 10 h CHX followed by 24 h of complete serum + 10 µM of imatinib (4th lanes).

Fig 5: Down-regulation of CrkI/II expression by siRNA in CrkL-deficient MEFs enhances pedestal formation.(A) WB with anti-CrkII Ab in WT and CrkL-deficient MEFs to show lower CrkII levels in cells treated using siRNA with an oligonucleotide against CrkI/II than in cells treated with control (Ctr.) oligonucleotide. The blot was probed with anti-CrkL Ab as a control for cell genotype, and with anti-actin MoAb as a loading control. The levels of Tir effector are shown. (B) Fluorescence images of WT and CrkL-deficient MEFs in which CrkI/II expression was inhibited by siRNA; cells were infected with preactivated EPEC at an MOI of 3. Actin was stained red with TRITC-phalloidin, while EPEC was stained blue using DAPI. Images were merged using Leica software. Insets are 4× digital zoom images. (C) Quantitation of the number of pedestals on WT and CrkL-deficient cells treated for siRNA using a scrambled control (Ctr.) oligonucleotide (white bar) or an oligonucleotide to reduce CrkI/II expression (black bar). Quantitation was done by counting the number of pedestals on 100 cells. The graph shows mean ± standard deviation (SD) for three independent experiments. The indicated groups differed significantly based on Student's t-test. **, p<0.01.