Journal: EBioMedicine

Article Title: Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice

doi: 10.1016/j.ebiom.2018.06.004

Figure Lengend Snippet: BC71 targets cell-surface GRP78 but not αvβ5 integrin to induce apoptosis. (a) BC71 induces HUVECs apoptosis in a dose-dependent manner. HUVECs were treated with BC71 (concentration range: 12.5, 25, 50, 100 μM) for 24 h and apoptosis was determined using the cell death ELISA kit (Roche). (b) Anti-GRP78 N-terminal domain antibody blocked the apoptosis function of BC71 in a dose-dependent manner. The apoptosis of the combined treatment with increasing amount of anti-GRP78 N-terminal domain antibody and 100 μM BC71 for 24 h was measured using the Cell Death Detection ELISA. (c) Anti-GRP78 C-terminal domain antibody and (d) anti-αvβ5 antibody did not block BC71 induced apoptosis. For clarity of presentation, data were normalized with that of non-treated (VEGF only) cells, which was set as 1. Data are expressed as mean ± standard error of the mean. The results are representative of at least three independent experiments. Statistical significance was determined using ANOVA. *P < 0.05; **P < 0.01, n ≥ 3.

Article Snippet: Antibodies against integrin αvβ5 (P1F76; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GRP78 (A-10, Santa Cruz Biotechnology) and GRP78 (C-20, Santa Cruz Biotechnology) were used for neutralization.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Acupuncture Delays Cartilage Degeneration through Upregulating SIRT1 Expression in Rats with Osteoarthritis

doi: 10.1155/2021/2470182

Figure Lengend Snippet: Acupuncture inhibited OA-associated inflammation, the NF- κ B signaling pathway, and ECM degradation through upregulating SIRT1 expression in rat articular cartilages. (a/b): The levels of TNF- α and IL-2 in the serum of acupuncture-treated OA rats injected with or without shSIRT1 lentiviruses were assessed by enzyme-linked immunosorbent assay. (c/d/e/f/g/h): The expressions of SIRT1, MMP-9, ADAMTS5, p-p65/p65, p-I κ B α /I κ B α , collagen II, and aggrecan in the articular cartilage of acupuncture-treated OA rats injected with or without shSIRT1 lentiviruses were analyzed by western blot, with GAPHD serving as a reference gene. ∧ P or ‡ P < 0.05; ∗∗ P or ∧∧ P or ## P or ‡‡ P < 0.01; ∗∗∗ P or ^^^ P or ### P or ‡‡‡ P < 0.001; ∗ vs. Sham; ∧ vs. Model + shNC; # vs. Model + Acupuncture + shNC; ‡ vs. Model + shSIRT1 (OA: Osteoarthritis; TNF- α : Tumor necrosis factor- α ; IL-2: interleukin-2; shNC: shRNA-negative control; MMP-9: matrix metallopeptidase-9; SIRT1: NAD-dependent deacetylase sirtuin-1; ADAMTS5: a disintegrin and metalloproteinase with thrombospondin motifs 5).

Article Snippet: The membranes were blocked by 5% nonfat milk (P2194, Sigma-Aldrich, USA) in tris buffered saline with 1% Tween 20 (TBST; TA-125-TT, ThermoFisher, USA) for 1 h and further probed with primary antibodies against SIRT1 (#9475, 120 kDa, 1 : 1000, Cell Signaling Technology, Danvers, MA, USA), matrix metallopeptidase (MMP)-9 (ab76003, 92 kDa, 1 : 1000, Abcam, USA), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5; ab41037, 73 kDa, 1 : 250, Abcam, USA), phosphorylated (p)-p65 (#3033, 62 kDa, 1 : 1000, Cell Signaling Technology, USA), p65 (#8242, 65 kDa, 1 : 1000, Cell Signaling Technology, USA), p-I κ B α (#2859, 40 kDa, 1 : 1000, Cell Signaling Technology, USA), I κ B α (#4812, 39 kDa, 1 : 1000, Cell Signaling Technology, USA), collagen II (ab188570, 141 kDa, 1 : 1000, Abcam, USA), aggrecan (orb624552, 250 kDa, 1 : 500, Biorbyt, Cambridge, UK), and GAPDH (ab8245, 36 kDa, 1 : 1000, Abcam, USA) at 4°C overnight.

Techniques: Expressing, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, shRNA, Negative Control, Histone Deacetylase Assay

Journal: The Journal of Biological Chemistry

Article Title: Oviductosome-Sperm Membrane Interaction in Cargo Delivery

doi: 10.1074/jbc.M114.633156

Figure Lengend Snippet: Localization of CD9 and the αv subunit of αvβ3 integrin on sperm and OVS. A, distribution of CD9 and αv integrin on sperm studied by immunofluorescence and conventional confocal microscopy. CD9 (green, a-b) and αv integrin (red, c-d) localized on the midpiece, the proximal principal piece, and the over the acrosome. Sperm nuclei were stained with DAPI (blue) and are seen in the merged images in b and d. IgG control did not give any signal (e-f).). Representative images from a total of n = ∼10 sperm in each group. B, Western blotting analysis performed with anti-CD9 (a) and anti-αv integrin (b) antibodies on OVS recovered from oviductal fluid, proestrus, and estrus combined and metestrus and diestrus combined, as well as hormonally induced estrus (I.E OVS), using epididymosomes (EPI) and uterus as positive controls. The 24-kDa CD9 and the 130-kDa αv integrin are present in OVS from all stages. C, co-localization of FM4–64FX-labeled OVS and αv integrin on caudal sperm following co-incubation and in vitro fusion, using three-dimensional SR-SIM. Immunofluorescence reveals that transferred αv integrin potentially co-localizes with OVS on the sperm membrane. The localization pattern of αv is similar to PMCA4a, over the acrosome, neck (blue arrow), mid-piece, and the proximal principal piece of the sperm flagellum. The yellow arrows show potential overlap between the red FM4–64FX and the green αv integrin signal giving yellow on the head (a) and on the midpiece (b), since the presence of endogenous αv integrin cannot be ruled out at these sites. The green signal on the head (b) and principal piece (a, b) is unequivocally endogenous αv integrin. Sperm nuclei were stained blue with DRAQ5. Images were captured with a 63× Plan-Apochromatic oil immersion objective (numerical aperture of 1.4). Representative images from a total of n = ∼15 sperm Scale bars, 5 μm.

Article Snippet: To perform a function-blocking assay, a rat monoclonal antibody against mouse αv integrin subunit (RM47) and its isotype IgG 1κ (R3–34) were obtained from BD Bioscience (San Diego, CA).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Western Blot, Labeling, Incubation, In Vitro

Journal: Cancer Science

Article Title: Significance of integrin αvβ5 and erbB3 in enhanced cell migration and liver metastasis of colon carcinomas stimulated by hepatocyte‐derived heregulin

doi: 10.1111/j.1349-7006.2010.01640.x

Figure Lengend Snippet: Liver metastasis of LS174T and LS‐LM6 and increased expression of integrin αvβ5 in LS‐LM6. (a) Livers at 6 weeks after intrasplenic injection of LS174T and LS‐LM6. (b) Flow cytometric analysis of surface integrins on LS174T and LS‐LM6. Three independent experiments were carried out and produced similar results.

Article Snippet: Rat mAb to integrin αv was from Immunotech.

Techniques: Expressing, Injection, Produced

Journal: Cancer Science

Article Title: Significance of integrin αvβ5 and erbB3 in enhanced cell migration and liver metastasis of colon carcinomas stimulated by hepatocyte‐derived heregulin

doi: 10.1111/j.1349-7006.2010.01640.x

Figure Lengend Snippet: Enhancement of integrin αvβ5‐dependent migration of LS‐LM6 by hepatocyte‐conditioned medium (HCM). (a) Migration of LS‐LM6 on vitronectin (VN) by HCM. Both LS174T and LS‐LM6 can migrate 100–300 cells on VN without stimulation. The migration of LS‐LM6 is enhanced up to sixfold by HCM (×1) and 6.9‐fold by the twice‐concentrated HCM (×2) compared with the control medium (*P < 0.05, two‐way anova). LS174T shows no significant migration by HCM. The filtrated fraction containing the low‐molecular weight proteins (FCM) has no effect on cell migration. (b) The enhanced migration of LS‐LM6 on VN by HCM is significantly inhibited by the neutralizing antibodies against integrins αvβ5 and αv, and VN (*P < 0.05, two‐way anova).

Article Snippet: Rat mAb to integrin αv was from Immunotech.

Techniques: Migration, Control, Molecular Weight

Journal: Cancer Science

Article Title: Significance of integrin αvβ5 and erbB3 in enhanced cell migration and liver metastasis of colon carcinomas stimulated by hepatocyte‐derived heregulin

doi: 10.1111/j.1349-7006.2010.01640.x

Figure Lengend Snippet: Abrogation of heregulin (HRG)‐induced migration by knock down of integrin αvβ5 and erbB3. (a) Reverse transcription (RT)‐PCR shows siRNAs of both integrin αv and erbB3 effectively suppressed the expression of their mRNAs in the transfected LS‐LM6 cells. (b) Western blot shows that synthesis of integrin αv and erbB3 are suppressed by siRNAs, but control siRNAs show no suppression (left panel). After HRG treatment, the phosphorylated erbB3 and Akt are suppressed in si‐erbB3 cells. Cells transfected with either integrin αv or erbB3 siRNAs show no response to HRG‐induced migration on vitronectin (VN) (right panel) (*P < 0.05, two‐way anova). (c) Liver metastasis lesions of both si‐integrin αv and si‐erbB3 cells are significantly suppressed compared with that of si‐control cells, but tumor sizes in the spleen are almost same. Arrows indicate metastatic nodules of LS‐LM6.

Article Snippet: Rat mAb to integrin αv was from Immunotech.

Techniques: Migration, Knockdown, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Control

Journal: Cancer Science

Article Title: Significance of integrin αvβ5 and erbB3 in enhanced cell migration and liver metastasis of colon carcinomas stimulated by hepatocyte‐derived heregulin

doi: 10.1111/j.1349-7006.2010.01640.x

Figure Lengend Snippet: Inhibition of hepatic metastasis of LS‐LM6 cells by si‐RNA

Article Snippet: Rat mAb to integrin αv was from Immunotech.

Techniques: Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Cell adhesion in cancer: Beyond the migration of single cells

doi: 10.1074/jbc.REV119.007759

Figure Lengend Snippet: Different drug discovery approaches to targeting integrin for cancer therapy. A, conjugate presenting a selective binder for the extracellular region of integrin aVb3 (black) covalently linked to sunitinib (blue) (79). B, amphiphilic dendrimer composed with a dual-targeting peptide bearing an Arg-Gly-Asp-Lys (RGDK) lipopeptide (red) (C) (81). D, atorvastatin identified as a novel candidate for drug repurposing for undruggable integrins (82).

Article Snippet: Some αV integrin antibodies have been evaluated in late-stage clinical trials, such as abituzumab (Merck KGaA, Darmstadt, Germany) and intetumumab (Centocor, Malvern, PA).

Techniques: Drug discovery