Journal: Scientific Reports

Article Title: Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction

doi: 10.1038/srep22363

Figure Lengend Snippet: ( a ) Surface proteins of RAW264.7 cells were biotinylated and separated by streptavidin beads. Integrin β1 and integrin β3 levels were detected by western blot (I: internalized proteins from cell surface; II: total cell lysates; III: cell surface proteins). ( b ) RAW264.7 cells were incubated with NaHS (100 μM) for the indicated time and surface levels of integrin β1 were labeled with the FITC-conjugated secondary antibody, followed by Flow cytometric analysis. ( c ) RAW264.7 cells were incubated with NaHS (100 μM) for the indicated time. Surface Integrin β1 was stained with FITC-conjugated antibody (green); F-actin bundles were detected with Rhodamine (Red); Nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. All the data are representative of three independent experiments. ( d ) After siRNA-integrin β1 or siRNA-NC transfection for 24 h, RAW264.7 cells were incubated without or with NaHS (100 μM) for 6 h, and then the migratory capacities of the cells were evaluated by transwell assay. Values are the mean ± SD from three independent experiments. ( e ) RAW264.7 cells transfected with siRNA-integrin β1 were treated with NaHS (100 μM) for 6 h and the phosphor-Src, -Pyk2, -FAK 397 , -FAK 925 , total Src and FAK were analyzed by western blot (NC, negative control; p, phosphorylated; t, total).

Article Snippet: And the siRNA to mouse integrin β1 were chemically synthesised by Shanghai GenePharma Co., Ltd.

Techniques: Western Blot, Incubation, Labeling, Staining, Transfection, Transwell Assay, Negative Control

Journal: Scientific Reports

Article Title: Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction

doi: 10.1038/srep22363

Figure Lengend Snippet: ( a ) After specific siRNA transfection for 24 h, RAW264.7 cells were treated with NaHS (100 μM) for 6 h, the interference efficiency of CSE was confirmed by quantitative real-time PCR. ( b , c ) After siRNA-CSE or siRNA-NC transfection for 24 h, RAW264.7 cells were incubated without or with NaHS (100 μM) for 6 h, and then the migratory capacities of the cells was evaluated by transwell assay. Values are the mean ± SD from three independent experiments. Scale bar, 50 μm. ( d ) RAW264.7 cells transfected with siRNA-CSE were treated with NaHS (100 μM) for 6 h and the expression of phospho-Pyk2, -FAK, total Src and FAK and integrin β1 in total cell lysates (I) or cell surface (II) were analyzed by western blot (NC, negative control; p, phosphorylated; t, total).

Article Snippet: And the siRNA to mouse integrin β1 were chemically synthesised by Shanghai GenePharma Co., Ltd.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Incubation, Transwell Assay, Expressing, Western Blot, Negative Control

Journal: Toxicology letters

Article Title: Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells

doi: 10.1016/j.toxlet.2016.11.021

Figure Lengend Snippet: Fascin2 Knockdown Increases Susceptibility to Cisplatin-Induced Nephrotoxicity. A. shRNA knockdown of fascin2 expression by four different targeting constructs; significant knockdown was seen with A1, B1, and D1 constructs. The impact on fascin1 and fascin2 expression is shown in B. C. Cell viability was determined by the MTT assay in cells treated 150 μM cisplatin for the indicated time periods. The results are presented as the percent viability of untreated NRK/V1 cells in SF media. D. The activities of caspase 3/7 in cells treated with 150 μM cisplatin for the indicated time periods were determined by luminescence. Data points represent the mean ± SE of four samples; *indicates a significant difference from control; similar results were seen in replicate experiments.

Article Snippet: The following antibodies were used: fascin1 (GeneTex, GTX63842; 1:1000), fascin2 (NOVUS; Ab78599, 1:1000), BH3 interacting-domain death agonist (BID; NOVUS, NB100–56106, 1:1000), B-cell lymphoma 2 (bcl-2; Cell Signaling, 2876, 1:1000), cleaved poly (ADP-ribose) polymerase (PARP; Sigma, SAB4500487, 1:1000), α(E)-catenin (GeneTex, GTX 61621, 1:1000) and anti-β-actin (Sigma, A2228, 1:2500).

Techniques: shRNA, Expressing, Construct, MTT Assay

Journal: Toxicology letters

Article Title: Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells

doi: 10.1016/j.toxlet.2016.11.021

Figure Lengend Snippet: Fascin2 Colocalizes with α-Catenin and the Actin Cytoskeleton; Overexpression of Fascin2 Rescues Stress Fibers in C2Cells. A. Immunofluorescence images (60x) of NRK-52E cells treated with rabbit anti-fascin2 (red), FITC-phalloidin (green) or anti-α-catenin (green) and DAPI (blue) demonstrates that fascin2 colocalizes with both actin and α-catenin. B. Overexpression of fascin1 (C2/Fscn1) or fascin2 (C2/Fscn2) in C2 cells. C. Phalloidin staining demonstrates increased actin stress fibers in C2/Fscn2 cells, while the bottom panel shows stress fibers using cell surface scanning with AFM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following antibodies were used: fascin1 (GeneTex, GTX63842; 1:1000), fascin2 (NOVUS; Ab78599, 1:1000), BH3 interacting-domain death agonist (BID; NOVUS, NB100–56106, 1:1000), B-cell lymphoma 2 (bcl-2; Cell Signaling, 2876, 1:1000), cleaved poly (ADP-ribose) polymerase (PARP; Sigma, SAB4500487, 1:1000), α(E)-catenin (GeneTex, GTX 61621, 1:1000) and anti-β-actin (Sigma, A2228, 1:2500).

Techniques: Over Expression, Immunofluorescence, Staining

Journal: American Journal of Translational Research

Article Title: Fascin-1 is released from proximal tubular cells in response to calcineurin inhibitors (CNIs) and correlates with isometric vacuolization in kidney transplanted patients

doi:

Figure Lengend Snippet: Urinary levels of fascin-1 are higher in kidney transplanted patients showing isometric vacuolization of tubular cells. Urinary fascin-1 (A), serum creatinine (B), proteinuria (C) and urinary KIM-1 (D) levels were analyzed in healthy volunteers, non-transplanted chronic kidney disease (CKD) patients and kidney transplanted patients presenting or not isometric vacuolization (IV) of tubular cells. Fascin-1 and KIM-1 levels were assessed using ELISA assays and the results were normalized with urinary creatinine levels. Each sample was tested twice (variation coefficient < 10%) and interplate controls were used. Data were analyzed using Kruskal-Wallis test and Dunn’s contrasts (*, P < 0.05). Fascin-1 levels in urine were significantly higher in kidney transplanted patients showing isometric vacuolization of tubular cells (A).

Article Snippet: ELISA assays Plasma and urine levels of fascin-1 and KIM-1 were determined with commercially available ELISA kits (fascin-1: E91757Hu, USCN Life Science Inc., Wuhan, PRC; KIM-1: DKM 100, R&D Systems, MN, USA) following the manufacturer instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: American Journal of Translational Research

Article Title: Fascin-1 is released from proximal tubular cells in response to calcineurin inhibitors (CNIs) and correlates with isometric vacuolization in kidney transplanted patients

doi:

Figure Lengend Snippet: An increase in urinary fascin-1 levels precedes kidney dysfunction in lung transplanted patients. The graphs show the levels of fascin-1 in serial urine samples and the corresponding serum creatinine levels in lung transplanted patients 1 (A), 2 (B), 3 (C), 4 (D). Fascin-1 levels in urine were assessed using an ELISA assay and the results were normalized with urinary creatinine levels. Each sample was tested twice (variation coefficient < 10%) and interplate controls were used. Patients 2, 3 and 4 showed an increase of urinary fascin-1 levels preceding a loss of kidney function (B, C and D, respectively). Levels of creatinine serum above 1.5 mg/ml were considered altered kidney function. Tp: Transplantation.

Article Snippet: ELISA assays Plasma and urine levels of fascin-1 and KIM-1 were determined with commercially available ELISA kits (fascin-1: E91757Hu, USCN Life Science Inc., Wuhan, PRC; KIM-1: DKM 100, R&D Systems, MN, USA) following the manufacturer instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Transplantation Assay