Journal: PLoS ONE

Article Title: Endotoxin-Induced Monocytic Microparticles Have Contrasting Effects on Endothelial Inflammatory Responses

doi: 10.1371/journal.pone.0091597

Figure Lengend Snippet: Endothelial protein expression of pSrc (Tyr416) and Src were examined after treatment with mMP. GAPDH was used as a loading control. Non-stimulated mMP had a more pronounced effect on resting (top left) rather than TNF-primed endothelial cells (top right). LPS mMP significantly decreased pSrc expression in both resting and TNF primed endothelial cells. Neither SN from the final NS or LPS induced mMP pellet had any effect. Non-stimulated (NS), TNF primed (TNF 0.2 ng/ml) and activated (TNF 100 ng/ml) endothelial cells are represented by open, grey and black bars respectively. Actual protein expression of pSrc, Src and GAPDH are shown in lower panels. Data represent three independent experiments. Data are mean ± SD. * p <0.05, ** p <0.01.

Article Snippet: Rabbit anti-pSrc-family (Tyr416) and mouse anti-Src antibodies were from Cell Signalling Technologies; mouse-anti-GAPDH (clone 6C5) antibody was from Millipore; rabbit anti-ZO-1 antibody was from Invitrogen and rabbit anti-VE-Cadherin was from Sigma (Saint Louis, MO, USA).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Tumor Suppressor SASH1 Interacts With the Signal Adaptor CRKL to Inhibit Epithelial–Mesenchymal Transition and Metastasis in Colorectal Cancer

doi: 10.1016/j.jcmgh.2018.08.007

Figure Lengend Snippet: SASH1 inhibits CRKL-mediated SRC signaling, which is required for the mesenchymal phenotype. ( A ) SASH1- and/or CRKL-deficient cells that adhered to fibronectin-coated plates for 3 hours ( left panel ) or for 24 hours ( right panel ) were subjected to immunoblot analysis. ( B ) Immunoblot quantification of SRC (Y416) and paxillin (Y118) phosphorylation after 3 hours of adhesion (Mann–Whitney test; n = 4 independent experiments; P = .0286). ∗ P ≤ .05. ( C ) Cells that adhered for 3 hours to fibronectin-coated plates were subjected to immunofluorescence staining (100× objective, scale bar : 10 μm) for Y118 phospho-paxillin (green), F-actin (red), and 4′,6-diamidino-2-phenylindole for nuclei (blue). ( D ) The same analysis was performed after 24-hour adhesion to fibronectin-coated plates. ( E ) Immunoblot analysis of parental HCT116 cells recombinantly expressing RFP as control or CRKL with a C-terminal RFP tag. In addition, CRKL-RFP–expressing cells were treated with 100 nmol/L dasatinib for 24 hours. ( F ) Parental (wild-type [WT]), CRKL-deficient (C2), SASH1-deficient (S1, S2), and compound SASH1/CRKL-deficient (S1C2, S2C2) cells were treated with 100 nmol/L dasatinib for 72 hours before they were subjected to immunoblot analysis. Mr (K), molecular range (kilodalton); pPaxillin, phospho-Paxillin; pSrc, phospho-Src; RFP, red fluorescent protein.

Article Snippet: The following antibodies were used: E-cadherin (3195; CST), β-actin (3700; CST), SASH1 (A302-265A; Bethyl, Montgomery, TX), GAB1 (A303-288A; Bethyl), C3G (Bethyl A301-965A), RFP (5F8; Chromotek, Martinsried, Germany), V5 epitope tag (MA5-15253; Thermo Scientific, Waltham, MA), Poly(ADP-Ribose)-Polymerase 1 (ab137653; Abcam, Cambridge, UK), GFP (3H9; Chromotek), CRK-II (sc-289; SCBT, Heidelberg, Germany), CRKL (05-414; EMD Millipore, Darmstadt, Germany), CRKL (sc-319; SCBT), ZEB1 (HPA027524; Sigma, Steinheim, Germany), vimentin (ab92547; Abcam), Y118 phospho-paxillin (2541; CST, Danvers, MA), paxillin (12065; CST), Y416 phospho-Src family (6943; CST), Src (2123; CST), human mitochondria (ab92824; Abcam), human E-cadherin (ab40772; Abcam), RFP-trap (rta-10; Chromotek), and GFP trap (gta-10; Chromotek).

Techniques: Western Blot, MANN-WHITNEY, Immunofluorescence, Staining, Expressing

Journal: Oncotarget

Article Title: The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells

doi: 10.18632/oncotarget.28300

Figure Lengend Snippet: ( A , B ) Migration of (A) INA-6 and (B) U266 matriptase overexpression (Matriptase) and control (Mock) cells. The promigratory cytokines HGF and/or SDF-1α were added to the lower wells as indicated. ( C ) Basal migration of matriptase knockdown (shMatriptase) and control cells (shMock) in RPMI-8226 and JJN-3. ( D ) Src activation in INA-6 Matriptase and Mock. Cells were starved for 6 h in serum-free medium. Subsequently, cells were stimulated with HGF and SDF-1α for 10 and 30 min and probed with antibodies as indicated. ( E ) Migration of INA-6 Matriptase and Mock cells. The combination of HGF and SDF-1α were added to the lower wells in all conditions. DMSO or PP2 Src inhibitor were added in concentrations as indicated to both upper and lower wells. (A–C and E) Cells were seeded in the upper well of a two-chamber transwell migration assay. After 24 h incubation, cells in the bottom wells were counted and the percentage of migrated cells calculated. Bars in (A), (B), (C) and (E) represent the mean (±SD) of at least two repeated counts in two independent measurements. Concentrations of HGF and SDF-1α was 150 ng/mL and 75 ng/mL, respectively, in all experiments. One representative of three independent experiments is shown in all figures. p -values were calculated by unpaired Student’s t -test. ns = not significant ( p > 0.05), * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Antibodies against phosphorylated Src (Tyr416; #6943), PAK1 (Ser144)/PAK2 (Ser141) (#2606), p44/42 MAPK (Erk1/2) (Thr202/Tyr204; #4370) and Akt (Ser473; #9271), total Src (#2123), p44/42 MAPK (Erk1/2) (#4695), Akt (#9272), p38 MAPK (#9212) and β-actin (#4970) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Migration, Over Expression, Activation Assay, Transwell Migration Assay, Incubation

Journal: Experimental Hematology & Oncology

Article Title: Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma

doi: 10.1186/2162-3619-2-4

Figure Lengend Snippet: PP2 and dasatinib inhibit constitutive phosphorylation of LYN and induce apoptosis of primary MCL cells. ( A ) Constitutive phosphorylation profiles of LYN in MCL patients’ samples. Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. The blots were stripped and re-probed for total LYN. ( B ) Total proteins from HBL-2 cells were immunoprecipitated with an anti LYN antibody (Ig-LYN) or with an irrelevant IgG control and immunobloted (IB) with either an anti-phosphotyrosine antibody (P-Tyr) or an anti-LYN antibody. ( C ) Primary MCL cells (UPN3, UPN14) were treated with variable concentrations of PP2 (2 to 20 μM) or dasatinib (1 to 200nM) for 2 h. Phospho-Tyr397 LYN and LYN total were analyzed by western-blot. ( D ) Primary MCL cells were treated with various concentrations of PP2 (UPN3, top panel ) or 10 μM of PP2 (UPN 1–3-7–8-9–14 , middle panel ) for 24 h and apoptosis was measured by flow cytometry after gating on CD19+ cells. All measurements were done in duplicate and the mean is provided. Results are also shown as median ± quartile ( box ) ± SE ( bars ) bottom panel ). Differences between groups were determined using the paired Student t test. ( E ) Primary MCL cells were treated with dasatinib for 24 h with various concentrations (top panel) or with 100nM (middle panel). Apoptosis was measured as described above. Results are also shown as median ± quartile ( box ) ± SE ( bars ) bottom panel ).

Article Snippet: Antibodies to EGR-1 (mAb 44D5), c-MYC (mAb 9B11), phospho-Src family (mAb 100 F9) also reactive with phospho-Tyr397 LYN (catalytic site) and phospho-JNK (Thr183/Tyr185) were from Cell Signaling (Beverley, MA).

Techniques: Immunoprecipitation, Western Blot, Flow Cytometry

Journal: Experimental Hematology & Oncology

Article Title: Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma

doi: 10.1186/2162-3619-2-4

Figure Lengend Snippet: PP2 and dasatinib inhibit BCR-induced LYN and JNK activation and EGR-1 upregulation. ( A ) Patients’ cells (UPN9) were pretreated with dasatinib (100nM) or SP600125 (10 μM) for 1 h and stimulated for 5 min or 15 min with soluble anti-IgM (10 μg/ml). Phospho-Tyr397 LYN was detected using a pan phospho-src family antibody. ( B ) The same experiment was done with PP2 (10 μM) on UPN 9 and UPN 13 under the same conditions of BCR stimulation for 10 min. Lines 1 and 2 have to be compared to evidence the effect of PP2 on the constitutive level of phosphorylation for Lyn. Similarly lines 3 and 4 reflect this effect upon BCR stimulation. ( C ) BCR-induced phospho-JNK (p54 and p46) was analyzed under treatment with dasatinib (100nM) or SP600125 (10 μM) used herein as a positive control of phospho-JNK inhibition. ( D ) Impact of dasatinib on BCR-induced EGR-1 expression. MCL cells were pretreated with various concentrations of dasatinib as indicated and stimulated with immobilized anti-IgM. EGR-1 mRNA and protein were analyzed by qRT-PCR at 1 h of stimulation ( left panel ) and western-blot at 3 h of stimulation ( right panel ). Relative mRNA expression was calculated compared with unstimulated cells.

Article Snippet: Antibodies to EGR-1 (mAb 44D5), c-MYC (mAb 9B11), phospho-Src family (mAb 100 F9) also reactive with phospho-Tyr397 LYN (catalytic site) and phospho-JNK (Thr183/Tyr185) were from Cell Signaling (Beverley, MA).

Techniques: Activation Assay, Positive Control, Inhibition, Expressing, Quantitative RT-PCR, Western Blot