Journal: Journal of Cellular and Molecular Medicine

Article Title: Transcriptomic analysis reveals cell apoptotic signature modified by heparanase in melanoma cells

doi: 10.1111/jcmm.14349

Figure Lengend Snippet: Gene ontology classification of differentially expressed genes regulated by silencing of heparanase gene (HPSE) expression. (A) Gene ontology (GO) analysis of up‐ and (B) down‐regulated genes after silencing of HPSE in MDA‐MB‐435s cells by terms of biological process and cellular component. X ‐axis i ndicates functional fold enrichment calculated by binomial test, P < 0.01. (C) Listing of an array of 28 pro‐apoptotic genes classified by GO term positive regulation of cell death and apoptotic process. Y ‐axis indicates fold change comparing HPSE silenced cells with control cells. Dashed line indicates 1.5‐fold change. (D) Validation of expression of the 28 pro‐apoptotic genes by real‐time PCR. n = 3 biological repeats, * indicates the selected genes for further validation by Western blots. Dashed line indicates 1.5‐fold change. (E) Validation of up‐regulation of selected genes including CYR61, EGR1 and TNFRSF12A on protein level by Western blots. N = 3 biological repeats, representative blots are shown

Article Snippet: Anti‐EGR1 (AF2818), CYR61 (MAB4055), TNFRSF12 (MAB1199) were from R&D Systems (Abingdon, UK), and anti‐GAPDH (AM4300) from Ambion.

Techniques: Expressing, Functional Assay, Control, Biomarker Discovery, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Cysteine‐rich protein 61 (Cyr61) levels are upregulated in plasma and bone marrow ( BM ) samples from patients with CML . A, Left panel: Levels of Cyr61 in the plasma from CML patients (n = 36) and normal plasma from age‐matched healthy individuals ( CON ; n = 66) were detected by ELISA . Right panel: Levels of Cyr61 in the BM supernatant from CML patients (n = 33) and the normal BM supernatant from age‐matched healthy transplant donors (n = 11) were detected by ELISA . B, Levels of Cyr61 in the plasma from CML patients in blast crisis ( BC ) (n = 5) and in chronic phase ( CP ) (n = 31) were detected by ELISA . Right panel: Levels of Cyr61 in the marrow from CML patients in BC (n = 4) and in CP (n = 29) were detected by ELISA . C, Relative levels of Cyr61 mRNA in a T acute lymphoblastic leukemia ( ALL ) cell line (Jurkat), B ALL cell line (Nalm‐6), and CML cell line (K562) were detected by qRT ‐ PCR , and the level of Cyr61 mRNA in Nalm‐6 cells was taken as the control to calculate the relative expression of Cyr61 in Jurkat and K562 cells. D, Levels of Cyr61 protein in Jurkat, Nalm‐6, and K562 cells were detected by western blotting. Band intensity of Cyr61 was quantified by densitometry and normalized to GAPDH . E, Concentration of Cyr61 in the culture supernatant of Jurkat, Nalm‐6, and K562 cells was detected by ELISA . Data represent mean ± SEM of at least 3 independent experiments. *P < 0.05, ** P < 0.01

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Expressing, Western Blot, Concentration Assay

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Role of cysteine‐rich protein 61 (Cyr61) in the chemosensitivity of CML cells to imatinib mesylate ( IM ). A, K562 cells were treated with Cyr61 (125, 250, 500, 1000 ng/mL) for 24 h, and then treated with 0.5 μmol/L IM for 24 h; the percentages of apoptotic K562 cells were determined by flow cytometric analysis. Average percentage of apoptotic cells is shown. B, K562 cells were collected, incubated with Cyr61 (1000 ng/ mL ) preincubated with the antihuman Cyr61 093G9 monoclonal antibody (5000 pg/ mL ) or murine isotype‐matched antibody (Con‐IgG) (5000 pg/ mL ), and then treated with 0.5 μmol/L IM for 24 h. Cell apoptosis was determined by flow cytometric analysis. C, Cyr61 knockdown by shCyr61 or sh NC (negative control) in K562 cells. Endogenous Cyr61 expression is shown in the upper panel, whereas the secreted Cyr61 level in culture medium was determined by ELISA and shown in the lower panel. D, Ratio of apoptotic K562‐shCyr61 and K562‐sh NC cells was determined by flow cytometry at 24 h post‐treatment with or without 0.5 μmol/L IM . E, K562 cells were incubated with BM supernatants from a mixture of different CML patients (Cyr61 concentration was 243 pg/ mL ) with preincubation with 1000 pg/ mL 093G9 antibody or murine isotype‐matched antibody (Con‐IgG) for 2 h, and then treated with 0.5 μmol/L IM for 24 h. F, Human CML cell line KCL 22 cells were treated with Cyr61 (1000 ng/mL) for 24 h and then treated with 0.5 μmol/L IM for 24 h; the percentages of apoptotic cells were determined by flow cytometric analysis. G, Primary leukemic cells from three patients with CP CML were isolated and treated with exogenous recombinant human Cyr61 (1000 ng/mL) for 24 h, and then treated with 0.5 μmol/L IM for 24 h. Data represent mean ± SEM of at least 3 independent experiments. * P < 0.05, ** P < 0.01

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Incubation, Knockdown, Negative Control, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Concentration Assay, Isolation, Recombinant

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Cysteine‐rich protein 61 (Cyr61) activates Bcl‐2 transcription in CML cells. A, Left panel: Bcl‐2, Bcl‐ xL , XIAP and Survivin mRNA expression in K562 cells treated by 1000 ng/mL Cyr61 for 8 h was detected by real‐time PCR . Right panel: Bcl‐2, Bcl‐ xL , XIAP and Survivin mRNA expression in K562‐shCyr61 cells and K562‐sh NC cells was detected by real‐time PCR . B, Left panel: Bcl‐2 protein in K562 cells stimulated by 1000 ng/mL Cyr61 for 48 h was detected by western blotting. Right panel: Bcl‐2 protein in K562‐shCyr61 cells and K562‐sh NC cells was detected by western blotting. The band intensity of Bcl‐2 was quantified by densitometry and normalized to GAPDH . C, K562 cells were treated with Cyr61 (1000 ng/mL), ABT 199 (1 μmol/L) (specific Bcl‐2 inhibitor), Cyr61 + ABT 199, or ABT 199 for 24 h, and then treated with 0.5 μmol/L imatinib mesylate ( IM ) for 24 h. Percentages of apoptotic K562 cells were determined by flow cytometric analysis. Data represent the mean ± SEM of at least 3 independent experiments. * P < 0.05, ** P < 0.01

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Cysteine‐rich protein 61 (Cyr61) inhibits imatinib mesylate ( IM )‐induced apoptosis through the nuclear factor kappa B ( NF ‐κB) signaling pathway. A, Effect of the inhibitors of signaling pathways on Cyr61 decreased CML cell apoptosis induced by IM . K562 cells were treated with 20 μmol/L LY 294002, 1 μmol/L PD 98059 or 4 μmol/L PDTC in combination with Cyr61 (1000 ng/mL) for 24 h and then treated with 0.5 μmol/L IM for 24 h; the percentages of apoptotic K562 cells were determined by flow cytometric analysis. B, NF ‐κB phosphorylation was detected by western blotting. Lane 1: stimulation of K562 cells with 0.5 μmol/L IM for 10 min; lane 2: stimulation of K562 cells with 1000 ng/ mL Cyr61 + 0.5 μmol/L IM for 10 min. C, K562 cells were treated with 1000 ng/ mL Cyr61 in combination with or without 4 μmol/L PDTC for 24 h, and then treated with 0.5 μmol/L IM for 24 h. Protein levels of Bcl‐2 in K562 cells were detected by western blotting. D, K562‐shCyr61 cells and K562‐sh NC cells were treated with 0.5 μmol/L IM for 24 h. Left panel: NF ‐κB phosphorylation was detected by western blotting. Right panel: Bcl‐2 protein levels in K562 cells were detected by western blotting. Band intensity of Bcl‐2 was quantified by densitometry and normalized to GAPDH . Data represent the mean ± SEM of at least 3 independent experiments. * P < 0.05, ** P < 0.01

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Protein-Protein interactions, Phospho-proteomics, Western Blot

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Inhibition of cysteine‐rich protein 61 (Cyr61) restores the chemosensitivity of CML cells to imatinib mesylate ( IM ) in vivo. NOD / SCID mice bearing s.c. K562‐shCyr61 or control K562‐sh NC cell xenografts (n = 6) were injected i.p. with IM or normal saline ( NS ) daily from 10 d after inoculation with 1.0 × 10 7 tumor cells for 20 d, and then the mice were killed. A, Representative images of tumors are shown. B, Tumor weight is shown. C, Average percentage of tumor volume is shown. * P < 0.05

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Inhibition, In Vivo, Control, Injection, Saline

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Proposed signaling pathway by which cysteine‐rich protein 61 (Cyr61) reduces imatinib mesylate ( IM )‐induced CML cell apoptosis. Increased Cyr61 in the bone marrow from CML patients stimulates nuclear factor kappa B ( NF ‐κB) phosphorylation, then upregulates Bcl‐2 production, finally leading to decrease of IM ‐induced CML cell apoptosis and insensitivity to IM

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: mTORC2-mediated direct phosphorylation regulates YAP activity promoting glioblastoma growth and invasive characteristics.

doi: 10.1016/j.neo.2021.07.005

Figure Lengend Snippet: Fig. 6. mTORC2-mediated YAP S436 phosphorylation can occur independent of Hippo pathway signaling in GBM. (A) Inhibition of YAP S436 phosphorylation by JR-AB2-011 (1 μM, 8 h) in the indicated lines. Immunoblots were probed for phospho-S436 YAP, total YAP and actin as shown. (B) CTGF and Cyr61 mRNA expression in LN229 shYAP1 cells expressing native YAP or LATS-resistant YAP-5SA mutant (C) following treatment with either JR-AB2-011 (1 μM, 8 h) or insulin (100 nM, 4 h) relative to control untreated cells. mRNA was isolated and subjected to qRT-PCR analyses. qRT-PCR measurements were performed in quadruplicate and the mean and + S.D. are shown. ∗P < 0.05.

Article Snippet: Antibodies to the following proteins were used: phospho-S 473 -AKT (#9271, CST), phospho-S 127 -YAP (#ab76252, Abcam), AKT (#9272, CST), Rictor (#A300-459A, Bethyl Laboratories), Raptor (A300-553A, Bethyl Laboratories), actin (#ab3280, Abcam), YAP1 (#12395S, CST), α-Flag (#TA50011, Origene), TEAD1 (#12292S, CST), TEAD2 (#ab92279, Abcam), TEAD3 (#13224S, CST), TEAD4 (#ab137833, Abcam), SMAD1 (#9743S, CST), p73 (#14620S, CST), FOS (#4384S, CST), TBX5 (#ab137833, Abcam), Mnk1 (#sc-133107, Santa Cruz Biotechnology), CTGF (#HPA031075, Sigma), Cyr61 (#NB100-356SS, Novus), Hsp90 (#SMC149B, StressMarq Biosciences), Lamin B2 (#12255S, CST) and mSin1 (#07-2276, MilliporeSigma).

Techniques: Phospho-proteomics, Inhibition, Western Blot, Expressing, Mutagenesis, Control, Isolation, Quantitative RT-PCR

Journal: Neoplasia (New York, N.Y.)

Article Title: mTORC2-mediated direct phosphorylation regulates YAP activity promoting glioblastoma growth and invasive characteristics.

doi: 10.1016/j.neo.2021.07.005

Figure Lengend Snippet: Fig. 7. Growth of YAP S436 mutant expressing GBM cells in vivo . (A) LN229 shYAP1 cells stably expressing native YAP1, nonphosphorylatable YAP1 S436A, or the phosphomimetic YAP1 S436E alleles were monitored for tumor growth for up to 52 days following establishment of ~200 cm 3 subcutaneous tumors in SCID mice ( n = 4-5 per group). (B) Overall survival of mice with harboring the indicated S436 YAP mutant subcutaneous implanted LN229 tumors. ∗, P < 0.05, n = 4-5 mice per group. (C) Weight of tumors harvested at autopsy from xenografted mice implanted with the indicated GBM cells. ∗P < 0.05. (D) CTGF and Cyr61 mRNA expression from harvested tumors cells expressing the indicated YAP1 alleles. mRNA was isolated and subjected to qRT-PCR analyses. qRT-PCR measurements were performed in quadruplicate and the mean and + S.D. are shown. ∗P < 0.05. (E) mTORC2 mediated phosphorylation of serine 436 of YAP1 leads to increases in protein stability, nuclear localization and TEAD association resulting in enhancement of YAP1 target gene expression.

Article Snippet: Antibodies to the following proteins were used: phospho-S 473 -AKT (#9271, CST), phospho-S 127 -YAP (#ab76252, Abcam), AKT (#9272, CST), Rictor (#A300-459A, Bethyl Laboratories), Raptor (A300-553A, Bethyl Laboratories), actin (#ab3280, Abcam), YAP1 (#12395S, CST), α-Flag (#TA50011, Origene), TEAD1 (#12292S, CST), TEAD2 (#ab92279, Abcam), TEAD3 (#13224S, CST), TEAD4 (#ab137833, Abcam), SMAD1 (#9743S, CST), p73 (#14620S, CST), FOS (#4384S, CST), TBX5 (#ab137833, Abcam), Mnk1 (#sc-133107, Santa Cruz Biotechnology), CTGF (#HPA031075, Sigma), Cyr61 (#NB100-356SS, Novus), Hsp90 (#SMC149B, StressMarq Biosciences), Lamin B2 (#12255S, CST) and mSin1 (#07-2276, MilliporeSigma).

Techniques: Mutagenesis, Expressing, In Vivo, Stable Transfection, Isolation, Quantitative RT-PCR, Phospho-proteomics, Targeted Gene Expression

Journal: International journal of molecular sciences

Article Title: Cilostazol Stimulates Angiogenesis and Accelerates Fracture Healing in Aged Male and Female Mice by Increasing the Expression of PI3K and RUNX2.

doi: 10.3390/ijms25020755

Figure Lengend Snippet: Figure 6. (a) Representative Western blots of BMP-2 and BMP-4 expression within the callus tissue of controls and cilostazol-treated mice at 2 weeks after fracture. (b,c) Expression of BMP-2 (b) and BMP-4 (c) within the callus tissue of controls (white bars, n = 4) and cilostazol-treated mice (black bars, n = 4) at 2 weeks after fracture. Mean ± SEM. (d) Representative Western blots of CYR61 and CD31 expression within the callus tissue of controls and cilostazol-treated mice at 2 weeks after fracture. (e,f) Expression of CYR61 (e) and CD31 (f) within the callus tissue of controls (white bars, n = 4) and cilostazol-treated mice (black bars, n = 4) at 2 weeks after fracture. Mean ± SEM; * p < 0.05 vs. control. (g) Representative Western blots of PI3K and RUNX2 expression within the callus tissue of controls and cilostazol-treated mice at 2 weeks after fracture. (h,i) Expression of PI3K (h) and RUNX2 (i) within the callus tissue of controls (white bars, n = 4) and cilostazol-treated mice (black bars, n = 4) at 2 weeks after fracture. Mean ± SEM; * p < 0.05 vs. control. (b,f) Non-parametric data; analysis performed by Mann–Whitney U-test. (c,e,h,i) Parametric data; analysis performed by unpaired Student’s t-test.

Article Snippet: After saving the whole protein fraction, the analysis was performed using the following monoclonal antibodies: goat anti-mouse BMP2 and BMP4 (1:300, R&D Systems, Wiesbaden, Germany), sheep anti-mouse CYR61 (1:300, R&D Systems), rabbit anti-mouse CD31 (1:300, Cell Signaling Technology, Danvers, MA, USA), mouse anti-mouse PI3K (1:100, Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit anti-mouse RUNX2 (1:300, Abcam).

Techniques: Western Blot, Expressing, Control, MANN-WHITNEY