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 medicine

Article Title: Nuclear receptor coactivator 6 (NCoA6) promotes cell proliferation, migration, and invasion in pancreatic cancer.

doi: 10.1002/cam4.6427

Figure Lengend Snippet: FIGURE 3 NCoA6 knockdown decreases the proliferation, cell cycle, and apoptosis of pancreatic cancer cells. CCK-8 assays (A, B) and colony formation assays (C, D) were used to test the proliferation of PDAC cells transfected with NCoA6 shRNAs. The representative images and statistical results of the cell cycle (E, F) and apoptosis (G, H) using flow cytometry. (I, J) Western blotting analysis of p21 and p27 expression in NCoA6-silenced PANC-1 and SW1990 cells. (K, L) Western blotting analysis of CDK4, CDK2, Cyclin D1, Cyclin E1, and Cyclin A2 expression in NCoA6-silenced PANC-1 and SW1990 cells.

Article Snippet: Protein extraction and western blotting were performed as described in our previous research.16 Antibodies against NCoA6 (1:1000), E- cadherin (1:5000), N- cadherin (1:3000), FBW7 (1:1000), CDX2 (1:1000), cyclin- dependent kinase 4 (CDK4, 1:2000), cyclin- dependent kinase 2 (CDK2, 1:5000), Cyclin D1 (1:5000), Cyclin E1 (1:1000), and Cyclin A2 (1:2000) were obtained from Proteintech.

Techniques: Knockdown, CCK-8 Assay, Transfection, Flow Cytometry, Western Blot, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen-Mediated Chemokine Secretion in Pancreatic Cancer.

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Figure 1. CCN1 is markedly upregulated in pancreatic cancer and associated with tumor progression. A) mRNA expression of CCN1 in human pancreatic cancer tissues (n = 182) and normal tissues (n = 165) were evaluated using the TCGA-PAAD (pancreatic adenocarcinoma) and GTEx datasets. B) Kaplan–Meier analysis of overall survival in PAAD patients based on CCN1 expression. CCN1 high expression (n = 91), CCN low expression (n = 91). C)

Article Snippet: The sections were then stained with antibodies against CCN1 (26689-1-AP, Proteintech), AACT (RAB-0011, MXB), AAT (RAB-0012, MXB), CA199 (MAB-0778, MXB), CD3 (MAB-0740, MXB), CD20 (Kit-0001, MXB), CD34 (Kit-0004, MXB), CD68 (Kit-0026, MXB), CD163 (MAB-0869, MXB), CD3 (MAB-0740, MXB), Ki-67 (MAB-0672, MXB), F4/80 (SC-377009, Santa Cruz), CD8 (SC-7970, Santa Cruz), CD3 (SC-20047, Santa Cruz), CD45 (SC-1178, Santa Cruz), CD86 (SC-28347, Santa Cruz), Gr (SC-393232, Santa Cruz), and GranzymeB (SC-8022, Santa Cruz) followed by incubation with secondary antibodies (SD3100, Celnovte) for 20 min at 37 °C in the dark.

Techniques: Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen-Mediated Chemokine Secretion in Pancreatic Cancer.

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Figure 2. Ccn1 expression in PDAC correlates with immunosuppression. A) Tumor growth curves of sgCtrl and sgCcn1 KPC cells subcutaneously in- oculated in C57B/L6 mouse. n = 6 mice per group. B) Representative images of tumors (left) and quantification of tumor weight (right) in sgCtrl and sgCcn1 KPC cell-inoculated mice. C) Representative images of H&E, and immunohistochemistry analysis for Ki67, CCN1, CD45, F4/80, CD4, CD8, and

Article Snippet: The sections were then stained with antibodies against CCN1 (26689-1-AP, Proteintech), AACT (RAB-0011, MXB), AAT (RAB-0012, MXB), CA199 (MAB-0778, MXB), CD3 (MAB-0740, MXB), CD20 (Kit-0001, MXB), CD34 (Kit-0004, MXB), CD68 (Kit-0026, MXB), CD163 (MAB-0869, MXB), CD3 (MAB-0740, MXB), Ki-67 (MAB-0672, MXB), F4/80 (SC-377009, Santa Cruz), CD8 (SC-7970, Santa Cruz), CD3 (SC-20047, Santa Cruz), CD45 (SC-1178, Santa Cruz), CD86 (SC-28347, Santa Cruz), Gr (SC-393232, Santa Cruz), and GranzymeB (SC-8022, Santa Cruz) followed by incubation with secondary antibodies (SD3100, Celnovte) for 20 min at 37 °C in the dark.

Techniques: Expressing, Immunohistochemistry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen-Mediated Chemokine Secretion in Pancreatic Cancer.

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Figure 3. Ccn1 loss reduces the expression of chemokines and collagens in PDAC. A) Volcano plot illustrates the distribution of differential genes between sgCtrl and sgCcn1 KPC cells. B) Heatmap showing the mRNA expression of chemokines and collagens between sgCtrl and sgCcn1 KPC cells. C) mRNA levels of chemokines, including Ccl2, Ccl7, Ccl20, Cxcl1, Cxcl3, Cxcl5, Csf1, Csf2, and Csf3 between sgCtrl and sgCcn1 in KPC cells. D) Spearman

Article Snippet: The sections were then stained with antibodies against CCN1 (26689-1-AP, Proteintech), AACT (RAB-0011, MXB), AAT (RAB-0012, MXB), CA199 (MAB-0778, MXB), CD3 (MAB-0740, MXB), CD20 (Kit-0001, MXB), CD34 (Kit-0004, MXB), CD68 (Kit-0026, MXB), CD163 (MAB-0869, MXB), CD3 (MAB-0740, MXB), Ki-67 (MAB-0672, MXB), F4/80 (SC-377009, Santa Cruz), CD8 (SC-7970, Santa Cruz), CD3 (SC-20047, Santa Cruz), CD45 (SC-1178, Santa Cruz), CD86 (SC-28347, Santa Cruz), Gr (SC-393232, Santa Cruz), and GranzymeB (SC-8022, Santa Cruz) followed by incubation with secondary antibodies (SD3100, Celnovte) for 20 min at 37 °C in the dark.

Techniques: Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen-Mediated Chemokine Secretion in Pancreatic Cancer.

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Figure 5. Ccn1 regulates the TME in PDAC. A) Schematic representation of macrophage isolation from mouse bone marrow. B) Macrophage invasion evaluated by the chemotactic effect of sgCtrl and sgCcn1 KPC cells in a Transwell invasion assay. C) Macrophage polarization assessed by flow cytometry in macrophages cocultured with sgCtrl and sgCcn1 KPC cells. D) Macrophage polarization assessed by flow cytometry in macrophages cocultured with

Article Snippet: The sections were then stained with antibodies against CCN1 (26689-1-AP, Proteintech), AACT (RAB-0011, MXB), AAT (RAB-0012, MXB), CA199 (MAB-0778, MXB), CD3 (MAB-0740, MXB), CD20 (Kit-0001, MXB), CD34 (Kit-0004, MXB), CD68 (Kit-0026, MXB), CD163 (MAB-0869, MXB), CD3 (MAB-0740, MXB), Ki-67 (MAB-0672, MXB), F4/80 (SC-377009, Santa Cruz), CD8 (SC-7970, Santa Cruz), CD3 (SC-20047, Santa Cruz), CD45 (SC-1178, Santa Cruz), CD86 (SC-28347, Santa Cruz), Gr (SC-393232, Santa Cruz), and GranzymeB (SC-8022, Santa Cruz) followed by incubation with secondary antibodies (SD3100, Celnovte) for 20 min at 37 °C in the dark.

Techniques: Isolation, Transwell Invasion Assay, Cytometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen-Mediated Chemokine Secretion in Pancreatic Cancer.

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Figure 6. Targeting Ccn1 enhances the efficacy of gemcitabine and anti-PD-1 in PDAC. A) Cell viability of sgCtrl and sgCcn1 KPC cells after treatment with indicated concentrations of gemcitabine for 48 h. B) ROS levels in sgCtrl and sgCcn1 KPC cells with gemcitabine for 48 h by Fluorescence-activated cell sorting (FACS) analysis. C) Representative images (left) and quantification (right) of ROS level in sgCtrl and sgCcn1 KPC cells treated with gemcitabine for 48 h. D,E) C57B/L6 mice were inoculated subcutaneously with sgCtrl and sgCcn1 KPC cells. Tumor-bearing mice were treated with isotype control, anti-PD1 mAb, gemcitabine, or a combination of both treatments (n = 5 mice per group). Tumor volume was monitored (D), and representative tumor images (left) were acquired with final tumor weights (right) shown (E). F) C57BL/6 mice were orthotopically implanted with sgCtrl or sgCcn1 KPC cells. Tumor-bearing mice were treated with isotype control, anti-PD1 monoclonal antibody, gemcitabine, or a combination of both (n = 5 mice per group). Representative tumor images (top) and final tumor weights (bottom) are shown. G) Representative images of Masson’s trichrome staining and immunohistochemical analysis for F4/80, CD8, CD45, GranzymeB (GrzB), and Gr in sgCtrl and sgCcn1 KPC orthotopic tumors.

Article Snippet: The sections were then stained with antibodies against CCN1 (26689-1-AP, Proteintech), AACT (RAB-0011, MXB), AAT (RAB-0012, MXB), CA199 (MAB-0778, MXB), CD3 (MAB-0740, MXB), CD20 (Kit-0001, MXB), CD34 (Kit-0004, MXB), CD68 (Kit-0026, MXB), CD163 (MAB-0869, MXB), CD3 (MAB-0740, MXB), Ki-67 (MAB-0672, MXB), F4/80 (SC-377009, Santa Cruz), CD8 (SC-7970, Santa Cruz), CD3 (SC-20047, Santa Cruz), CD45 (SC-1178, Santa Cruz), CD86 (SC-28347, Santa Cruz), Gr (SC-393232, Santa Cruz), and GranzymeB (SC-8022, Santa Cruz) followed by incubation with secondary antibodies (SD3100, Celnovte) for 20 min at 37 °C in the dark.

Techniques: Fluorescence, FACS, Control, Staining, Immunohistochemical staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen-Mediated Chemokine Secretion in Pancreatic Cancer.

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Figure 7. Proposed model for CCN1-induced tumor immunosuppression in pancreatic cancer. This schematic diagram illustrates the role of CCN1 in pancreatic cancer. In control pancreatic cancer cells, the tumor cells exhibit resistance to TNF𝛼-mediated cell death. In contrast, CCN1- KO pancreatic tumor cells are more sensitive to TNF𝛼-induced apoptosis. Loss of CCN1 leads to reduced collagen expression, which further downregulates the secretion of chemokines, ultimately promoting immune cell infiltration into the TME.

Article Snippet: The sections were then stained with antibodies against CCN1 (26689-1-AP, Proteintech), AACT (RAB-0011, MXB), AAT (RAB-0012, MXB), CA199 (MAB-0778, MXB), CD3 (MAB-0740, MXB), CD20 (Kit-0001, MXB), CD34 (Kit-0004, MXB), CD68 (Kit-0026, MXB), CD163 (MAB-0869, MXB), CD3 (MAB-0740, MXB), Ki-67 (MAB-0672, MXB), F4/80 (SC-377009, Santa Cruz), CD8 (SC-7970, Santa Cruz), CD3 (SC-20047, Santa Cruz), CD45 (SC-1178, Santa Cruz), CD86 (SC-28347, Santa Cruz), Gr (SC-393232, Santa Cruz), and GranzymeB (SC-8022, Santa Cruz) followed by incubation with secondary antibodies (SD3100, Celnovte) for 20 min at 37 °C in the dark.

Techniques: Control, Expressing

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

Journal: Journal of Cell Communication and Signaling

Article Title: Cellular communication network 1 promotes CASP2 mRNA expression but suppresses its protein translation in esophageal adenocarcinoma

doi: 10.1002/ccs3.12046

Figure Lengend Snippet: The effect of CCN1 on caspase gene expression based on RT_PCR (fold change).

Article Snippet: The following antibodies were used in this study: CCN1 (TA349858, OriGene), CASP2 (ab179519, Abcam), CASP3 (TA374282, OriGene), p21 (TA808128, OriGene), p16 (ab270058, Abcam), E2F1 (TA384125, OriGene), RB1 (ab32513, Abcam), phosphorylated RB1 (ab277774, Abcam), tripartite motif 25 (TRIM25) (ab167154, Abcam), HuR (ab200342, Abcam), and β‐actin (sc69879, Santa Cruz Biotechnology).

Techniques: Gene Expression

Journal: Journal of Cell Communication and Signaling

Article Title: Cellular communication network 1 promotes CASP2 mRNA expression but suppresses its protein translation in esophageal adenocarcinoma

doi: 10.1002/ccs3.12046

Figure Lengend Snippet: CCN1 upregulates CASP2 mRNA expression but downregulates CASP2 protein expression in EAC cells. (A) Northern blot analyses of CASP2 mRNA expression in response to treatment with rCCN1. (B) OE19 and OE33 cells were both transfected with pCCN1 or the vector. CASP2 and CCN1 expression were analyzed by Western blotting after a week of cell selection with neomycin and normalized to β‐actin. (C) The relative levels of CASP2 expression in the CCN1‐transfected cells were quantified against the vector‐transfected control, based on at least five replicates. *Statistical significance at p < 0.01. (D) OE19 and OE33 cells were both incubated with rCCN1 or BSA at 1 μg/mL for 6 h. CASP2 and CCN1 expression were analyzed by Western blotting and normalized to β‐actin. (E) The relative levels of CASP2 expression in the CCN1‐treated cells were quantified against the BSA‐treated control, based on at least 5 replicates. *Statistical significance at p < 0.01. BSA, bovine serum albumin; CCN1, cellular communication network 1; EAC, esophageal adenocarcinoma; pCCN1, pcDNA3.1‐CCN1; rCCN1, recombinant CCN1 protein.

Article Snippet: The following antibodies were used in this study: CCN1 (TA349858, OriGene), CASP2 (ab179519, Abcam), CASP3 (TA374282, OriGene), p21 (TA808128, OriGene), p16 (ab270058, Abcam), E2F1 (TA384125, OriGene), RB1 (ab32513, Abcam), phosphorylated RB1 (ab277774, Abcam), tripartite motif 25 (TRIM25) (ab167154, Abcam), HuR (ab200342, Abcam), and β‐actin (sc69879, Santa Cruz Biotechnology).

Techniques: Expressing, Northern Blot, Transfection, Plasmid Preparation, Western Blot, Selection, Control, Incubation, Recombinant

Journal: Journal of Cell Communication and Signaling

Article Title: Cellular communication network 1 promotes CASP2 mRNA expression but suppresses its protein translation in esophageal adenocarcinoma

doi: 10.1002/ccs3.12046

Figure Lengend Snippet: CCN1 downregulates p16 and p21 and thereby suppresses RB1 phosphorylation, allowing E2F1 to transcribe CASP2. (A) OE19 cells were transfected with pCCN1 or the vector (control) and selected with neomycin for a week. Expression of CASP2, CCN1, p16, p21, RB1, phosphorylated‐RB1, and E2F1 was analyzed by Western blotting and normalized to β‐actin. (B) RB1 was pulled down by immunoprecipitation from the transfected OE19 cells and analyzed for RB1, phosphorylated‐RB1, and E2F1 by Western blotting. (C) RB1 phosphorylation and RB1‐E2F1 binding (B) were quantified against the input protein (A), based on 3 replicates. *Statistical significance at p < 0.01. (D) OE19 and OE33 cells were transfected with pcDNA3.1‐CDKN1A or pcDNA3.1‐CDKN2A or the vector and selected for a week with neomycin and then incubated with rCCN1 or BSA at 1 μg/mL for 6 h. RNA was isolated and examined for CASP2 expression by Northern blot analysis. (E) CDKN2A‐transfected OE19 cells were incubated with rCCN1 or BSA at 1 μg/mL for 6 h. Protein extracts were analyzed for the expression of CASP2 and p16 by Western blotting and normalized to β‐actin. The transfected cells expressed flagged p16 along with the endogenous p16. (F) CDKN1A‐transfected OE19 cells were incubated with rCCN1 or BSA at 1 μg/mL for 6 h. Protein extracts were analyzed for the expression of CASP2 and p21 by Western blotting and normalized to β‐actin. The transfected cells expressed flagged p21 along with the endogenous p21. (G) OE19 and OE33 cells were transfected with pRS‐E2F1.shRNA (shRNA‐E2F1) or the vector and selected for a week with puromycin and then incubated with rCCN1 or BSA at 1 μg/mL for 6 h. RNA was isolated and examined for CASP2 expression by Northern blot analysis. (H) Western blot analyses for E2F1 were performed after E2F1 knockdown in OE19 cells. (I) Western blot analyses for E2F1 were performed after E2F1 knockdown in OE33 cells. BSA, bovine serum albumin; CCN1, cellular communication network 1; pCCN1, pcDNA3.1‐CCN1; rCCN1, recombinant CCN1 protein.

Article Snippet: The following antibodies were used in this study: CCN1 (TA349858, OriGene), CASP2 (ab179519, Abcam), CASP3 (TA374282, OriGene), p21 (TA808128, OriGene), p16 (ab270058, Abcam), E2F1 (TA384125, OriGene), RB1 (ab32513, Abcam), phosphorylated RB1 (ab277774, Abcam), tripartite motif 25 (TRIM25) (ab167154, Abcam), HuR (ab200342, Abcam), and β‐actin (sc69879, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Immunoprecipitation, Binding Assay, Incubation, Isolation, Northern Blot, shRNA, Knockdown, Recombinant

Journal: Journal of Cell Communication and Signaling

Article Title: Cellular communication network 1 promotes CASP2 mRNA expression but suppresses its protein translation in esophageal adenocarcinoma

doi: 10.1002/ccs3.12046

Figure Lengend Snippet: Overexpression of p16 or p21 attenuates CCN1‐upregulated CASP2 mRNA expression based on RT_PCR (fold change).

Article Snippet: The following antibodies were used in this study: CCN1 (TA349858, OriGene), CASP2 (ab179519, Abcam), CASP3 (TA374282, OriGene), p21 (TA808128, OriGene), p16 (ab270058, Abcam), E2F1 (TA384125, OriGene), RB1 (ab32513, Abcam), phosphorylated RB1 (ab277774, Abcam), tripartite motif 25 (TRIM25) (ab167154, Abcam), HuR (ab200342, Abcam), and β‐actin (sc69879, Santa Cruz Biotechnology).

Techniques: Over Expression, Expressing

Journal: Journal of Cell Communication and Signaling

Article Title: Cellular communication network 1 promotes CASP2 mRNA expression but suppresses its protein translation in esophageal adenocarcinoma

doi: 10.1002/ccs3.12046

Figure Lengend Snippet: Knockdown of E2F1 attenuates CCN1‐induced CASP2 mRNA expression based on RT_PCR (fold change).

Article Snippet: The following antibodies were used in this study: CCN1 (TA349858, OriGene), CASP2 (ab179519, Abcam), CASP3 (TA374282, OriGene), p21 (TA808128, OriGene), p16 (ab270058, Abcam), E2F1 (TA384125, OriGene), RB1 (ab32513, Abcam), phosphorylated RB1 (ab277774, Abcam), tripartite motif 25 (TRIM25) (ab167154, Abcam), HuR (ab200342, Abcam), and β‐actin (sc69879, Santa Cruz Biotechnology).

Techniques: Knockdown, Expressing

Journal: Journal of Cell Communication and Signaling

Article Title: Cellular communication network 1 promotes CASP2 mRNA expression but suppresses its protein translation in esophageal adenocarcinoma

doi: 10.1002/ccs3.12046

Figure Lengend Snippet: CCN1 downregulates CASP2 protein expression by increasing HuR expression. (A) OE19 cells were transfected with pCCN1 or the vector and selected for a week with neomycin and analyzed for expression of TRIM25, HuR, CASP2, and CCN1 and normalized to β‐actin. (B) TRIM25, HuR, and CASP2 expression in CCN1‐transfected cells were quantified against the vector‐transfected cells (control). *Statistical significance at p < 0.01. (C) HuR was pulled down from an equal amount of protein extracts of the transfected cells by immunoprecipitation and analyzed by either Western blotting for HuR (lower panel) or RT_PCR for CASP2 mRNA (upper panel). (D) HuR expression in OE19 cells was knocked down using specific shRNA against HuR and selected for a week with puromycin and then incubated with rCCN1 or BSA at 1 μg/mL for 6 h. The protein extracts were analyzed for expression of CASP2 and HuR by Western blotting and normalized to β‐actin. (E) CASP2 and HuR expression were quantified against to control. *Statistical significance at p < 0.01. BSA, bovine serum albumin; CCN1, cellular communication network 1; pCCN1, pcDNA3.1‐CCN1; rCCN1, recombinant CCN1 protein.

Article Snippet: The following antibodies were used in this study: CCN1 (TA349858, OriGene), CASP2 (ab179519, Abcam), CASP3 (TA374282, OriGene), p21 (TA808128, OriGene), p16 (ab270058, Abcam), E2F1 (TA384125, OriGene), RB1 (ab32513, Abcam), phosphorylated RB1 (ab277774, Abcam), tripartite motif 25 (TRIM25) (ab167154, Abcam), HuR (ab200342, Abcam), and β‐actin (sc69879, Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Reverse Transcription Polymerase Chain Reaction, shRNA, Incubation, Recombinant

Journal: Journal of Cell Communication and Signaling

Article Title: Cellular communication network 1 promotes CASP2 mRNA expression but suppresses its protein translation in esophageal adenocarcinoma

doi: 10.1002/ccs3.12046

Figure Lengend Snippet: CASP3, not CASP2 mediates CCN1‐induced EAC cell apoptosis. (A) OE19 cells were transfected with specific shRNA to knock down CASP2 or CASP3 and selected for a week with puromycin, and then treated with rCCN1 for 6 h. Expression of CASP2 and CASP3 were analyzed by Western blotting and normalized to β‐actin. (B) Apoptosis in the transfected OE19 cells was assessed using Apopxin under rCCN1 treatment compared to BSA (control), based on five independent experiments. *Statistical significance at p < 0.01. (C) OE33 cells were transfected with specific shRNA to knock down CASP2 or CASP3 and selected for a week with puromycin, and then treated with rCCN1 for 6 h. Expression of CASP2 and CASP3 were analyzed by Western blotting and normalized to β‐actin. (D) Apoptosis in the transfected OE33 cells was assessed using Apopxin under rCCN1 treatment compared to BSA (control), based on five independent experiments. *Statistical significance at p < 0.01. (E) OE19 cells were transfected with specific shRNA to knock down CASP2 or CASP3 and selected for a week with puromycin. Then cells were treated with rCCN1 for 0, 2, 6, or 12 h and analyzed for cell proliferation using a CCK‐8 kit. (F) OE33 cells were transfected with specific shRNA to knock down CASP2 or CASP3 and selected for a week with puromycin. Then cells were treated with rCCN1 for 0, 2, 6, or 12 h and analyzed for cell proliferation using a CCK‐8 kit. BSA, bovine serum albumin; CCN1, cellular communication network 1; EAC, esophageal adenocarcinoma; rCCN1, recombinant CCN1 protein.

Article Snippet: The following antibodies were used in this study: CCN1 (TA349858, OriGene), CASP2 (ab179519, Abcam), CASP3 (TA374282, OriGene), p21 (TA808128, OriGene), p16 (ab270058, Abcam), E2F1 (TA384125, OriGene), RB1 (ab32513, Abcam), phosphorylated RB1 (ab277774, Abcam), tripartite motif 25 (TRIM25) (ab167154, Abcam), HuR (ab200342, Abcam), and β‐actin (sc69879, Santa Cruz Biotechnology).

Techniques: Transfection, shRNA, Knockdown, Expressing, Western Blot, Control, CCK-8 Assay, Recombinant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Silencing of LINC01013 inhibited hypoxia-induced hPASMC proliferation and inflammation. A , B hPASMC proliferation was determined by CCK8 and EdU incorporation assays ( n = 6). EdU (red), DAPI (blue), scale bar, 50 μm. C Western blotting analysis of PCNA, cyclin A and cyclin D in hPASMCs ( n = 6). D RT‒qPCR analysis showed the mRNA levels of TNF-α and IL-6 after LINC01013 knockdown, β-actin was used as a internal reference gene ( n = 6). E Western blotting analysis of TNF-α and IL-6 in hPASMCs. ( n = 6). All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Western Blot, Knockdown, Negative Control