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

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: CCN1 expression in Panc1 is increased by co-culturing with mPSCs. ( A , B ) The expression level of CCN1/Ccn1 in Panc1-WT ( A ) and mPSCs ( B ) from monospheroids and heterospheroids on day 5 was analyzed via RNA-seq and RT-PCR. Represented are RPKM (Reads Per Kilobase per Million mapped reads) values from the published expression profiling , and the mRNA fold changes presented here were normalized to Panc1 or mPSCs monospheroids. All data shown are means with SEM from three independent biological replicates. ( C ) The representative Western blot illustrates the expression of CCN1 protein levels in Panc1-WT and 3 different Panc1-CCN1-KO clones (C3, C7, and F3) cultured in 2D. ( D ) Representative pictures of Panc1-WT and Panc1-CCN1-KO clones C3, C7, and F3 monospheroids. Scale bars represent 500 μm. M, monospheroid; H, heterospheroid. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Expressing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Clone Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: CCN1 negatively regulates stemness and positively affects gemcitabine transporter and activating genes. ( A ) The mRNA expression of the stemness marker gene CD24, the epithelial marker genes CK19 and CDH1, matricellular protein CTGF/CCN2, SHH signaling mediator GLI1, proliferation marker MKI67, gemcitabine transporter SLC29A1, and gemcitabine-activating enzyme DCK in Panc1-WT and Panc1-CCN1-KO clones (C3, C7, and F3) from monospheroids were analyzed with qRT-PCR. The mRNA fold changes were normalized to Panc1-WT monospheroids. All data shown are means with SEM from three independent biological replicates. ( B ) The protein level of DCK in Panc1-WT and Panc1-CCN1-KO clones (C3, C7, and F3) from 2D culture was analyzed with Western blot (WB). The relative ratio of DCK was quantified via DCK amount per lane/Tubulin amount per lane and then normalized to Panc1-WT. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. n = 4, M = protein weight marker. ( C ) The associations between the expression of these marker genes and CCN1 were fitted using linear regression based on the microarray dataset GSE71729. n = 145 patient samples. ( D ) Protein levels of CCN1, DCK (yellow arrow), and Tubulin for Panc1 WT, Panc1-CCN1-KO C7, and the CCN1-rescued Panc1-CCN1-KO C7 carried out with adenoviral infection. The control for adenoviral infection was carried out with Panc1-CCN1-KO C7 cells infected with adenovirus containing an eGFP expression cassette.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Expressing, Marker, Clone Assay, Quantitative RT-PCR, Western Blot, Microarray, Infection, Control

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: CCN1 increases chemotherapy sensitivity for gemcitabine but not paclitaxel and SN38. ( A ) The relative cell viability of Panc1-WT and Panc1-CCN1-KO (C3 and C7) cells grown in 2D culture after 72 h treatment with different doses of gemcitabine were measured via CellTiter-Glo ® 3D Cell Viability Assay and normalized to NC (negative control). ( B ) The relative cell viability of Panc1-WT and Panc1-CCN1-KO (C3 and C7) cells in monospheroids after 72 h treatment with different concentrations of gemcitabine were measured via CellTiter-Glo ® 3D Cell Viability Assay and normalized to NC. ( C ) The ratios of epithelial specific caspase-cleaved cytokeratin 18 (ccCK18) for Panc1-WT and Panc1-CCN1-KO (C3, C7, and F3) cells from monospheroids and heterospheroids after 72 h treatment with 50 µM gemcitabine were measured via M30 Apoptosense ® CK18 kit. ( D ) The relative ratios of the epithelial-specific cleaved ccCK18 following 72 h gemcitabine treatment of Panc1-WT and Panc1-CCN1-KO (C3, C7, and F3) cells from heterospheroid versus monospheroid culture are shown. ( E ) The mRNA expression of Ccn1 in mPSCs from heterospheroids co-cultured with either Panc1-WT or Panc1-CCN1-KO (C3, C7, and F3) cells is depicted. The mRNA fold changes were normalized to mPSCs co-cultured with Panc1 WT. ( F ) The relative cell viability of Panc1-WT and Panc1-CCN1-KO (C3, C7, and F3) cells from monospheroids after 72 h treatment with paclitaxel [1 μM] were measured via CellTiter-Glo ® 3D Cell Viability Assay and normalized to NC. ( G ) The relative cell viability of Panc1-WT and Panc1-CCN1-KO (C3, C7, and F3) cells from monospheroids after 72 h treatment with SN38 [1 μM] were measured via CellTiter-Glo ® 3D Cell Viability Assay and normalized to NC. All data shown are means with SEM of at least three independent biological replicates. *, p < 0.05; **, p < 0.01. M, monospheroid; H, heterospheroid; NC, negative control; GEM, gemcitabine; PAC, paclitaxel, SN38, active form of irinotecan.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Viability Assay, Negative Control, Expressing, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: LPA and TGFB1 upregulate CCN1 expression in Panc1. ( A ) Analyses of mRNA expression of selected genes in Panc1-WT monospheroids cultured in low-serum condition following stimulation with 20 μM LPA or 5 μg/mL recombinant TGFB1 for 72 h. ( B ) Analyses of mRNA expression of selected genes in Panc1-WT monospheroids cultured in high-serum condition following stimulation with 5 μg/mL recombinant TGFB1 for 72 h. ( C ) The mRNA expressions of CCN1, DCK, and SCL29A1 in Panc1 cells from heterospheroids cultured in high-serum condition stimulated with 5 ng/mL TGFB receptor kinase inhibitor, or 10 μM ATX inhibitor PF8380, or a combination of both inhibitors for 72 h are depicted. The mRNA fold changes presented were normalized NC. All data shown are means with SEM of three biological replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001. ( D ) The associations between ENPP2, TGFB1, and CYR61(CCN1) were fitted using linear regression based on the microarray dataset GSE71729. n = 145 patient samples. ATX, autotaxin; INH, inhibitor.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Expressing, Cell Culture, Recombinant, Microarray

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: TGFB1 and LPA positively regulate Ccn1 in mPSCs and shift mPSCs to the myCAF subtype. ( A ) Analyses of mRNA expression of genes in mPSCs monospheroids stimulated with 5 μg/mL recombinant TGFB1 for 72 h. ( B ) Analyses of mRNA expression of genes in mPSCs monospheroids stimulated with 20 μM LPA for 72 h. The fold changes in the expression of selected genes were normalized to NC. ( C ) Analysis of the mRNA expression of genes in mPSCs from heterospheroid co-cultured with Panc1-WT cells stimulated with 10 μM ATX/ Enpp2 inhibitor PF8380 on day 5 for 72 h is depicted. The mRNA fold changes were normalized to NC. All data shown are means with SEM of three biological replicates. ( D ) The associations between ACTA2 and ENPP2 using linear regression based on the microarray dataset GSE71729. n = 145 patient samples. NC, negative control. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Expressing, Recombinant, Cell Culture, Microarray, Negative Control