Journal: International Journal of Oncology

Article Title: The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

doi: 10.3892/ijo.2018.4608

Figure Lengend Snippet: EHMT2 is overexpressed in breast cancer (BRC). (A) Heatmap of gene expression related to histone methylation/demethylation in the in silico histone methyltransferase/demethylase panel, sorted by fold change in the BRC/normal FPKM value. In the heatmap, yellow indicates normal liver samples, while red indicates BRC samples in TCGA. The threshold was set to a 2.0-fold-change. (B) Expression levels of EHMT2 in BRC using normal and BRC samples. P-values were calculated using Wilcoxon’s tests ( *** P<0.001). (C) Immunohistochemical analysis of EHMT2 in a BRC tissue microarray. Breast cancer tissues were purchased from BioChain. Scale bar, 200 µ m. (D) Cell growth assay. After knocking down EHMT2 for 72 h, RT-qPCR analysis of EHMT2 was performed. Actin (ACTB) was used as the internal control (left panel). Cell fixation with 100% methanol and cell staining with crystal violet was performed. Scale bar, 100 µ m (right panel); siRNA duplexes against EHMT2 (siEHMT2; 5′-GCAAAUAUUUCACCUGCCATT-3′, 5′-UGGCAGGUGAAAUAUUUGCTT-3′) were purchased from ST Pharm. Negative control siRNA was also used (siCont; 5′-AUGAACGUGAAUUGCUCAATT-3′, 5′-UUGAGCAAUUCACGUUCACTT-3′). (E) Western blot analysis of EHMT2, PARP, caspase-3 and β-catenin. ACTB was used as the internal control. (F) Spheroid formation. MCF7 cells treated with siEHMT2 and siCont were loaded onto a spheroid plate and incubated for 96 h. The cells were imaged under a microscope. Scale bar, 500 µ m.

Article Snippet: The samples were stained with anti-HSPD1 (#12165S), poly(ADP-ribose) polymerase (PARP; #9542S) and caspase-3 (#9662S) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), EHMT2 (CSB-PA007497GA01HU) antibodies from Cusabio and ACTB (SC-47778) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 4°C (overnight).

Techniques: Expressing, Methylation, In Silico, Immunohistochemical staining, Microarray, Growth Assay, Quantitative RT-PCR, Staining, Negative Control, Western Blot, Incubation, Microscopy

Journal: International Journal of Oncology

Article Title: The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

doi: 10.3892/ijo.2018.4608

Figure Lengend Snippet: EHMT2 expression and breast cancer (BRC) prognosis. (A) Expression of EHMT2 in patients with BRC from the UNC500 cohort. The median value of EHMT2 expression was considered the cut-off in the patient cohort. (B and C) Kaplan-Meier curves of (B) overall survival and (C) recurrence-free survival in the UNC500 cohort. (D and E) Kaplan-Meier curves of (D) overall survival and (E) metastasis-free survival in the KFSYSCC cohort.

Article Snippet: The samples were stained with anti-HSPD1 (#12165S), poly(ADP-ribose) polymerase (PARP; #9542S) and caspase-3 (#9662S) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), EHMT2 (CSB-PA007497GA01HU) antibodies from Cusabio and ACTB (SC-47778) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 4°C (overnight).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

doi: 10.3892/ijo.2018.4608

Figure Lengend Snippet: RNA-seq analysis after siEHMT2 treatment. (A) Functional enrichment analysis of RNA-seq EHMT2 knockdown results obtained from the Ingenuity Pathway Knowledge Base. (B) Gene Ontology (GO) pathway term enrichment networks. GO pathway term networks in the EHMT2 knockdown and control groups were functionally grouped by ClueGO (top panel). The Venn diagram shows the distribution of the functionally grouped GO terms (bottom panel). Terms in the functionally grouped networks were cut-off at P<0.05.

Article Snippet: The samples were stained with anti-HSPD1 (#12165S), poly(ADP-ribose) polymerase (PARP; #9542S) and caspase-3 (#9662S) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), EHMT2 (CSB-PA007497GA01HU) antibodies from Cusabio and ACTB (SC-47778) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 4°C (overnight).

Techniques: RNA Sequencing Assay, Functional Assay

Journal: International Journal of Oncology

Article Title: The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

doi: 10.3892/ijo.2018.4608

Figure Lengend Snippet: EHMT2 controls the expression and subcellular localization of HSPD1. (A) Phosphor array with the lysates of cells in which EHMT2 was knocked down. The phosphor array (ARY003B) was purchased from R&D Biosystems. Approximately 200 µ g of cell lysate was used. (B and C) Expression levels of HSPD1 in breast cancer samples (TCGA); P-values were calculated using Wilcoxon’s tests ( *** P<0.001) (B) and RNA-seq results (C). (D) RT-qPCR analysis of HSPD1 after EHMT2 knockdown in MDA-MB-231 cell lines. ACTB was used as the internal control (top panel). Western blot analysis was performed using the indicated antibodies (bottom panel). (E) Cell growth assay. After knocking down HSPD1 for 72 h, the cells were fixed with 100% methanol and stained with crystal violet. Scale bar, 200 μm (top panel); P-values were calculated using Student’s t-tests ( ** P<0.01) (bottom panel). (F) Western blot analysis after HSPD1 knockdown using anti-HSPD1, anti-PARP, anti-caspase3 and anti-β-catenin antibodies. ACTB was used as the internal control in MB231 cells. (G) Immunocytochemical analysis of HSPD1. MB231 cells treated with siCont or siEHMT2 were fixed with 100% methanol and stained with anti-HSPD1 (Alexa Fluor 488, green) and DAPI (blue). Scale bar, 200 µ m. (H) Cell growth assay. After MB231 cells were treated with BIX01294 for 24 h, the cells were fixed with 100% methanol and stained with crystal violet. Scale bar, 200 μm (top panel); P-values were calculated using Student’s t-tests ( ** P<0.01). (I) RT-qPCR analysis of HSPD1 after treatment with BIX01294. ACTB was used as the internal control (top panel), and the signal intensities corresponding to HSPD1 were quantified using ImageJ software (bottom panel). The P-values were calculated using Student’s t-test ( ** P<0.01). (J) Western blot analysis after treatment with BIX01294 using anti-HSPD1, anti-PARP, anti-caspase3 and anti-β-catenin antibodies. ACTB was used as the internal control in MB231 cells.

Article Snippet: The samples were stained with anti-HSPD1 (#12165S), poly(ADP-ribose) polymerase (PARP; #9542S) and caspase-3 (#9662S) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), EHMT2 (CSB-PA007497GA01HU) antibodies from Cusabio and ACTB (SC-47778) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 4°C (overnight).

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Growth Assay, Staining, Software

Journal: International Journal of Oncology

Article Title: The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

doi: 10.3892/ijo.2018.4608

Figure Lengend Snippet: Gene expression pattern of the EHMT2 signature and patient survival of two clusters from the UNC500, KFSYSCC and TCGA cohorts (n=388, 327 and 775, respectively). Follow-up time and survival data were used in this analysis. (A) Gene expression patterns of EHMT2 and its associated genes in the UNC500 cohort. A total of 1,765 genes having at least a 2-fold difference between siEHMT2 and siCont cells were involved in the EHMT2 signature. Among these genes, 1,410 were used in the hierarchical clustering analysis of the UNI500 cohort. The patients were divided into 2 groups: an EHMT2 -low cluster group (ELC) and an EHMT2 -high cluster group (EHC). (B and C) Kaplan-Meier plot depicting (B) overall and (C) recurrence-free survival rates. The survival rates of the patients in the EHC group were significantly increased compared with those of the patients in the ELC group (each P<0.05 as determined by log-rank test). (D-G) Several survival rates of the two patient subgroups in the (D and E) KFSYSCC and (F and G) TCGA cohorts.

Article Snippet: The samples were stained with anti-HSPD1 (#12165S), poly(ADP-ribose) polymerase (PARP; #9542S) and caspase-3 (#9662S) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), EHMT2 (CSB-PA007497GA01HU) antibodies from Cusabio and ACTB (SC-47778) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 4°C (overnight).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

doi: 10.3892/ijo.2018.4608

Figure Lengend Snippet: Gene expression pattern of the EHMT2 signature and its association with molecular features in the TCGA cohort (n=817). Histological and molecular subtypes of breast cancer displayed according to the patient clusters divided by EHMT2 signature. * P-value was obtained by an χ 2 test and the remaining P-values were obtained by Fisher’s exact tests.

Article Snippet: The samples were stained with anti-HSPD1 (#12165S), poly(ADP-ribose) polymerase (PARP; #9542S) and caspase-3 (#9662S) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), EHMT2 (CSB-PA007497GA01HU) antibodies from Cusabio and ACTB (SC-47778) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 4°C (overnight).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

doi: 10.3892/ijo.2018.4608

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of overall survival in breast cancer (combined with the UNC500 and KFSYSCC cohorts).

Article Snippet: The samples were stained with anti-HSPD1 (#12165S), poly(ADP-ribose) polymerase (PARP; #9542S) and caspase-3 (#9662S) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), EHMT2 (CSB-PA007497GA01HU) antibodies from Cusabio and ACTB (SC-47778) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 4°C (overnight).

Techniques:

Journal: International Journal of Oncology

Article Title: The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer

doi: 10.3892/ijo.2018.4608

Figure Lengend Snippet: Association between EHMT2 and HSPD1 in the breast cancer (BRC) clinical cohorts. (A) Comparison of expression levels between the patients in the EHC and ELC groups. A two-group box plot comparing the expression levels of EHMT2 and HSPD1 in EHC and ELC patients is illustrated. P-values were obtained by two-sample t-tests between the EHC and ELC subgroups. The r value indicates the correlation coefficient value of the gene compared with the EHC and ELC subgroup categories. (B) Gene-to-gene networks of EHMT2 and HSPD1 associated with BRC prognosis. Up- and downregulated genes in the high-EHMT2 cluster (EHC) group are indicated in red and green, respectively. The intensity of the color is indicative of the degree of over- or under-expression. Each line and arrow represent functional and physical interactions between the genes and the direction of regulation reported in the literature, respectively. (C) Schematic summary of the effects of EHMT2 on breast cancer.

Article Snippet: The samples were stained with anti-HSPD1 (#12165S), poly(ADP-ribose) polymerase (PARP; #9542S) and caspase-3 (#9662S) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), EHMT2 (CSB-PA007497GA01HU) antibodies from Cusabio and ACTB (SC-47778) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), at 4°C (overnight).

Techniques: Expressing, Functional Assay

Journal: PLoS ONE

Article Title: Preferential activation of HIF-2α adaptive signalling in neuronal-like cells in response to acute hypoxia

doi: 10.1371/journal.pone.0185664

Figure Lengend Snippet: The forward (F) and reverse (R) oligonucleotides used in this study.

Article Snippet: Protein expression was analysed via immunoblotting using anti-NSE (AbCam, #AB16808), anti-KCC2 (Merck Millipore, #07–432), anti-actin (Santa Cruz, #SC-1615), anti-HIF1α (BD Sciences, #610958; Abcam, #ab1), anti-HIF-2α (1:500, R&D Systems, #AF2886), anti-HIF-3α (Acris antibodies, #AP20606PU-N), anti-CD44 (CST, #5640) or anti-Vimentin (BD Sciences, #550513) at a dilution of 1:1000 in 3% non-fat milk in 1x TBS-tween (0.1% v/v).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Preferential activation of HIF-2α adaptive signalling in neuronal-like cells in response to acute hypoxia

doi: 10.1371/journal.pone.0185664

Figure Lengend Snippet: A: Relative HIF1-3α mRNA expression was analysed in MCF7 and differentiated PC12 and NT2 cells exposed to 8 hours of hypoxia by qPCR. Data is expressed as mean ± SEM; n = 3; *p<0.05, **p≤0.01, ***p≤0.001. The dotted line represents basal gene expression. B-C: Representative immunoblots of HIF-1α ( B ), HIF-2α ( Ci ), HIF-3α ( Cii ) protein expression in MCF7 and differentiated PC12 and NT2 cells, 1, 2, 4, 8 or 24 hours after exposure to hypoxia. B and C : Equal protein loading was assessed by immunoblotting for actin.

Article Snippet: Protein expression was analysed via immunoblotting using anti-NSE (AbCam, #AB16808), anti-KCC2 (Merck Millipore, #07–432), anti-actin (Santa Cruz, #SC-1615), anti-HIF1α (BD Sciences, #610958; Abcam, #ab1), anti-HIF-2α (1:500, R&D Systems, #AF2886), anti-HIF-3α (Acris antibodies, #AP20606PU-N), anti-CD44 (CST, #5640) or anti-Vimentin (BD Sciences, #550513) at a dilution of 1:1000 in 3% non-fat milk in 1x TBS-tween (0.1% v/v).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Preferential activation of HIF-2α adaptive signalling in neuronal-like cells in response to acute hypoxia

doi: 10.1371/journal.pone.0185664

Figure Lengend Snippet: A: Under physiological oxygen concentrations, HIF-1α/2α are hydroxylated by prolyl hydroxylases (PHD), promoting HIF-1α/2α binding to the E3 ligase, von Hippel-Lindau protein (pVHL), and their ubiquitination . This maintains very low basal expression of HIF-1α/2α due to rapid proteasomal degradation. B: Under hypoxic conditions, PHD activity is inhibited . This stabilises HIF-1α/2α expression, enhancing binding to HIFβ and translocation to the nucleus and transcription of various hypoxia-responsive genes. HIF-1α and -2α share regulation of several genes, yet also regulate distinct subsets [ , ]. HIF-3α function is not yet fully understood. Abbreviations: CHOP, CCAAT-enhancer-binding protein homologous protein; EPO, Erythropoietin; Grp, Glucose regulated protein; HIF: Hypoxia Inducible Factor; PDI, protein disulphide isomerase; PHD, Prolyl hydroxylase; VEGF, Vascular epithelial growth factor; VHL, Von Hippel Lindau.

Article Snippet: Protein expression was analysed via immunoblotting using anti-NSE (AbCam, #AB16808), anti-KCC2 (Merck Millipore, #07–432), anti-actin (Santa Cruz, #SC-1615), anti-HIF1α (BD Sciences, #610958; Abcam, #ab1), anti-HIF-2α (1:500, R&D Systems, #AF2886), anti-HIF-3α (Acris antibodies, #AP20606PU-N), anti-CD44 (CST, #5640) or anti-Vimentin (BD Sciences, #550513) at a dilution of 1:1000 in 3% non-fat milk in 1x TBS-tween (0.1% v/v).

Techniques: Binding Assay, Expressing, Activity Assay, Translocation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

doi: 10.1681/ASN.2014040320

Figure Lengend Snippet: The kidney macrophage subtype evolution mirrors kidney function. (A) The elevated BUN levels observed at day 2 (Figure 1F) decreased by day 8, indicating ongoing kidney repair (n=6–11). (B–D) Analysis of macrophages obtained from kidney cell suspensions. Three regions were discriminated according to the expression levels of CD11b and F4/80 as follows: F4/80-CD11b+ (R0), F4/80lowCD11bhigh (R1), and F4/80highCD11b+ (R2). CD11b corresponds to R0+R1+R2 sum. Macrophage distribution among the three regions was affected by glycerol injection at day 2 and day 8. (B) Representative dotplot gated on 30,000 live CD45+ cells. (C) R0, R1, R2, and total CD11b+ cell counts in kidney samples (n=3–5; **P<0.01 and ***P<0.001 compared with day 0; #P<0.05 and ####P<0.0001 compared with day 2). (D) Distribution of CD11b+ cells in the three regions (n=3–5). Macrophage subpopulations were characterized by expression of surface markers using flow cytometry. Mean fluorescence intensity (MFI) is reported on the yaxis for the following markers: (E) CD206 (M2 marker) and (F) CD36 (M2 marker). (n=3–9; *A star above a line displays P<0.05; #displays significant difference between R2 at day 2 or day 8 compared with R2 at day 0 [p<0.05]; $displays significant difference between R1 at day 8 compared with R1 at day 2 [p<0.05]. Lines display means±SEM for individual data).

Article Snippet: Specific primary antibodies were incubated on mouse tissue sections for the detection of F4/80 (rat anti-mouse, 1/100, clone BM8, MF48000; Invitrogen, Saint Aubin, France), collagen III (rabbit anti-mouse, BP8014; Acris Antibodies GmbH, Herford, Germany) or fibronectin (rabbit anti-human/mouse, F3648; Sigma-Aldrich, Saint-Quentin Fallavier, France).

Techniques: Expressing, Injection, Flow Cytometry, Fluorescence, Marker

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

doi: 10.1681/ASN.2014040320

Figure Lengend Snippet: CL-mediated macrophage depletion attenuates kidney lesions 2 days after glycerol injection. Mice received EL or lCL in the saline (NaCl) or the glycerol (Gly) condition according to the protocol shown in Figure 6E. (A and B) CL reduced glycerol-induced fibronectin (Original magnification, ×400 in A) and collagen III (Original magnification, ×200 in B) accumulation at the protein level (left panel; n=3–9) and at the mRNA level in the whole kidney (right panel; n=5–6). (C) Hmox1 was upregulated by glycerol treatment and not modified by CL. CL reduced glycerol-induced Ccl2 and Ccl7 mRNA expression (n=5–6). (Versus NaCl EL: ***P<0.001 and ****P<0.0001. Versus glycerol EL: ##P<0.01; ###P<0.001; ####P<0.0001.) (D) CL reduced glycerol-induced lesions on periodic acid-Schiff staining (Original magnification, ×200 in representative examples). (E) CL did not affect rhabdomyolysis intensity (n=4–8).

Article Snippet: Specific primary antibodies were incubated on mouse tissue sections for the detection of F4/80 (rat anti-mouse, 1/100, clone BM8, MF48000; Invitrogen, Saint Aubin, France), collagen III (rabbit anti-mouse, BP8014; Acris Antibodies GmbH, Herford, Germany) or fibronectin (rabbit anti-human/mouse, F3648; Sigma-Aldrich, Saint-Quentin Fallavier, France).

Techniques: Injection, Modification, Expressing, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

doi: 10.1681/ASN.2014040320

Figure Lengend Snippet: CL-mediated macrophage depletion attenuates kidney fibrosis. (A–D) One month after rhabdomyolysis. CTL, age-matched control group; Gly, glycerol-treated group; Gly CL, CL-treated mice according to the pre, post, and glycerol CL protocol depicted in Figure ​Figure6C.6C. (A) BUN levels (n=2–6). (B) CD206+ expression was significantly increased in the glycerol CL condition among CD11b+ cells (R0, R1, and R2) (n=5–8). (C) Collagen III and Masson trichrome staining (representative examples are shown). (D) Collagen III deposit quantification (n=5–7). (Versus control: **P<0.01 and ****P<0.0001. Versus Gly: #P<0.05 and ##P<0.01.) (E–H) Seven months after rhabdomyolysis. NaCl EL, glycerol EL, and pre-glycerol early CL refer to mice treated according to the protocol depicted in Figure 6A. (E) BUN levels (n=6–14). (F) Kidney mass indicated global atrophy after glycerol injection. (n=6–14) (G) Collagen III and Masson trichrome staining (representative examples are shown). (H) Collagen III deposits were still significantly increased in the glycerol condition, which was partially prevented by CL (n=6–14). (Versus NaCl EL: *P<0.05 and ****P<0.0001. Versus glycerol: #P<0.05, ##P<0.01, ####P<0.0001.)

Article Snippet: Specific primary antibodies were incubated on mouse tissue sections for the detection of F4/80 (rat anti-mouse, 1/100, clone BM8, MF48000; Invitrogen, Saint Aubin, France), collagen III (rabbit anti-mouse, BP8014; Acris Antibodies GmbH, Herford, Germany) or fibronectin (rabbit anti-human/mouse, F3648; Sigma-Aldrich, Saint-Quentin Fallavier, France).

Techniques: Expressing, Staining, Injection

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

doi: 10.1681/ASN.2014040320

Figure Lengend Snippet: Proposed early mechanisms involved in rhabdomyolysis-induced kidney injury. (1) In response to myoglobin, tubular cells secrete macrophage chemoattractants (Ccl2, Ccl7). (2) In response to these chemoattractants blood monocytes migrate to the renal interstitium. (3) In addition to its effect on tubular cells, myoglobin polarizes these macrophages toward a proinflammatory phenotype. (4) CD11bhighF4/80lowLy6-Bhigh subtype macrophages enhance renal injury by secreting extracellular matrix compounds (fibronectin, collagen III), proinflammatory cytokines (Il1b, Il12p40) and by increasing recruitment of newly generated macrophages (secretion of Ccl2 and Ccl7).

Article Snippet: Specific primary antibodies were incubated on mouse tissue sections for the detection of F4/80 (rat anti-mouse, 1/100, clone BM8, MF48000; Invitrogen, Saint Aubin, France), collagen III (rabbit anti-mouse, BP8014; Acris Antibodies GmbH, Herford, Germany) or fibronectin (rabbit anti-human/mouse, F3648; Sigma-Aldrich, Saint-Quentin Fallavier, France).

Techniques: Generated