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: 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: Human molecular genetics

Article Title: Abnormal activation of Yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.

doi: 10.1093/hmg/ddab374

Figure Lengend Snippet: Figure 1. YAP and TAZ levels and YAP/TAZ downstream targets are elevated in human cortical tuber samples. (A) Western blots for the human cortical tuber samples (n = 2) and normal age-matched controls (n = 2). (B) Quantification of protein level using densitometry show elevations in in YAP and TAZ, CCN2 and CYR61 (two YAP/TAZ target genes), and pS6 (a readout of mTORC1 activity) in the human cortical tuber samples versus the control samples. Data are presented as the mean. (C) qRT-PCR demonstrates that there is no significant difference in YAP mRNA levels, but the mRNA levels of two known YAP/TAZ target genes, CCN2 and CYR61, are elevated in human cortical tuber samples (n = 2) in comparison to normal age-matched controls (n = 3).

Article Snippet: Cux1 (sc-13 024, Santa Cruz, IHC: 1:400), Ctip2 (ab18465, Abcam, IHC: 1:200), CCN2 (23936-1-AP, Proteintech, WB:1:500), Cyr61 (26689-1-AP, Proteintech, WB:1:500), GAPDH (60004-1-Ig, Proteintech, WB:1:3000), GAPDH (10494-1-AP, Proteintech, WB 1:3000), GFAP (RB-087, Thermo Fisher Scientific, IHC: 1:200), MBP (#836504, Biolegend, IHC: 1:2000), N-cadherin (#610920, BD Biosciences, IHC: 1:400), pS6240/244 (#2215, Cell Signaling Technology, WB:1:1000, IHC: 1:500), pYap (#4911, Cell Signaling, WB:1:1000), Taz (#4883, Cell Signaling Technology, WB:1:1000), TSC2 (#4308, Cell Signaling Technology, WB: 1:2000, IHC: 1:200), Yap (ab56701, Abcam, WB: 1:1000, IHC: 1:500), Yap (#4912, Cell Signaling, IHC: 1:100).

Techniques: Western Blot, Activity Assay, Control, Quantitative RT-PCR, Comparison

Journal: bioRxiv

Article Title: Cyr61 delivery promotes angiogenesis during bone fracture repair

doi: 10.1101/2024.04.05.588239

Figure Lengend Snippet: ( A ) Experimental setup. ( B ) Representative images of Safranin-O-staining and ( C ) quantitative measurement of chondrogenic pellet diameter at 2 weeks. ( D ) Relative mRNA expression of RUNX2, SP7, COL1A1 and CYR61 after 3 weeks of osteogenic differentiation normalized to housekeeping gene and control. ( E ) 3D in vitro angiogenesis assay combing HUVECs and hMSCs. ( F ) Exemplary images of tube formation at 3 days. ( G ) Quantification of relative GFP+ cell area, relative tube number and tube length. Mean ± SEM and individual data points. One-way ANOVA was used to determine the statistical significance; p-values are indicated with *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001 . Scale bars indicate 500 μm (B) and 200 μm (F).

Article Snippet: Different concentrations of endogenous Cyr61 (0-200 ng/ml; Recombinant Human Cyr61/CCN1 Fc Chimera Protein, carrier-free; catalog no.: 4055-CR; R&D Systems, Minneapolis, MN) and TGFβ (0 or 10 ng/ml; rhTGF-beta1; R&D Systems, Minneapolis, MN) were added to the medium.

Techniques: Staining, Expressing, Control, In Vitro, Angiogenesis Assay

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: ( 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

Journal: Stem Cells International

Article Title: ECM Protein CYR61 Promotes Migration and Osteoblastic Differentiation of Irradiation BMSCs via Migrasomes

doi: 10.1155/sci/8825935

Figure Lengend Snippet: Proteomic technology identified the key molecule influencing the diminished osteogenic and migratory abilities of IR BMSCs. (a) Heat map revealed the differential expression proteins between BMSCs and IR BMSCs. CYR61 was found to be downregulated in IR BMSCs. (b) Western blot verified the proteomics data. (c) GO analysis of analysis on molecular functions suggested the significance of migrasome-related integrin binding ( n = 3).

Article Snippet: To investigate the requirement of ERK in CYR61 function, 10 μM CYR61 (HY-P7842, MCE, USA) in serum-free medium for 24 h and 10 μM specific ERK inhibitor U0126 (HY-12031A, MCE, USA) for 1 h were employed [ ].

Techniques: Quantitative Proteomics, Western Blot, Binding Assay

Journal: Stem Cells International

Article Title: ECM Protein CYR61 Promotes Migration and Osteoblastic Differentiation of Irradiation BMSCs via Migrasomes

doi: 10.1155/sci/8825935

Figure Lengend Snippet: Potential role and molecular mechanism of CYR61 mediate the migration and osteogenesis of IR BMSCs. (a) Sequences of the siRNAs targeting CYR61. (b) Efficacy of CYR61 siRNAs at reducing mRNA levels. (c) Protein levels confirming the inhibitory efficiency of CYR61 siRNAs. (d) Results of the wound healing assay. Scalar bar = 100 µm. (e) Cell proliferation assessed by CCK-8 assay. (f) Western blot analysis showing levels of ALP, OSX, and RUNX2 following CYR61 knockdown. (g) Impact of CYR61 knockdown on integrin αvβ3 and ERK signaling pathway. (h) Results of the molecular docking assay. (i) Immunofluorescence localization of CYR61 and integrin αvβ3. (j) Co-IP analysis demonstrated the specific interaction between CYR61 and integrin αvβ3. (k) Diagram showing the potential binding site of CYR61 with integrin αvβ3. Scalar bar = 50 µm. (l) Rescue assay showing that CYR61-induced increases in osteogenic markers (ALP and RUNX2) and ERK phosphorylation are partially reversed by U0126-mediated ERK inhibition. Western blot detection. (m) Wound healing assay revealing that CYR61-enhanced cell migration is suppressed by U0126 treatment. Data are represented as mean ± SEM, n = 3, ⁣ ∗∗ p < 0.01, ⁣ ∗ p < 0.05.

Article Snippet: To investigate the requirement of ERK in CYR61 function, 10 μM CYR61 (HY-P7842, MCE, USA) in serum-free medium for 24 h and 10 μM specific ERK inhibitor U0126 (HY-12031A, MCE, USA) for 1 h were employed [ ].

Techniques: Migration, Wound Healing Assay, CCK-8 Assay, Western Blot, Knockdown, Docking Assay, Immunofluorescence, Co-Immunoprecipitation Assay, Binding Assay, Rescue Assay, Phospho-proteomics, Inhibition

Journal: Stem Cells International

Article Title: ECM Protein CYR61 Promotes Migration and Osteoblastic Differentiation of Irradiation BMSCs via Migrasomes

doi: 10.1155/sci/8825935

Figure Lengend Snippet: CYR61 could be secreted into the extracellular space via migrasomes. Extraction and characteristics of migrasomes derived from BMSCs. (a) Migrasomes from BMSCs (highlighted by yellow arrows), showing green fluorescence of contractile filaments stained with WGA-488, as observed by laser confocal microscopy. Scalar bar = 5 µm. (b) A schematic representation of the process for extracting migrasomes. (c) Transmission electron microscopy (TEM) captured the appearance of the pomegranate-like vesicles. Scalar bar = 1 µm. (d) Western blot analysis confirmed the presence of specific markers of migrasomes. (e) CYR61 was expressed in both migrasomes and origin cells. Scalar bar = 5 µm. One representative experiment is shown ( n = 3).

Article Snippet: To investigate the requirement of ERK in CYR61 function, 10 μM CYR61 (HY-P7842, MCE, USA) in serum-free medium for 24 h and 10 μM specific ERK inhibitor U0126 (HY-12031A, MCE, USA) for 1 h were employed [ ].

Techniques: Extraction, Derivative Assay, Fluorescence, Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Western Blot

Journal: Stem Cells International

Article Title: ECM Protein CYR61 Promotes Migration and Osteoblastic Differentiation of Irradiation BMSCs via Migrasomes

doi: 10.1155/sci/8825935

Figure Lengend Snippet: Migrasomes originating from BMSCs can enhance both the cell mobility and osteogenic capacity of IR BMSCs by transporting CYR61. (a) ALP activity of IR BMSC stimulated by varying concentrations of migrasomes, displayed a dose-dependent increase, with 2 µg/mL identified as the optimal concentration. (b) Addition of migrasomes could compensate for the CYR61 deficiency in IR BMSCs. (c) Transwell assay showed the upregulated migratory ability. (d) Quantitative analysis of transwell assay. (e) Wound healing assay further illustrated the increased migration ability induced by migrasomes. (f) Quantitative analysis based on relative RNA expression levels of ALP , OSX , and RUNX2 . (g) Western blot analysis and statistical evaluation of migrasomes-mediated expression levels of ALP, OSX, and RUNX2 when compared to IR BMSCs alone. (h) Result of ALP staining. (i) ARS staining results. Scalar bar = 100 µm. Data are represented as mean ± SEM, n = 3, ⁣ ∗∗ p < 0.01, ⁣ ∗ p < 0.05.

Article Snippet: To investigate the requirement of ERK in CYR61 function, 10 μM CYR61 (HY-P7842, MCE, USA) in serum-free medium for 24 h and 10 μM specific ERK inhibitor U0126 (HY-12031A, MCE, USA) for 1 h were employed [ ].

Techniques: Activity Assay, Concentration Assay, Transwell Assay, Wound Healing Assay, Migration, RNA Expression, Western Blot, Expressing, Staining