Journal: Cancer Science

Article Title: Ligand‐Receptor Interactions Between Squamous and Endothelial Cells Induce Head and Neck Squamous Cell Carcinoma

doi: 10.1111/cas.70085

Figure Lengend Snippet: Double immunofluorescence staining. (A) High‐grade squamous dysplasia, H&E staining. (B) High‐grade squamous dysplasia. ITGAV and FN1 proteins are displayed in red and green, respectively. (C) High‐grade squamous dysplasia. ITGAV and CCN1 proteins are displayed in red and green, respectively. Yellow markings indicate cells expressing both proteins. (D) Normal squamous epithelium, H&E staining. (E) Normal squamous epithelium. ITGAV and FN1 proteins are displayed in red and green, respectively. (F) Normal squamous epithelium. ITGAV and CCN1 proteins are displayed in red and green, respectively. Yellow markings indicate cells expressing both proteins. (G) Low‐grade squamous dysplasia, H&E staining. (H) Low‐grade squamous dysplasia. ITGAV and FN1 protein are displayed in red and green, respectively. (I) Low‐grade squamous dysplasia. ITGAV and CCN1 proteins are displayed in red and green, respectively. Yellow markings indicate cells expressing both proteins.

Article Snippet: Immunofluorescence staining was performed for ITGAV (clone: sc‐9969 AF594, mouse monoclonal antibody, Santa Cruz Biotechnology, CA, US, 1:50), Cyr61/CCN1 (clone: NB100‐356, rabbit polyclonal antibody, NOVUS, 1:200), and FN1 (clone: sc‐8422 AF488, mouse monoclonal antibody, Santa Cruz Biotechnology, 1:50).

Techniques: Double Immunofluorescence Staining, Staining, Expressing

Journal: Cancer Science

Article Title: Ligand‐Receptor Interactions Between Squamous and Endothelial Cells Induce Head and Neck Squamous Cell Carcinoma

doi: 10.1111/cas.70085

Figure Lengend Snippet: Immunohistochemistry of normal tissue and low‐grade squamous dysplasia. (A) Normal tissue, H&E staining (B) Normal tissue, ITGAV (C) Normal tissue, CCN1 (D) Normal tissue, FN1 (E) Low‐grade squamous dysplasia, H&E staining (F) Low‐grade squamous dysplasia, ITGAV (G) Low‐grade squamous dysplasia, CCN1 (H) Low‐grade squamous dysplasia, FN1. The box in the upper right corner is an enlarged image of an IPCL.

Article Snippet: Immunofluorescence staining was performed for ITGAV (clone: sc‐9969 AF594, mouse monoclonal antibody, Santa Cruz Biotechnology, CA, US, 1:50), Cyr61/CCN1 (clone: NB100‐356, rabbit polyclonal antibody, NOVUS, 1:200), and FN1 (clone: sc‐8422 AF488, mouse monoclonal antibody, Santa Cruz Biotechnology, 1:50).

Techniques: Immunohistochemistry, Staining

Journal: Cancer Science

Article Title: Ligand‐Receptor Interactions Between Squamous and Endothelial Cells Induce Head and Neck Squamous Cell Carcinoma

doi: 10.1111/cas.70085

Figure Lengend Snippet: Immunohistochemistry analysis of high‐grade squamous dysplasia and invasive SCC. (A) High‐grade squamous dysplasia, H&E staining (B) High‐grade squamous dysplasia, ITGAV (C) High‐grade squamous dysplasia, CCN1 (D) High‐grade squamous dysplasia, FN1(E) Invasive SCC, H&E staining (F) Invasive SCC, ITGAV (G) Invasive SCC, CCN1. Arrowheads indicate keratinized SCC cells. (H) Invasive SCC, FN1.

Article Snippet: Immunofluorescence staining was performed for ITGAV (clone: sc‐9969 AF594, mouse monoclonal antibody, Santa Cruz Biotechnology, CA, US, 1:50), Cyr61/CCN1 (clone: NB100‐356, rabbit polyclonal antibody, NOVUS, 1:200), and FN1 (clone: sc‐8422 AF488, mouse monoclonal antibody, Santa Cruz Biotechnology, 1:50).

Techniques: Immunohistochemistry, Staining

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Apelin enhances integrin αvβ3-dependent prostate cancer motility. (A) Cells were treated with apelin for 24 hours, the indicated integrin expression was examined by qPCR (n=3). (B&C) Cells were treated with integrin αvβ3 or α5β1 antibody then with apelin, the wound healing and cell migration was examined (n=3). (D) Integrin αv and β3 gene levels in normal and prostate cancer patients retrieved from the GEO database. (E&F) Representative images showing the results of IHC staining for integrin αvβ3 in tissue samples from healthy individuals (n=3) and prostate cancer patients (n=3). (G&H) Cells were co-transfected with integrin αv and β3 siRNA then with apelin, the wound healing and cell migration was examined (n=3). * p < 0.05 compared with the control group. # p < 0.05 compared with the apelin-treated group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Migration, Immunohistochemistry, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: MAPK pathway is regulated apelin-induced integrin expression and prostate cancer migration. (A&B) IPA pathway enrichment figure showing pathways that were changed in the GSE7930 dataset (Orange color indicated upregulated gene profile; blue color indicated downregulated gene profile). (C-H) Cells were treated with ERK (ERK II inhibitor; 10 μM), p38 (SB203580; 10 μM) and JNK (SP600125; 10 μM) inhibitors or transfected with ERK, p38 and JNK siRNA for 24 hours then with apelin, the wound healing, cell migration and integrin mRNA expression was examined (n=3). (I) PC3 cells were stimulated with apelin and ERK, p38 and JNK phosphorylation was examined by Western blotting (n=3). * p < 0.05 compared with the control group. # p < 0.05 compared with the apelin-treated group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Migration, Transfection, Phospho-proteomics, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Apelin promotes integrin production and cell motility via the STAT3 pathway. (A-F) Cells were treated with STAT3 inhibitor (10 μM) or transfected with STAT3 siRNA for 24 hours then with apelin, the wound healing, cell migration and integrin mRNA expression was examined (n=3). (G&H) PC3 cells were treated with apelin or pretreated with ERK, p38 and JNK inhibitor then with apelin, the STAT3 phosphorylation was examined by Western blotting (n=3). * p < 0.05 compared with the control group. # p < 0.05 compared with the apelin-treated group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Transfection, Migration, Expressing, Phospho-proteomics, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Apelin enhances integrin expression and promotes cell migration by inhibiting miR-8070 expression. (A) The diagrams depict the selection of miRNA candidates targeting integrin αv and β3. (B) PC3 cells were treated with apelin, the miRNAs expression was examined by qPCR (n=3). (C) Cells were treated with apelin for 24 hours, the miR-8070 expression was examined by qPCR (n=3). (D-F) Cells were transfected with miR-8070 mimic for 24 hours then with apelin, the wound healing, cell migration and integrin mRNA expression was examined (n=3). (G-I) Cells were treated with ERK, p38 and JNK inhibitor or siRNA then with apelin, the 3'UTR activity and miRNA expression was examined by luciferase activity and qPCR (n=3). * p < 0.05 compared with the control group. # p < 0.05 compared with the apelin-treated group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Migration, Selection, Transfection, Activity Assay, Luciferase, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Apelin blockade inhibits prostate cancer metastasis in vivo . (A-C) Cells were transfected with apelin siRNA, the integrin expression, wound healing and cell migration was examined (n=3). (D&E) IHC analysis of prostate cancer tissue samples stained with integrin αvβ3 antibody (n=3). (F) IHC analysis of leg bone (n=3), liver (n=3), and lung (n=3) sections stained with apelin and integrin αvβ3 antibodies. * p < 0.05 compared with the control group.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: In Vivo, Transfection, Expressing, Migration, Staining, Control

Journal: International Journal of Biological Sciences

Article Title: Apelin facilitates integrin αvβ3 production and enhances metastasis in prostate cancer by activating STAT3 and inhibiting miR-8070

doi: 10.7150/ijbs.113161

Figure Lengend Snippet: Schematic diagram illustrating the mechanism underlying the effects of apelin in prostate cancer metastasis. Apelin stimulation enhances integrin αvβ3-dependent prostate cancer migration and metastasis. The activation of STAT3 and inhibition of miR-8070 via the ERK, p38 and JNK pathways mediate apelin-facilitated integrin synthesis and cell motility.

Article Snippet: JNK (SC-474), p38 (SC-4972), ERK (SC-1647), STAT3 (SC-482), p-JNK (SC-6254), p-p38 (SC-166182), p-ERK (SC-7383) and integrin αvβ3 (SC-7312) antibodies as well as JNK (sc-29380), p38 (sc-29433), STAT3 (sc-29493), integrin αv (sc-29373), integrin β3 (sc-29375) and apelin (sc-44741) siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Migration, Activation Assay, Inhibition

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: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Representative IHC images of tumour samples from patients with low and high α V expression in tumour cells. Objective: 20×. b Kaplan−Meier curve of OS for stage I treatment-naïve lung cancer patients according to the α V expression by IHC analysis of FFPE tumours. c Kaplan−Meier curve of PFS of PD-1 blockade-treated patients with tumours harbouring low and high expression of α V integrin. d Percentages of anti-PD-(L)1-treated patients displaying α V high tumours among long-responders (LR: PFS > 6 months and OS > 12 months) or fast progressors (FP: defined by “early death” occurring within 12 weeks of treatment initiation). e Representative digital mark-up image of fluorescent IHC of CD8 (green), cytokeratin (turquoise), and dapi (blue) staining in α V low and α V high tumour sections. d = CD8 + cell density. Left, the density of CD8 + TIL in α V low and α V high tumours. The numbers of tumours in each group are indicated (* p = 0.046). Scale bar, 2 cm. f Representative digital mark-up image of CD8 + CD103 neg (green), CD8 + CD103 + (orange), CD8 - CD103 + (red), cytokeratin (turquoise) and dapi (blue) staining in α V low and α V high tumour sections. d = CD8 + CD103 + cell density. Left, the density of CD8 + CD103 + (* p = 0.016) and CD8 + CD103 neg ( p = 0.120) cells in tumour regions of α V low and α V high tumours. Scale bar, 2 cm. Each symbol represents an individual cell type from tumour samples; horizontal lines correspond to mean ± standard error of the mean (SEM) ( e , f ). Data were calculated with the log-rank test ( b , c ) and Welch’s two-sided t -test ( e , f ). Source data are provided as a Source Data file.

Article Snippet: The cells were double transfected with integrin α V CRISPR-Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400506) and integrin α V HDR plasmid (Santa Cruz Biotechnology, sc-400506-HDR).

Techniques: Expressing, Staining

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Representative flow cytometry plots (bi-exponential scale) of α V expression in EpCAM + E-cadherin + and EpCAM neg E-cadherin neg cells from a lung tumour. Right, percentage of α V expression in EpCAM + E-cadherin + and EpCAM neg E-cadherin neg cells ( n = 18, *** p = 0.0002). b Representative flow cytometry plots of β 6 subunit expression in EpCAM + E-cadherin + α V + and EpCAM neg E-cadherin neg α V + cells from a tumour sample. Right, expression of β 6 integrin in EpCAM + E-cadherin + α V + and EpCAM neg E-cadherin neg α V + cells ( n = 16), * p = 0.013. c Surface expression of α V , β 6 , and β 8 subunits in the IGR-B2 cell line. d Concentration of total TGF-β in CM from IGR-B2, IGR-B2T, and IGR-B2T-KO cells measured by ELISA (*** p = 0.0004). Results are presented as mean ± SEM of six independent experiments. Right, relative luciferase activity in the Mu.1LV cell line transfected with (CAGA)9-Lux reporter plasmid and treated with CM from IGR-B2, IGR-B2T, and IGR-B2T-KO cells, normalized to luciferase activity in Mu.1LV cell treated with CM from IGR-B2. Results are presented as mean ± SEM of six independent experiments (* p = 0.011, **** p < 0.0001). e Expression of α V integrin on IGR-B2T and IGR-B2T-KO cells. An isotype control was included. f Representative photos of the morphology of IGR-B2T and IGR-B2T-KO cells by phase-contrast light microscope from one experiment out of five. Objective: 20×. Each symbol represents the individual cell type from tumour samples ( a , b ); horizontal lines correspond to mean ± SEM ( a , b , d ). Data were calculated with paired Student t -tests ( a , b ) and one-way ANOVA with Tukey’s correction ( d ). ns: non-significant. Source data are provided as a Source Data file.

Article Snippet: The cells were double transfected with integrin α V CRISPR-Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400506) and integrin α V HDR plasmid (Santa Cruz Biotechnology, sc-400506-HDR).

Techniques: Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Control, Light Microscopy

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: αvβ3 integrin cell surface expression on untransduced, scrambled shRNA, and αv stable knocked down SK-Mel-28 cells. p<0.001 (***). αvβ5 integrin expression was not examined since we demonstrated that SK-Mel-28 cells do not express the β5 subunit (Seoane et al. 2010).

Article Snippet: Integrin αv gene expression knockdown Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Expressing, shRNA

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: r-Moj-DL, r-Moj-DM, and r-Moj-DN peptides induced apoptosis of SK-Mel-28 cells by binding to the αv integrin. p=0.05 (*), p=0.01 (**), p<0.001 (***).

Article Snippet: Integrin αv gene expression knockdown Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Binding Assay

Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): a second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells

doi: 10.1016/j.toxicon.2016.04.038

Figure Lengend Snippet: All r-Moj-D_ mutant peptides inhibited proliferation of SK-Mel-28 cells by binding to the αv integrin. The scrambled shRNA control treated cells are not shown, since these cells grew at much slower rate than untransduced or αv knocked down cells.

Article Snippet: Integrin αv gene expression knockdown Inhibition of integrin αv expression was performed in SK-Mel-28 cells using integrin αv shRNA (h) lentiviral particles (Santa Cruz Biotech, sc-29373-v).

Techniques: Mutagenesis, Binding Assay, shRNA, Control