Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: NOX2 enhances FADDosome-induced gastric epithelial mitochondrial fission and dysfunction in PHG. (A) Two-dimensional hierarchical clustering results for the enzyme NADPH oxidase (NOX) family were presented for the comparison between PHG patients and healthy volunteers (n = 3 per group). The fold changes and P values of the indicated mRNAs in PHG tissues relative to those in normal (Uninvolved) tissues from the microarray experiment were also presented. (B) NOX1, NOX2 and NOX4 IHC staining (brown) showed that NOX2, but not NOX1 or NOX4, was upregulated in the gastric mucosal samples of PHG patients, and the NOX1, NOX2 and NOX4 areas were also analyzed. n = 6 per group. * P < 0.05. (C) Representative images of IHC staining (brown) for NOX1, NOX2 and NOX4 in mouse models revealed that Nec-1 (an inhibitor of RIPK1) repressed NOX2 expression in mice with PVL. n = 6 per group. (D) The NOX1, NOX2 and NOX4 areas detected by IHC staining in (C) were presented. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (E) The colocalization of TOMM20 (red) and NOX2 (green) determined by confocal imaging in GES-1 cells confirmed that TNF-α increased the mitochondrial localization of NOX2 (white arrows indicate colocalization). Nuclei (blue) were counterstained with DAPI. (F) The levels of and interaction between NOX2 and TOMM20 were determined by western blotting, and IP showed that Nec-1 decreased the epithelial expression of total and mitochondrial NOX2 in the primary epithelial cells of mice with PVL. TEM also revealed that Nec-1 partly normalized the abnormal or damaged mitochondria in the mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (G) The ATP concentration in the mouse models verified that Nec-1 and GSK2795039 (GSK, a NOX2 inhibitor) improved the production of ATP in mice with PVL. n = 6 per group. * P < 0.05. (H) Representative 4-HNE IHC staining (brown) and quantification analysis in SO mice and mice with PVL suggested that GSK2795039 (GSK) reduced the level of 4-HNE in mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (I) Representative MitoSox staining (red) and the MitoSox intensity in mouse model revealed that GSK2795039 alleviated mitochondrial ROS (mtROS) accumulation in the primary epithelial cells of mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (J) Related protein levels in primary epithelial cells from mouse models treated with the indicated agents ( shFADD , Nec-1 or BAY) showed interactions among TAK1, NF-κBp65, FADD and NOX2 signaling. (K) GES-1 cells were transfected with either the control vector or shFADD and then treated with TNF-α or vehicle, and the NOX2 luciferase reporter activities were presented (left panel). * P < 0.05 versus the vehicle group and TNF-α-treated cells transfected with shFADD . The NOX2 luciferase reporter activities in GES-1 cells transfected with pcDNA3.1-p65 -vector or pcDNA3.1 -vector were also shown (right panel), * P < 0.05.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Comparison, Microarray, Immunohistochemistry, Expressing, Imaging, Western Blot, Concentration Assay, Staining, Transfection, Control, Plasmid Preparation, Luciferase

Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: The alteration of glycolysis associated with dysfunction of the mitochondrial electron transport chain contributes to oxidative stress in the PHG. (A) Western blotting showed that the protein levels of LDHA and HK2 were increased in mice with PVL but were decreased by GSK2795039 (GSK, a NOX2 inhibitor) in mice with PVL. The ratios of the normalized LDHA/β-actin and HK2/β-actin densitometric units and the relative level of lactic acid were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL not treated with GSK2795039. (B) Oxygen consumption rate (OCR) analysis of primary epithelial cells isolated from mouse models suggested that increased nonmitochondrial oxygen consumption and reduced maximum respiration were present in mice with PVL but were reversed by GSK2795039. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (C) Examination of the glycolytic rate (for the extracellular acidification rate [ECAR]) showed that GSK2795039 treatment reduced basal and compensatory glycolysis in mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (D) Two-dimensional hierarchical clustering results of the genes of electron transport chain (ETC)-related elements between PHG patients (as PHG) and healthy volunteers (as Uninvolved) (n = 3 per group). (E) Western blotting analysis (left panel) of protein levels of representative ETC complex subunits in the primary epithelial cells of PHG patients and healthy volunteers (as Uninvolved) revealed that the levels of the mitochondrial complex I, II, III, IV and V subunits were decreased in PHG patients. The activity of the antioxidant enzyme SOD and the ratio of reduced (GSH) to oxidized (GSSG) states (GSH/GSSG) detected by assay kits (right panel) were found to be decreased in PHG. n = 6 per group. * P < 0.05. (F) Western blotting analysis of representative ETC complex subunits from primary epithelial cells isolated from mouse models suggested that Nec-1 (an inhibitor of RIPK1) mitigated the defects in ETC subunit expression and decreased ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (G) SOD activity and the GSH/GSSG ratio, as detected by assay kits, were inhibited in mice with PVL but were restored by Nec-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (H) Western blotting analysis and quantification of protein levels for representative ETC complex subunits from primary epithelial cells isolated from mouse models showed that Mdivi-1 reversed the decrease in mitochondrial complex I, II, III, IV and V subunit levels and reduced ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also presented. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment. (I) Decreases in SOD activity and the GSH/GSSG ratio detected by assay kits were found in mice with PVL but were reversed by Mdivi-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Western Blot, Isolation, Activity Assay, Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Association between CRBP1 gene gain copy number and its expression in cervical cancer samples

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Immunodetection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Correlation between CRBP1 expression and clinic pathological variables in cervical cancer

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Methylation, Molecular Weight, Marker

Journal: Cell Reports Methods

Article Title: Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

doi: 10.1016/j.crmeth.2023.100465

Figure Lengend Snippet:

Article Snippet: Mouse α-MYH7 monoclonal , Santa Cruz Biotech , Cat# Sc-53090; RRID: AB_2147279.

Techniques: Recombinant, Membrane, Transfection, SYBR Green Assay, DNA Methylation Assay, Mutagenesis, CRISPR, Software, Functional Assay, Microarray, In Vivo, Comparison, Next-Generation Sequencing

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Current Biology

Article Title: Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a ( Ink4a/Arf )

doi: 10.1016/j.cub.2017.09.007

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (clone 197G2) , Cell Signaling Technology , Cat# 4377; RRID: AB_331775.

Techniques: Recombinant, Produced, Reverse Transcription, Microarray, Labeling, DNA Purification, Methylation, TA Cloning, Sequencing, SYBR Green Assay, Software

Journal: Current Biology

Article Title: Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a ( Ink4a/Arf )

doi: 10.1016/j.cub.2017.09.007

Figure Lengend Snippet:

Article Snippet: Primary antibodies were from the following sources:anti-STAT3, anti-phospho-Akt (Ser 473), anti-Akt, anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-phospho Src (Y418), anti-Src from Cell Signaling Technology, (Buckinghamshire, UK); rabbit anti-phospho-Stat3 (Tyr705) from Abcam (Cambridge, UK), mouse anti-β Actin (loading control) was from Sigma–Aldrich (Gillingham, UK).

Techniques: Recombinant, Produced, Reverse Transcription, Microarray, Labeling, DNA Purification, Methylation, TA Cloning, Sequencing, SYBR Green Assay, Software

Journal: Stem cell research & therapy

Article Title: Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

doi: 10.1186/s13287-017-0507-4

Figure Lengend Snippet: Fig. 2 Expression and promoter methylation of DNA methyltransferases in intestinal epithelium. a, b Decreased Dnmt1 expression is detected in db/db crypts and intestinal epithelium stem cells (IESCs) by qRT-PCR using the FACS cell population (a) and Western blot using the MACS cell population (b left) and quantification of immunoblot bands from Western blot (b right). mRNA and protein levels are expressed relative to β-actin. Mean ± SE; n ≥6; *P < 0.05, **P < 0.01 by t test. C represents the control db/+ group; D represents the diabetic db/db group. c Promoter methylation microarray of Dnmt3b indicates promoter hypermethylation not only in db/db mice (orange bars) but also in db/+ mice (light blue bars). Values in brackets indicate the peak score that reflects the probability of positive enrichment and average P value scores. d, e Validation of Dnmt3b microarray results by bisulfite sequence. The table indicates the results of bisulfite sequencing analyses of 10 bacterial colonies per group (d). The CpG sites validated are the sites that exhibit a peak signal in the microarray. Black circles represent methylated CpG sites; white hollow circles represent unmethylated CpG sites; crosses represents methylation status not determined . Bar graph showing the ratio of methylated cyto- sine in CpG within the regions analyzed (e). f, g Immunohistochemical staining of Dnmt1 (f) and Dnmt3a (g). Scale bars = 50 μm. Diff differenti- ated cells, N.S. not significant

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot, Control, Microarray, Biomarker Discovery, Sequencing, Methylation Sequencing, Immunohistochemical staining, Staining

Journal: Stem cell research & therapy

Article Title: Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

doi: 10.1186/s13287-017-0507-4

Figure Lengend Snippet: Fig. 3 Promoter methylation levels and gene expression of Wnt signaling-related genes. a (left) Hierarchical clustering analysis on the basis of the DMEP-related genes related with Wnt signaling. Each row indicates a DMEP-related gene, and the corresponding gene name is indicated on the right. Each column indicates a sample analyzed. Methylation levels range from unmethylated (green) to fully methylated (red), as indicated by the color legend at the top of the graph. a (right) The degree of methylation of Wnt signaling pathway-related genes among DMEP-related genes. The bar height reflects the degree of methylation. b, c The mRNA levels of Wnt signaling-related genes in crypts and intestinal epithelium stem cells (IESCs) using the FACS cell population. d (upper) Western blot analysis of Sox9 using the MACS cell population, and d (lower) quantification of immunoblot bands from three repetitions of western blot experiments. e Immunohistochemical staining of Sox9 in the small intestinal epithelium. The arrows indicate cells expressing low levels of Sox9, which were considered IESCs. Scale bars = 50 μm. mRNA and protein levels are expressed relative to β-actin. Mean ± SE; n ≥6 (mRNA); n ≥3 (protein); *P < 0.05, **P < 0.01, ***P < 0.001 by t test. N.S. not significant

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Methylation, Gene Expression, Western Blot, Immunohistochemical staining, Staining, 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: 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: Gene Expression, Methylation, In Silico, Expressing, Immunohistochemical staining, Microarray, Growth Assay, Quantitative RT-PCR, Control, 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, Functional Assay, Knockdown, Control

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, Quantitative RT-PCR, Knockdown, Control, 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: Gene Expression

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: Gene Expression

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: Comparison, Expressing, Functional Assay