Journal: Neurotherapeutics

Article Title: Pyruvate Kinase M2 Increases Angiogenesis, Neurogenesis, and Functional Recovery Mediated by Upregulation of STAT3 and Focal Adhesion Kinase Activities After Ischemic Stroke in Adult Mice

doi: 10.1007/s13311-018-0635-2

Figure Lengend Snippet: rPKM2 promoted angiogenesis after ischemic stroke. Immunohistochemical staining was applied to examine vascular markers in the peri-infarct cortical region. A Immunostaining images of the vascular endothelial cell marker Glut-1 (green) and the proliferating marker BrdU (red) in sham control, stroke, and experimental groups 14 days after stroke. Arrows point to representative BrdU and Glut-1-positive cells. B A confocal image shows the colabeling of Glut-1 (green) and BrdU (red), indication of proliferating endothelial cells. C Quantified data of Glut-1/BrdU-colabeled cells. rPKM2 treatment (stroke + PKM2) increased the number of proliferating vascular cells, indicating significantly enhanced angiogenesis in stroke animals. The STAT3 inhibitor BP-1-102 eliminated the effects of rPKM2. Mean ± SEM, n = 6 in sham group, n = 9 in stroke group, n = 6 in stroke animals that received rPKM2 treatment (stroke + PKM2) group, n = 6 in stroke animals received rPKM2 and STAT3 inhibitor BP-1-102 (stroke + PKM2 + BP) group. Asterisk, p < 0.05 versus sham; number sign, p < 0.05 versus stroke; delta, p < 0.05 versus stroke + PKM2. D The immunostaining of integrin αvβ3 (red) as an angiogenic factor in the peri-infarct region at 14 days after stroke. E Costaining of integrin αvβ3 (red) and the extracellular matrix marker collagen IV. F The expression of integrin αvβ3 was quantified by the area fraction function using the NIH image J software. rPKM2 treatment significantly increased the level of integrin αvβ3 in the peri-infarct region, which was blocked by coapplied BP-1-102. n = 6 in each group; asterisk, p < 0.05 versus sham; number sign, p < 0.05 versus stroke; delta, p < 0.05 versus stroke + PKM2

Article Snippet: Sections were dried on a slide warmer for 30 min, fixed with 10% buffered formalin for 10 min, permeabilized with 0.2% Triton-X 100 (in PBS) for 5 min, blocked for 1 h with 1% fish gel at room temperature, and then incubated with the primary antibodies Glut-1 (1:800; Millipore), NeuN (1:400; Millipore), collagen IV (Millipore), and integrin α v β 3 (Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: Immunohistochemical staining, Staining, Immunostaining, Marker, Control, Expressing, Software

Journal: Neurotherapeutics

Article Title: Pyruvate Kinase M2 Increases Angiogenesis, Neurogenesis, and Functional Recovery Mediated by Upregulation of STAT3 and Focal Adhesion Kinase Activities After Ischemic Stroke in Adult Mice

doi: 10.1007/s13311-018-0635-2

Figure Lengend Snippet: rPKM2 increased migration factors and migration of neural progenitor cells. In vitro assays were performed to examine the neural progenitor cells (NPCs) dissected from the SVZ region. A The protein level of integrin β1 and FAK activation was examined in NPC cultures using Western blotting after 24 and 48 h exposure to rPKM2 (0.4 and 4 nM). B Quantification of Western blot data showed that NPCs exposed to 24 h of rPKM2 (4 nM) expressed a higher level of integrin β1. C The level of FAK phosphorylation (p-FAK) increased in NPCs exposed to 48 h of rPKM2 (0.4 and 4 nM). 3 replicates in each group and experiments were repeated 3 times. Asterisk, p < 0.05 versus control group. D and E Immunochemistry staining showed that these NPCs expressed immature neuronal markers Nestin (green) and DCX (red). F Transwell migration assay was performed to detect the effect of rPKM2 on the migration of NPCs. NPCs were treated with rPKM2 at different concentrations for 48 h prior to the migration measurement. 24 h after plating in transwell inserts, NPCs of the bottom membrane of inserts were fixed and stained with nuclei marker Hoechst 33342 and cytoskeleton marker Acti-stain 555 phalloidin. rPKM2 (0.4–4 nM) was added in the inserts (upper chamber) and the chemoattractant factor SDF-1 was added to the bottom inset to attract cell migration. There were more NPCs migrated to the bottom membrane of the inserts with 0.4 and 4 nM rPKM2. n = 3 independent assays; asterisk, p < 0.05 versus control group

Article Snippet: Sections were dried on a slide warmer for 30 min, fixed with 10% buffered formalin for 10 min, permeabilized with 0.2% Triton-X 100 (in PBS) for 5 min, blocked for 1 h with 1% fish gel at room temperature, and then incubated with the primary antibodies Glut-1 (1:800; Millipore), NeuN (1:400; Millipore), collagen IV (Millipore), and integrin α v β 3 (Santa Cruz, Dallas, TX) overnight at 4 °C.

Techniques: Migration, In Vitro, Activation Assay, Western Blot, Phospho-proteomics, Control, Staining, Transwell Migration Assay, Membrane, Marker

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: 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: 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: 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: 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

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