Journal: Nature Communications

Article Title: Direct cysteine sulfenylation drives activation of the Src kinase

doi: 10.1038/s41467-018-06790-1

Figure Lengend Snippet: Cysteine oxidation to Cys-277-SOH promotes unfolding of the Src A-loop α-helix. a A final snapshot of Cys-277-SOH system (purple blue) from MD simulation superposed with it from metadynamics simulation that displays the unfolded conformation of the A-loop (residues 406–423, yellow). Detailed structures compared with wild-type group are shown in Supplementary Fig. . Key residues that interact with the A-loop region are labeled. b Percent α-helix fold of residues 406–423 throughout the MD simulation of Cys-277-SOH system (two replicas labeled as #1 and #2 are in red and pink) in comparison with control groups (green and cyan). c Free-energy landscapes of Cys-277-SOH system and Cys-277-SH system, respectively. Two collective variables (CVs), pair distances of Tyr-416-Asp-386 and Arg-419-Asp-386, are used in metadynamics simulations. Color scale: Cys277-SOH white = 7.0 kcal/mol, blue = 0.0 kcal/mol (left) and Cys277-SH white = 11.0 kcal/mol, blue = 0.0 kcal/mol

Article Snippet: Derivatized cell lysates were separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes, and probed using antibodies against p-Src Tyr 416 (1:400; 2101S), p-Src Tyr 527 (1:400; 2105S), Src (L4A1; 1:1000; 2110S), FLAG (DDK; 1:1000; 2368S) (Cell Signaling), or streptavidin peroxidase polymer ultrasensitive (1:10,000; S2438; Sigma).

Techniques: Labeling

Journal: Oncotarget

Article Title: IL-6 influences the polarization of macrophages and the formation and growth of colorectal tumor

doi: 10.18632/oncotarget.24734

Figure Lengend Snippet: ( A – B ) NIH3T3/Src cells (500) were seeded in six-well plates and treated with supernatants of Ana-1 or polarized Ana-1 macrophages. The colonies were stained two weeks later. Data from representative experiment is shown. Bars represent the colony number of NIH3T3/Src cells. Data are expressed as mean ± SD ( n = 2), * p < 0.05, ** p < 0.01. ( C ) NIH3T3/Src cells (4 × 10 5 ) with and without polarized Ana-1 macrophages (8 × 10 4 ) were subcutaneously injected into each flank of 4-week old nude mice; mice were sacrificed 16 days later and tumors were shown. ( D ) Xenograft tumor sizes were measured every 2 days with a digital caliper. Data are expressed as mean ± SD ( n = 5), ** p < 0.01. ( E ) Bars represent the weights of xenograft tumors. Data are expressed as mean ± SD ( n = 5), *** p < 0.001. ( F ) The expression of p-Src (Y416), Src and Arg-1 in tumors were analyzed by Western blotting. Intensity was quantified and normalized to β-actin. ( G ) F4/80 and CD206 expression in xenograft tumor tissues. Representative immunofluorescence images showed the expression and localization of F4/80 (red) and CD206 (green). DAPI is shown in blue. The arrows indicated M2 macrophages. (Scale bar: 30 μm).

Article Snippet: The membranes were blocked with 5% milk and then incubated with antibodies against p-Src (Y416) (2101) (1:1000; Cell Signaling), total-Src (36D10) (1:1000; Cell Signaling), p-STAT3(D3A7) (1:1000; Cell Signaling), total-STAT3 (9132) (1:1000; Cell Signaling), NF-κB(p65) (D14A12) (1:1000; Cell Signaling), Arg-1 (sc-271430) (1:1000; Santa Cruz), or iNOS (sc-650) (1:1000; Santa Cruz), total IκBα (44D4) (1:1000; Cell Signaling), p-IκBα (14D4) (1:1000; Cell Signaling).

Techniques: Staining, Injection, Expressing, Western Blot, Immunofluorescence

Journal: Oncotarget

Article Title: A cytoplasmic C-terminal fragment of syndecan-1 is generated by sequential proteolysis and antagonizes syndecan-1 dependent lung tumor cell migration

doi:

Figure Lengend Snippet: A–H. A549 cells were transduced to express SDC-1 cCTF or empty vector. A-C) Transduced cells were investigated for adhesion to collagen G (A) or fibronectin (B). The number of adherent cells was determined using the IncuCyte ZOOM and expressed in relation to the number of seeded cells. Images of a representative cell adhesion experiment to fibronectin and collagen G are shown (C). D-E) Immunocytochemistry of paxillin recruitment. Transduced cells were grown on collagen G, fixed, permeabilized and stained with paxillin antibody (green signal). Expression of mCherry (red signal) was visualized as transduction control. Paxillin recruitment was quantified as lengths of the focal adhesions (D). Focal adhesions are shown as representative images (E). F–H) Cell lysates were analyzed for phosphorylation of Src at Tyr416 (F), FAK at Tyr397 (G) and activation of Rho GTPase (H) by Western blotting. Signals were quantified by densitometry as phosphorylated or activated versus total forms and calculated in relation to the control cells expressing empty vector. Data shown in A, B, D, F–H represent means + SD of three independent experiments and representative Western blots are shown F–H. Statistically significant differences compared to corresponding empty vector control are indicated by asterisks ( p < 0.05).

Article Snippet: Rabbit mAb against human Akt (clone C67E7), rabbit mAb against human p-Akt (Ser473) (clone D9E), polyclonal rabbit Ab against human p38, mouse mAb against human p-p38 (Thr180/Tyr182) (clone D3F9) and rabbit Ab against human p-Src (Tyr416) (clone 2101) were from Cell Signaling (Danvers, MA, USA).

Techniques: Plasmid Preparation, Immunocytochemistry, Staining, Expressing, Transduction, Activation Assay, Western Blot

Journal: Oncotarget

Article Title: A cytoplasmic C-terminal fragment of syndecan-1 is generated by sequential proteolysis and antagonizes syndecan-1 dependent lung tumor cell migration

doi:

Figure Lengend Snippet: A. Schematic representation of the human SDC-1 cCTF peptide and the scrambled control peptide. B-D) A549 cells were treated with SDC-1 cCTF peptide or the scrambled peptide without B. or with C. carrier reagent (Chariot delivery reagent). A549 cells were grown to confluence on collagen G coated wells and wounded by a defined scratch. After wounding, cells were treated with SDC-1 cCTF peptide or scrambled peptide (0.1 μM, 1 μM or 10 μM) and investigated for wound closure over 24 h (left). In parallel, cells were seeded at a lower density and proliferation was quantified as change in density (right) over a period of 24 h using the IncuCyte ZOOM. D–E. A549 cells were treated with 10 μM SDC-1 cCTF or 10 μM scramble SDC-1 cCTF with carrier reagent for 2 h. Cell lysates of treated A549 cells were analysed for phosphorylation of Src at Tyr416 (D) and FAK at Tyr397 (E) by Western blotting. Signals were quantified by densitometry as phosphorylated versus total forms and calculated in relation to the control cells. All data were expressed as means + SD and statistically significant differences compared to corresponding scramble syndecan-1 cCTF treatment are indicated by asterisks ( p < 0.05).

Article Snippet: Rabbit mAb against human Akt (clone C67E7), rabbit mAb against human p-Akt (Ser473) (clone D9E), polyclonal rabbit Ab against human p38, mouse mAb against human p-p38 (Thr180/Tyr182) (clone D3F9) and rabbit Ab against human p-Src (Tyr416) (clone 2101) were from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot