Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: The podosome marker protein Tks5 regulates macrophage invasive behavior.

doi: 10.1002/cm.20545

Figure Lengend Snippet: Fig. 1. Differentiated THP-1 macrophages form proteolytically active podosome adhesions. (A–C) THP-1 cells were treated with 25 nM PMA for 72 h, then fixed and stained with phalloidin to visualize F-actin (red) and with antibodies to either WASp (A), cortactin (B), or vinculin (C) (green). Shown are larger composite images; the demarcation of which depicts the results for each individual detection agent in the inset images. (D–F) THP-1 cells were cultured on fluorescently labeled gelatin for 24 h in the presence of 25 nM PMA (A), PMA plus 10 lM GM6001 (B), or DMSO as a negative control (C). Cells were fixed and stained with Hoechst 33258 to visualize nuclei (blue). Holes (dark regions) within the fluorescent gelatin monolayer (green) are indicative of gelatin degradation (arrows).

Article Snippet: Primary antibodies were used to detect WASp (1:50; #sc-8353; Santa Cruz Biotechnology, Santa Cruz, CA), cortactin (1:100; #05-180; Millipore, Billerica, MA), vinculin (1:100; #05-386; Millipore, Billerica, MA), phosphotyrosine (1:250; #05-321; Cell Signaling), Tks5 (1:250), and Src (1:200; cst.1) in 5% donkey serum (The Jackson Laboratory, Bar Harbor, ME)/PBS for 2 h at room temperature or for overnight at 4 C. Tks5 antibodies to the first (D7771, polyclonal) and fourth (2F4G8D2, monoclonal) SH3 domains were generated in the laboratory of Dr. Sara Courtneidge (Sanford-Burnham Medical Research Institute).

Techniques: Staining, Cell Culture, Labeling, Negative Control

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: The podosome marker protein Tks5 regulates macrophage invasive behavior.

doi: 10.1002/cm.20545

Figure Lengend Snippet: Fig. 2. Tks5 is a podosome marker protein that accumulates during macrophage differentiation. (A and B) THP-1 cells (A) were differentiated with 25 nM PMA for 72 h, while PBMCs (B) were differentiated in the presence of 10% FBS for 6 days. All cells were fixed and stained with phalloidin to visualize F-actin (red) and with a Tks5 antibody (green). Shown are larger composite images; the demarcation of which depicts the results for each individual detection agent in the inset images. (C–E) THP-1 cells were treated with or without 25 nM PMA for the indicated times (C) or for 72 h with the indicated PMA concentrations (D). Protein levels for Tks5, cortactin, WASp, and GAPDH (loading control) were determined from total cell lysates by immunoblot analysis with specific antibodies. Densitometry in D was used to determine the fold change in Tks5 protein levels relative to untreated (0 nM) cells. (E) Human PBMCs differentiated for the indicated times were analyzed for Tks5, cortactin, WASp, and GAPDH (loading control) protein levels by immunoblot analysis of total cell lysates.

Article Snippet: Primary antibodies were used to detect WASp (1:50; #sc-8353; Santa Cruz Biotechnology, Santa Cruz, CA), cortactin (1:100; #05-180; Millipore, Billerica, MA), vinculin (1:100; #05-386; Millipore, Billerica, MA), phosphotyrosine (1:250; #05-321; Cell Signaling), Tks5 (1:250), and Src (1:200; cst.1) in 5% donkey serum (The Jackson Laboratory, Bar Harbor, ME)/PBS for 2 h at room temperature or for overnight at 4 C. Tks5 antibodies to the first (D7771, polyclonal) and fourth (2F4G8D2, monoclonal) SH3 domains were generated in the laboratory of Dr. Sara Courtneidge (Sanford-Burnham Medical Research Institute).

Techniques: Marker, Staining, Control, Western Blot

Journal: eLife

Article Title: Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

doi: 10.7554/eLife.83710

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse anti-M13 pVIII-HRP, clone RL-pH1 , Santa Cruz Biotech , Cat# sc53004 , .

Techniques: Construct, Transfection, Recombinant, Avidin-Biotin Assay, Virus, Sequencing, Cloning, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation

doi: 10.1186/s13046-019-1211-2

Figure Lengend Snippet: Effects of PDGF-B knockdown on metastasis, chemosensitization and vascular maturity and functionality. ( a ) Double staining for CD31 (green) and α-SMA (red) of 4T1 tumor sections. shRNA-PDGF-B- and shRNA-Control-transfected 4 T1 tumor-bearing mice received control, metformin (225 mg/kg), imatinib (60 mg/kg) for the combined treatment. Fluorescent signaling of series thin-layer scanning were reconstructed for 3D observation of VSMCs on vessels. VSMCs No. is at the bottom of the panel and indicated as mean ± SEM (n = 8). ( b ) Quantification of ratio of α-SMA + area/CD31 + area (upper), CD31 + area per μm 2 (middle) and percentage of α-SMA + VSMCs associated with vessels (bottom) in 4 T1 cancers (n = 8). ( c ) Representative images showing decreased vascular leakage in shRNA-PDGF-B- than shRNA-Con-transfected 4T1 tumors. Fitc-dextran (green) was injected through tail vein 10 mins before sacrifice. Tumor sections was counterstained with anti-CD31 antibody (Red). White arrows indicate the dextran leaking outside the vessel wall. Bars: 100 μm. ( d ) Representative images showing more lectin-perfused CD31 + vessels in shRNA-PDGF-B- than shRNA-Con-transfected 4T1 tumors (n = 8). Red: perfused lectin; Green: CD31 + vessels. White arrows indicate CD31 + vessels with lectin perfusion. Percentage of Lectin + /CD31 + vessels (of CD31 + vessels) is indicated as mean ± SEM (at the bottom of the panel). ( e ) H&E staining of sections of 4T1 cancers (Left) and quantification of necrotic and hemorrhagic areas (Right; n = 8). shRNA-PDGF-B or shRNA-Con-transfected 4T1 tumor-bearing mice received low dose CTX (20 mg/kg•day). “N” indicates “necrosis”; black arrows indicate tumor hemorrhage. ( f ) Decreased primary tumor lung metastasis in mice bearing shRNA-PDGF-B 4T1 cancer cells than that in mice bearing shRNA-Con 4T1 cells. (Upper) H&E staining for 4T1 tumor sections; (Lower) quantification of 4T1 metastatic index (metastatic nodules per gram of primary tumor; n = 8). Red asterisk indicates lung metastasis nodule. Magnification: 200Х. ( g ) Schematic diagram of metformin-induced vascular remodeling in metastatic breast cancers. Metastatic breast cancers are angiogenic with hypoperfusion and vascular immaturity, which contribute to vascular leakage, chemoresistance, hypoxia and distant metastasis. By decreasing PDGF-B of those metastatic breast cancers, metformin inhibits angiogenesis and improved the vascular maturity and functionality, therefore improving the chemosensitivity and reducing the distant metastasis

Article Snippet: Metformin (No.13118), cyclophosphamide (CTX, No.13849), cisplatin (CPT, No.13119) and imatinib (No.13139) were purchased from Cayman Chemical.

Techniques: Knockdown, Double Staining, shRNA, Control, Transfection, Injection, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CX3CR1 positively regulates BCR signaling coupled with cell metabolism via negatively controlling actin remodeling

doi: 10.1007/s00018-019-03416-7

Figure Lengend Snippet: Loss of CX3CR1 enhances the accumulation of F-actin mediated by WASP and WIP. Confocal analysis of pWASP and F-actin staining (a) and pEzrin (i) from WT and CX3CR1 KO splenic B cells upon antigenic stimulation. The Pearson’s correlation coefficients between BCR and pEzrin (j) were determined using the ZEN 2.3 (blue edition) software. Western blot analysis of the expression levels of pWASP, WASP and WIP (b) and pEzrin (k) in splenic B cells upon antigenic stimulation. Shown are representative blots, blots’ relative band intensities and the ratio of pWASP, WASP, pWASP to WASP, WIP and pEzrin from three independent experiments (c–f, l). Splenic B cells were incubated with Percp-anti-B220 for 30 min at 4 °C. Then the cells were either incubated with streptavidin or with medium alone (0 min) as a control at 37 °C for indicated time. After fixation and permeabilization the cells were stained for pWASP and F-actin for phosflow cytometry. Relative MFI of pWASP and F-actin in splenic B cells was quantified using Flow Jo software (g, h). Shown are representative images and mean values (± SEM) from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Lysates were analyzed by SDS-PAGE, and transferred onto a nitrocellulose membrane and probed with anti-pCD19 (3571S, Cell Signaling Technology), anti-pY (05-321, Merck Millipore), anti-pBTK (5082S, Cell Signaling Technology), anti-pSHIP1 (3941S, Cell Signaling Technology), anti-pAkt (4060L, Cell Signaling Technology), anti-pS6 (4856S, Cell Signaling Technology), anti-pFoxO1 (9461S, Cell Signaling Technology), anti-pmTOR (5536S, Cell Signaling Technology), anti-pPI3K p85/p55 (4228S, Cell Signaling Technology), anti-WASP (sc-13139 SantaCruz), anti-pWASP (A300-205A, Bethyl), anti-WIP (sc-271113SantaC), anti-BTK (8547S, Cell Signaling Technology), anti-SHIP1 (2728S, Cell Signaling Technology), anti-PI3K (4292S,Cell Signaling Technology), anti-FoxO1 (2880S,Cell Signaling Technology), anti-Akt (9272S,Cell Signaling Technology), anti-S6 (2217S,Cell Signaling Technology), anti-mTOR (2983S,Cell Signaling Technology), anti-HIF-1α (39665, Active Motif), anti-NF-κB (D14E12, Cell Signaling Technology), anti-pNF-κB (3033S, Cell Signaling Technology), anti-pIKKα/β (2697S, Cell Signaling Technology), anti-IKKβ (D30C6, Cell Signaling Technology) and β-actin (60008-1-IG-10 proteintech).

Techniques: Staining, Software, Western Blot, Expressing, Incubation, Cytometry