Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: Heart failure and cardiomyocyte injury caused by various pathological factors lead to decreased DDX17 expression. a , b The average data of mouse cardiac function as shown by the left ventricular EF and FS of the sham and TAC-induced chronic heart failure mice ( n = 6). c – e Ddx17 mRNA and protein expression in the myocardium from the sham or TAC-induced chronic heart failure mouse models ( n = 6). f Representative images of hearts showing the cardiac morphology of the control (Con) and Dox-treated (Dox) mice ( n = 6); scale bar, 2 mm. g Representative images of H&E-stained heart sections from the Con and Dox-treated mice ( n = 6); scale bar, 2 mm. h , i Mouse cardiac function as shown by the left ventricular EF and FS of Con and Dox-treated mice ( n = 6). j – l Ddx17 mRNA ( n = 6) and protein ( n = 6) expression in Con and Dox-treated mouse hearts. m , n DDX17 expression in NMVMs treated with different injury factors: serum-free medium (SF), hypoxia for 16 h (HP), 2 μg/mL TNF-α, 100 μM H 2 O 2 , and 0.5 μM Dox ( n = 3). o , p DDX17 protein expression in NMVMs treated with normoxia or hypoxia for 16 h ( n = 5). q , r DDX17 protein levels and the average data of NMVMs treated with different concentrations of H 2 O 2 for 24 h ( n = 3). s , t DDX17 protein levels and the average data of NMVMs treated with 0.1, 0.5, and 1 μM Dox for 24 h ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Expressing, Control, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: Cardiomyocyte-specific Ddx17 knockout leads to reduced cardiac function and exacerbates Dox-induced heart failure in mice. a , b Western blot and the average data of DDX17 in isolated NMVMs of control (Con) and Ddx17- cKO mice ( n = 6). c Western blot of DDX17 levels in heart, skeletal muscle, liver and kidney of Con and Ddx17 -cKO mice ( n = 3). d Mice were injected intraperitoneally with saline or 7.5 mg/kg Dox 3 times every other day. Survival curve of the mice in the four groups ( n = 18). e Body weight of mice in the control + saline (Con + Saline), Ddx17- cKO + saline ( Ddx17- cKO + Saline), control + doxorubicin (Con + Dox) and Ddx17- cKO + doxorubicin ( Ddx17- cKO + Dox) groups ( n = 5). f Representative images of the hearts from the mice in Con + Saline, Ddx17- cKO + Saline, Con + Dox, and Ddx17- cKO + Dox groups, the scale bar represents 2 mm. g Representative H&E-stained heart sections from mice in the four groups ( n = 5); scale bar, 50 μm. h , i Representative images of Sirius Red-stained hearts from mice in the four groups and semiquantitative analysis of the fibrosis area ratio ( n = 5); scale bar, 200 μm. j – l Representative images of echocardiography of mouse hearts and the average data of cardiac function of left ventricular EF ( k ) and FS ( l ) in the four groups; n = 10 for each group. m TUNEL staining quantification results of myocardial tissue in the four groups; n = 5 for each group. n , o Mouse serum LDH ( n ) and CK-MB ( o ) levels in the four groups ( n = 6). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Knock-Out, Western Blot, Isolation, Control, Injection, Saline, Staining, TUNEL Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: Overexpression of Ddx17 in cardiomyocytes attenuates myocardial injury and improves cardiac function under pathological conditions. a Western blot of DDX17 protein expression from control (Con) and two cardiac-specific Ddx17 -overexpressing mouse lines ( Ddx17 -Tg-H and Ddx17 -Tg) ( n = 5). b DDX17 expression in heart, skeletal muscle, liver and kidney of control (Con) and Ddx17 -Tg mice ( n = 3). c Doxorubicin (7.5 mg/kg) or an equivalent volume of saline was administered to mice by intraperitoneal injection every other day for a total of three injections in the control (Con) and Ddx17 -Tg mice. Survival curves of mice in the control + saline (Con + Saline), Ddx17 -transgene + saline ( Ddx17 -Tg + Saline), control + doxorubicin (Con + Dox), and Ddx17 -transgene + doxorubicin ( Ddx17 -Tg + Dox) groups ( n = 18). d Body weight of the mice in the Con + Saline, Ddx17 -Tg + Saline, Con + Dox, and Ddx17 -Tg + Dox groups ( n = 6). e – g Representative images of echocardiography of mouse heart and the average data of cardiac function of left ventricle EF ( f ) and FS ( g ) in the four groups ( n = 11). h , i Representative images of heart morphologies and H&E-stained heart sections in the four groups ( n = 6). j – k Representative images of Sirius Red-stained mouse heart sections and quantification of myocardial fibrosis area ratio in the four groups ( n = 5); scale bar, 200 μm. l TUNEL staining of myocardial tissue in Con + Saline, Ddx17 -Tg + Saline, Con + Dox, and Ddx17 -Tg + Dox, ( n = 5); scale bar, 50 μm. m Quantification of TUNEL staining of myocardial tissue in the four groups ( n = 5). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Over Expression, Western Blot, Expressing, Control, Saline, Injection, Staining, TUNEL Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: DDX17 plays an important role in maintaining mitochondrial morphology and function in cardiomyocytes. a Transmission electron microscopy (TEM) of LVs from control (Con) and Ddx17 -cardiomyocyte-specific knockout (cKO) mice (scale bars: low-1 μm, medium-500 nm, high-200 nm) ( n = 5). b , c Statistical analysis of mitochondrial length and mitochondrial area in control (Con) and Ddx17 -cKO mice ( n = 48). d , e Mitochondrial membrane potential (ΔΨm) analyzed by JC-1 red/green fluorescence intensity in control (Con) and Ddx17 -overexpressing ( Ddx17 -OE) HL-1 cells treated with normoxic and hypoxic conditions ( n = 3); scale bar: 50 μm. f , g Mitochondrial permeability transition pore (mPTP) analyzed by calcein-AM fluorescence intensity in control (Con) and Ddx17 -overexpressing ( Ddx17 -OE) HL-1 cells ( n = 4); scale bar: 90 μm. h , i NMVMs were infected with Ddx17 -overexpressing adenovirus ( Ddx17 -OE) or its control (Con) for 24 h and then treated with PBS or Dox for 24 h. Mitochondria of cardiomyocytes were stained with Mito-Tracker Red and nuclei were stained with DAPI, and the rate of mitochondrial fission was analyzed by confocal microscopy ( n = 4); scale bar corresponds to 20 μm. j Cellular ATP concentration in the NMVMs of control (Con) and Ddx17 -cKO mice treated with PBS or Dox ( n = 4) . k Cellular ATP concentration in the NMVMs of control (Con) and Ddx17 -Tg mice treated with PBS or Dox ( n = 4) . l , m 8-OHdG and MDA levels in NMVMs from each group ( n = 3). n – r HL-1 cardiomyocytes were infected with Ddx17 overexpressing adenovirus ( Ddx17 -OE) or its control (Con) for 24 h and then treated with PBS or 0.5 μM Dox for 24 h. Based on the measured mitochondrial OCR of HL-l cells in response to 1 μM oligomycin, 1 μM FCCP and 0.5 μM rotenone/antimycin A, the basal respiration, maximal respiration, ATP production and spare respiratory capacity were measured using a Seahorse flux analyser ( n = 4). s Western blot of DRP1, MFN1 and MFN2 in the left ventricle of mice in the control + saline (Con + Saline), Ddx17 -cKO + saline (Ddx17 -cKO + Saline), control + doxorubicin (Con + Dox) and Ddx17 -cKO + doxorubicin ( Ddx17 -cKO + Dox) groups ( n = 6). t Western blot of DRP1, MFN1 and MFN2 in LVs from mice in the control + saline (Con + Saline), Ddx17 -transgene + saline ( Ddx17 -Tg + Saline), control + doxorubicin (Con + Dox) and Ddx17 -transgene+doxorubicin ( Ddx17 -Tg + Dox) groups ( n = 6). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Transmission Assay, Electron Microscopy, Control, Knock-Out, Membrane, Fluorescence, Permeability, Infection, Staining, Confocal Microscopy, Concentration Assay, Western Blot, Saline

Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: DDX17 coordinates with BCL6 in the transcriptional repression of the Drp1 gene in cardiomyocytes. a , b GO terms responding to promoters with higher ( a ) and lower ( b ) methylation levels in NMVMs from Ddx17 -Tg mice compared to controls (Con) ( n = 3). Representative genes are indicated below. c Motif enrichment analysis of proximal NDRs in Ddx17 -Tg and control (Con) NMVMs ( n = 3). d , e Co-IP experiments were performed with DDX17 ( d ) and BCL6 ( e ) antibodies to analyze the interaction of DDX17 and BCL6 in control (Con) and Ddx17 -cKO NMVMs ( n = 4). f Immunofluorescence staining of DDX17 (red), BCL6 (green) and nuclei (DAPI, blue) in cultured wild-type NMVMs; scale bar, 20 μm ( n = 3). g To investigate the regulatory effects of DDX17 and BCL6 overexpression on Drp1 promoter activity, HEK293A cells were transfected with Ddx17 (pcDNA- Ddx17 ) and/or Bcl6 (pcDNA- Bcl6 ) expression plasmids and simultaneously cotransfected with the Drp1 0.8-kb wild-type promoter (pGL3- Drp1 -WT) luciferase reporter plasmid, and Drp1 promoter activity was analyzed by luciferase assay ( n = 3). h HEK293A cells were transfected with Ddx17 (pcDNA- Ddx17 ) and/or Bcl6 (pcDNA- Bcl6 ) expression plasmids and simultaneously cotransfected with the 0.8 kb Drp1 promoter mutation plasmid (pGL3- Drp1 -MUT) with mutated BCL6 binding sites using the pGL3-basic plasmid ( n = 3). i HEK293A cells were cotransfected with the Drp1 promoter plasmid (pGL3- Drp1 -WT) and different concentrations of the Bcl6 expression plasmid (pcDNA- Bcl6 ) to detect Drp1 promoter activity ( n = 3). j ChIP analysis of NMVMs revealed the recruitment of BCL6 to regions containing BCL6 binding sites within the promoter region of Drp1 by quantitative real-time PCR ( n = 3). k – n HL-1 cells were transfected with Ddx17 siRNA and/or Drp1 siRNA for 24 h and then treated with doxorubicin for 24 h. Expression of DDX17, DRP1 and c-CASP-3 was analyzed by western blot, and GAPDH was used as a protein loading control ( n = 3). In all co-transfection experiments, pcDNA3.1 was used as the equilibrium plasmid in the different transfection mixtures to balance the total amount of DNA, and NCi was used as the equilibrium RNA in the different transfection mixtures to balance the total amount of RNA ( n = 3). o – q HL-1 cardiomyocytes were transfected with NCi (Con) or Ddx17 siRNA ( Ddx17 -KD) for 24 h and then treated with PBS or 0.5 μM Dox for 24 h. Cardiomyocyte mitochondria and cytoplasm were isolated and the expression of mitochondrial DRP1 (Mito-DRP1) and cytoplasmic DRP1 (Cyto-DRP1) was analyzed by western blot. COX IV and β-tubulin were used as protein loading controls for mitochondria and cytoplasm, respectively ( n = 4). r HL-1 cells were transfected with NCi (Con) and Ddx17 siRNA ( Ddx17 -KD) for 24 h, then treated with PBS or 0.5 μM Dox for 24 h and divided into Con, Ddx17 -KD, Con + Dox and Ddx17 -KD + Dox groups. Cardiomyocyte mitochondria and cytoplasm were extracted separately using a mitochondrial isolation kit. Cytochrome c levels in mitochondria and cytoplasm were analyzed by western blot. COX IV was used as a protein loading control for mitochondria and β-tubulin as a protein loading control for cytoplasmic proteins ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Methylation, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Cell Culture, Over Expression, Activity Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Cotransfection, Isolation

Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: DDX17 expression levels are positively correlated with cardiac function in patients with different stages of heart failure. a H&E staining of myocardial tissue from the control (Con) and heart failure (HF) patients ( n = 3); scale bar, 100 μm. b Wheat germ agglutinin staining of tissue samples isolated from Con and HF patients ( n = 3); scale bar, 100 μm. c Transmission electron microscopy images of myocardial tissue from Con and HF patients ( n = 3); scale bar, 2 μm. d Correlation of the DDX17 mRNA levels with left ventricular EF in myocardial tissue from heart failure patients. e Correlation of DRP1 mRNA levels with left ventricular EF in myocardial tissue from heart failure patients. f Correlation of DDX17 with DRP1 mRNA levels in the myocardium of heart failure patients. g Graphic summary of DDX17 protecting cardiac function by promoting mitochondrial homeostasis through the BCL6-DRP1 pathway in heart failure

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Expressing, Staining, Control, Isolation, Transmission Assay, Electron Microscopy

Journal: Journal of the American Society of Nephrology

Article Title: The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects against Podocyte Oxidative Stress and Renal Injury in Diabetes

doi: 10.1681/asn.2014090898

Figure Lengend Snippet: Figure 1. EZH2 depletion with DZNep augments podocyte TxnIP expression and oxi- dative stress. Effect of DZNep or high glucose on EZH2 and TxnIP expression, ROS levels, and programmed cell death in cultured mouse podocytes. (A and B) Immunoblotting mouse podocytes for (A) EZH2 and (B) H3K27me3 under control conditions or after DZNep treatment for 48 hours. (C) CFDA fluorescence intensity in mouse podocytes exposed to control (5.6 mM glucose), high glucose (25 mM) for 1, 3, or 12 hours, or mannitol (osmotic control). (D) CFDA fluorescence intensity in mouse podocytes under control conditions or treated with DZNep, high glucose, high glucose and DZNep, or mannitol. All values are normalized to control. (E) Quantitation of TUNEL-positive nuclei (%) from cultured mouse podocytes incubated under conditions of normal glucose, DZNep, high glucose, high glucose and DZNep, or mannitol. (F and G) Change in TxnIP expression by (F) real-time PCR for TxnIP mRNA and (G) Western blot for TxnIP protein in cultured mouse podocytes incubated for 48 hours under the following conditions: control, DZNep, high glucose, a combination of high glucose and DZNep, or mannitol. (H) CFDA fluorescence intensity in podocytes transfected with scrambled shRNA, TxnIP shRNA, or a constitutively active vector overexpressing Trx and incubated with either normal glucose or a combination of high glucose (12 hours) and DZNep for 48 hours. All values are normalized to control or scrambled as indicated. AU, arbitrary unit; HG, high glucose. *P,0.05 versus control; †P,0.01 versus control; ‡P,0.05 versus control or DZNep; §P,0.001 versus all other groups except high glucose (P,0.01); |P,0.001 versus control by two-way ANOVA; ¶P,0.001 versus all other groups except mannitol (P,0.001); **P,0.05 for an interaction

Article Snippet: For shRNA–mediated gene knockdown or vector– based gene overexpression experiments, podocytes were transfected (Lipofectamine 2000; Life Technologies, Carlsbad, CA) with 2.5 mg sequence-specific shRNA targeting TxnIP (Origene, Rockville, MD) or EZH2 (Qiagen, Valencia, CA) or a DEST40 vector (Life Technol- ogies) containing the mouse Trx gene with constitutive expression driven by a cytomegalovirus promoter (Origene).

Techniques: Expressing, Cell Culture, Western Blot, Control, Quantitation Assay, TUNEL Assay, Incubation, Real-time Polymerase Chain Reaction, Transfection, shRNA, Plasmid Preparation

Journal: Journal of the American Society of Nephrology

Article Title: The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects against Podocyte Oxidative Stress and Renal Injury in Diabetes

doi: 10.1681/asn.2014090898

Figure Lengend Snippet: Figure 2. EZH2 knockdown augments TxnIP expression and EZH2 augmentation through miR-101 inhibition attenuates TxnIP ex- pression. Effect of EZH2 knockdown or upregulation through miR-101 inhibition on podocyte TxnIP expression and oxidative stress. (A–C) Effect of shRNA–mediated EZH2 knockdown. (A) TxnIP gene expression by real-time PCR, (B) CFDA fluorescence intensity, and (C) quantitation of TUNEL-positive nuclei (%) in mouse podocytes transfected with scrambled shRNA, incubated with 25 mM (high) glucose (control), or transfected with EZH2 shRNA in the presence of high glucose. (D–F) Effect of miR-101 inhibition. (D) Western blot for EZH2 expression in mouse podocytes treated with vehicle (control) or miR-101 inhibitor for 48 hours. (E) TxnIP protein expression by Western blot and (F) CFDA fluorescence intensity in podocytes under the following conditions: normal glucose, miR-101 inhibitor, high glucose for 12 hours, and high glucose (12 hours) in the presence of miR-101 inhibitor. All values are normalized to control except TUNEL. AU, arbitrary unit; HG, high glucose. *P,0.01 versus scrambled; †P,0.001 versus scrambled or high glucose (control); ‡P,0.001 versus scrambled or high glucose (control); §P,0.01 versus control; |P,0.001 versus high glucose; ¶P,0.05 versus control; **P,0.001 versus high glucose; ††P,0.01 versus high glucose.

Article Snippet: For shRNA–mediated gene knockdown or vector– based gene overexpression experiments, podocytes were transfected (Lipofectamine 2000; Life Technologies, Carlsbad, CA) with 2.5 mg sequence-specific shRNA targeting TxnIP (Origene, Rockville, MD) or EZH2 (Qiagen, Valencia, CA) or a DEST40 vector (Life Technol- ogies) containing the mouse Trx gene with constitutive expression driven by a cytomegalovirus promoter (Origene).

Techniques: Knockdown, Expressing, Inhibition, shRNA, Gene Expression, Real-time Polymerase Chain Reaction, Quantitation Assay, TUNEL Assay, Transfection, Incubation, Control, Western Blot

Journal: Journal of the American Society of Nephrology

Article Title: The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects against Podocyte Oxidative Stress and Renal Injury in Diabetes

doi: 10.1681/asn.2014090898

Figure Lengend Snippet: Figure 3. DZNep treatment augments proteinuria, podocytopathy, glomerular TxnIP expression, and oxidative stress in diabetic rats. (A) Western blot analysis of H3K27me3 and total histone H3 expression in rat kidney homogenates in control and DZNep- treated animals after 5 days (n=4 per group). (B) Measurement of urinary protein excretion in control and diabetic rats treated with vehicle or DZNep for 3 weeks (n=12 per group). (C–F) Representative podocyte ultrastructure by transmission electron microscopy from (C and D) control and (E and F) diabetic rats treated with (C and E) vehicle or (D and F) DZNep. The asterisks in D and F mark the presence of (D) adsorption droplets or (F) vacuoles in podocytes from (D) a control rat treated with DZNep and (F) a diabetic rat treated with DZNep. (G–J) Transmission electron micrographs of podocyte foot processes from (G and H) control and (I and J) diabetic rats treated with (G and I) vehicle or (H and J) DZNep. The arrows in I and J mark areas of foot process effacement in diabetic rats treated with (I) vehicle or (J) DZNep. (K) Quantitation of podocyte abnormalities (%). (L–O) Glomerular TxnIP im- munostaining of rat kidney sections in (L and M) control and (N and O) diabetic rats treated with (L and N) vehicle or (M and O) DZNep for 3 weeks. Original magnification, 3400. (P) Quantitation of glomerular TxnIP immunostaining. (Q) Urinary 8-hydroxy-29- deoxyguanosine (8-OHdG) excretion. AU, arbitrary unit. *P,0.05 versus control; †P,0.001 versus control and vehicle, P,0.001 versus control and DZNep, and P,0.05 versus diabetes; ‡P,0.05 versus all other groups; §P,0.001 versus control and vehicle, P,0.05 versus control and DZNep, and P,0.05 versus diabetes; |P,0.001 versus control and vehicle or control and DZNep; ¶P,0.01 versus control and vehicle or control and DZNep; **P,0.01 versus diabetes and vehicle.

Article Snippet: For shRNA–mediated gene knockdown or vector– based gene overexpression experiments, podocytes were transfected (Lipofectamine 2000; Life Technologies, Carlsbad, CA) with 2.5 mg sequence-specific shRNA targeting TxnIP (Origene, Rockville, MD) or EZH2 (Qiagen, Valencia, CA) or a DEST40 vector (Life Technol- ogies) containing the mouse Trx gene with constitutive expression driven by a cytomegalovirus promoter (Origene).

Techniques: Expressing, Western Blot, Control, Transmission Assay, Electron Microscopy, Adsorption, Quantitation Assay, Immunostaining

Journal: Journal of the American Society of Nephrology

Article Title: The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects against Podocyte Oxidative Stress and Renal Injury in Diabetes

doi: 10.1681/asn.2014090898

Figure Lengend Snippet: Figure 4. The transcription factor Pax6 mediates the regulation of TxnIP expression by EZH2 in podocytes. (A) Luciferase promoter reporter assay of podocytes incubated under control conditions or after treatment with DZNep for 48 hours. (B) ChIP sequencing of the Pax6 promoter and gene regions after enrichment by immunoprecipitation with an anti-H3K27me3 antibody. Increased H3K27me3 at the Pax6 promoter is shown in control podocytes compared with either input DNA or podocytes treated with DZNep for 48 hours. (C) ChIP of the Pax6 promoter after H3K27me3 enrichment. (D and E) Increased Pax6 (D) mRNA and (E) protein after treatment with DZNep for 48 hours. (F) ChIP of the TxnIP promoter after Pax6 enrichment. (G) Increased TxnIP mRNA in podocytes after treatment with DZNep for 48 hours and prevention of TxnIP upregulation by transfection of cells with siRNA directed against Pax6. (H–K) Immunoblotting glomeruli isolated from control rats (n=4) or rats after 3 weeks of STZ-induced diabetes (n=7) for (I) EZH2, (J) Pax6, and (K) TxnIP. AU, arbitrary unit; IP, immunoprecipitation. *P,0.01 versus control; †P,0.05 versus IgG; ‡P,0.001 versus IgG; §P,0.01 versus all other conditions; ¶P,0.05 versus control.

Article Snippet: For shRNA–mediated gene knockdown or vector– based gene overexpression experiments, podocytes were transfected (Lipofectamine 2000; Life Technologies, Carlsbad, CA) with 2.5 mg sequence-specific shRNA targeting TxnIP (Origene, Rockville, MD) or EZH2 (Qiagen, Valencia, CA) or a DEST40 vector (Life Technol- ogies) containing the mouse Trx gene with constitutive expression driven by a cytomegalovirus promoter (Origene).

Techniques: Expressing, Luciferase, Reporter Assay, Incubation, Control, ChIP-sequencing, Immunoprecipitation, Transfection, Western Blot, Isolation

Journal: Journal of the American Society of Nephrology

Article Title: The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects against Podocyte Oxidative Stress and Renal Injury in Diabetes

doi: 10.1681/asn.2014090898

Figure Lengend Snippet: Figure 5. TxnIP upregulation in a gene expression array of podocytes exposed to DZNep and high glucose (HG). Gene expression analysis of podocytes after exposure to DZNep, high glucose or HG and DZNep for 48 hours compared with control conditions. (A) Venn diagram of pairwise differential comparisons: 18 genes are dif- ferentially expressed (adjusted P value ,0.05) between control and the three other conditions. (B) Using the 18-gene intersection, the individual arrays were clustered. DZNep- and HG- and DZNep-treated podocytes are observed to cluster together; HG clusters closer to these groups than it does to control. TxnIP is one of seven genes to be upregulated in the three treatment states.

Article Snippet: For shRNA–mediated gene knockdown or vector– based gene overexpression experiments, podocytes were transfected (Lipofectamine 2000; Life Technologies, Carlsbad, CA) with 2.5 mg sequence-specific shRNA targeting TxnIP (Origene, Rockville, MD) or EZH2 (Qiagen, Valencia, CA) or a DEST40 vector (Life Technol- ogies) containing the mouse Trx gene with constitutive expression driven by a cytomegalovirus promoter (Origene).

Techniques: Gene Expression, Control

Journal: Journal of Biological Chemistry

Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma

doi: 10.1074/jbc.m110.191361

Figure Lengend Snippet: FIGURE 3. Knockdown of KLK-5 expression reduces processing of Dsg1. A, immunocytochemical analysis of KLK5 expression in control (left panel) or KLK5-KD (right panel) SCC25 cells. Cells were cultured on glass coverslips, fixed and incubated with antibodies against KLK5 (1:50 dilution) followed by Al- exa-Fluor-labeled secondary antibodies and counterstained with DAPI. Yellow scale bar, 100 m. B, quantification of immunofluorescent staining with NIH ImageJ for average intensity. C, quantitative real time PCR analysis of KLK5 levels in control and KLK5-KD SCC25 cells. Relative quantification normalized against the housekeeping gene PGK-1 mRNA levels. Graph depicts KLK5 levels in SCC25-KLK5-KD cells normalized relative to SCC25 parental cells (desig- nated as 100). D, analysis of Dsg1 processing in SCC25-KLK5-KD cells. Lysates from duplicate cultures of parental SCC25 cells (lanes 1 and 2) or two clones of SCC25-KLK5-KD cells (lanes 3 and 4) were electrophoresed on 9% SDS-polyacrylamide gels, transferred to PVDF membrane and immunoblotted with anti- Dsg1 (1:1000; upper panel), anti-E-cadherin (1:1000; middle panel), or anti-GAPDH (1:4000; lower panel) followed by peroxidase-conjugated secondary anti- body (1:4000) and peroxidase substrate. Arrow denotes migration position of full-length Dsg1 (165 kDa); arrowhead denotes migration position of cleavage product (130 kDa). E, densitometric quantitation of band density of Dsg1 cleavage product denoted by arrowhead in D. F, densitometric quantitation of band density of corresponding blots shown in D.

Article Snippet: To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFPV-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

Techniques: Knockdown, Expressing, Control, Cell Culture, Incubation, Labeling, Staining, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Clone Assay, Membrane, Migration, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma

doi: 10.1074/jbc.m110.191361

Figure Lengend Snippet: FIGURE 4. Ultrastructural analysis of desmosomes. SCC25 or SCC25- KLK5-KD cells were grown on coverslips to confluence, then fixed and pro- cessed for transmission electron microscopy. Ultrathin sections were exam- ined with a JEOL 1400 Transmission Electron Microscope. Representative TEM image from (A) SCC25 and (B) SCC25-KLK5-KD cells. Scale bar, 0.2 m. C, quantitation of desmosome number/field from a minimum of 70 images each of SCC25 and SCC25-KLK5-KD cells.

Article Snippet: To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFPV-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

Techniques: Transmission Assay, Electron Microscopy, Microscopy, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma

doi: 10.1074/jbc.m110.191361

Figure Lengend Snippet: FIGURE 5. Overexpression of KLK-5 expression induces processing of Dsg1. A, immunocytochemical analysis of KLK5 expression in control (left panel) OKF/6 cells or OKF/6 cells transfected with a KLK5 expression vector to generate OKF/6-KLK5 cells (right panel). Cells were cultured on glass coverslips, fixed, and incubated with antibodies against KLK5 (1:50 dilution) followed by Alexa-Fluor-labeled secondary antibodies and counterstained with DAPI. Scale bar 100 m. B, quantification of immunofluorescent staining with NIH ImageJ for average intensity. C, quantitative real time PCR analysis of KLK5 lev- els in control and KLK5 OKF/6 cells. Relative quantification normalized against the housekeeping gene PGK-1 mRNA levels. Graph depicts KLK5 levels in OKF/6-KLK5 cells normalized relative to OKF/6 parental cells (designated as 100). D, analysis of Dsg1 processing in OKF/6-KLK5 cells. Lysates from paren- tal OKF/6 cells (lane 1), vector-transfected OKF/6 cells (lane 2), and OKF/6-KLK5 cells (lane 3) were electrophoresed on 9% SDS-polyacrylamide gels, trans- ferred to PVDF membrane, and immunoblotted with anti-Dsg1 (1:1000; upper panel), anti-E-cadherin (1:1000; middle panel) or anti-GAPDH (1:4000; lower panel) followed by peroxidase-conjugated secondary antibody (1:4000) and peroxidase substrate. The arrow denotes migration position of full-length Dsg1 (165 kDa); arrowhead denotes migration position of cleavage product (130 kDa). E, densitometric quantitation of band density of Dsg1 cleavage product denoted by arrowhead in D. F, densitometric quantitation of band density of corresponding blots shown in D.

Article Snippet: To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFPV-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

Techniques: Over Expression, Expressing, Control, Transfection, Plasmid Preparation, Cell Culture, Incubation, Labeling, Staining, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Membrane, Migration, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma

doi: 10.1074/jbc.m110.191361

Figure Lengend Snippet: FIGURE 6. Effect of KLK5 expression on cell-cell aggregation dynamics. Single cell suspensions of (A) SCC25, (B) SCC25-KLK5-KD, (C) OKF/6, or (D) OKF/6- KLK5 were incubated in culture medium containing 0.5% BSA and rotated for 7 h. At the indicated time points, aliquots were removed and photographed to visualize cell-cell aggregation. E and H, quantitation of aggregation kinetics. The number of single cells remaining in the suspension at each time point was enumerated and is shown relative to time 0 (100% single cells). (Closed circle) SCC25, (open circle) SCC25-KLK5-KD, (closed triangle) OKF/6, (open triangle) OKF/6-KLK5. F, G, I, J, distribution of cellular aggregates. Within a high-powered field, the number of cellular clusters comprised of (black bar) 10 cells, (white bar) 10–50 cells, and (gray bar) 50 cells was enumerated at the designated time points. (F) SCC25, (G) SCC25-KLK5-KD, (I) OKF/6, (J) OKF/6-KLK5.

Article Snippet: To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFPV-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

Techniques: Expressing, Incubation, Quantitation Assay, Suspension

Journal: Journal of Biological Chemistry

Article Title: Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma

doi: 10.1074/jbc.m110.191361

Figure Lengend Snippet: FIGURE 7. Effect of KLK5 expression on monolayer cohesion. Cell-cell adherent monolayers were separated from culture dishes by pulsing with dispase as described under “Experimental Procedures,” transferred to conical tubes affixed to a rocking platform, and subjected to 50 inversion cycles. A, B, D, E, aliquots were photographed to visualize relative monolayer cohesion or dissociation. C and F, total number of fragments present following mechanical dis- ruption was quantified.

Article Snippet: To generate SCC25 cells with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD), pGFPV-RS plasmid containing a gene-specific shRNA sequence silencing KLK5 (GTG TTG GTG CAT CCA CAG TGG CTG CTC AC) (OriGene Technologies, Rockville, MD) was used to transfect SCC25 cells using the Human Keratinocyte Nucleofector kit (Amaxa GmbH, Germany).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Autophagy inhibition impairs the epithelial-mesenchymal transition and enhances cisplatin sensitivity in nasopharyngeal carcinoma

doi: 10.3892/ol.2017.5963

Figure Lengend Snippet: Cisplatin induces autophagy, while the inhibition of autophagy by CQ elevates the cytotoxicity of cisplatin in 6–10B and 5–8F nasopharyngeal carcinoma cells. (A) The 6–10B cells were incubated with (Aa) vehicle or with (Ab and Ac) 2 µg/ml cisplatin for 24 h and then subjected to transmission electron microscopy. White triangles denote an autophagic vacuole, the asterisk denotes an early autophagic vacuole and the black triangle denotes a degradative autophagic vacuole. (B) The 6–10bB and 5–8F cells were treated with 0, 1, 2 or 4 µg/ml cisplatin for 24 or 48 h. Western blot analysis revealed a dose- and time-dependent increase in LC3B-II expression in the cisplatin-treated 6–10B and 5–8F cells. (C) The 6–10B and 5–8F cells were incubated with 2 µg/ml cisplatin and/or 10 µM CQ for 24 h. Western blot analysis showed that cisplatin enhanced the expression of LC3B-II, and the combination of cisplatin and CQ resulted in more LC3B-II expression. (D) The 6–10B and 5–8F cells were treated with increasing concentrations of cisplatin (0.31–10 µg/ml) in the presence or absence of 10 µM CQ for 48 h. The CCK-8 assay revealed that the IC 50 value decreased in the CQ group compared with the blank group (6–10B cells: 2.17±0.01 vs. 2.67±0.13, P<0.01; 5–8F cells: 1.01±0.08 vs. 1.35±0.03, P<0.01). Cis, cisplatin; CQ, chloroquine; LC3B, microtubule-associated protein 1 light chain 3B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IC 50 , half-maximal inhibitory concentration.

Article Snippet: LC3B small interfering RNA (siRNA; catalogue no. sc-43390) and control siRNA (catalogue no. sc-37007) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Inhibition, Incubation, Transmission Assay, Electron Microscopy, Western Blot, Expressing, CCK-8 Assay, Concentration Assay

Journal: Oncology Letters

Article Title: Autophagy inhibition impairs the epithelial-mesenchymal transition and enhances cisplatin sensitivity in nasopharyngeal carcinoma

doi: 10.3892/ol.2017.5963

Figure Lengend Snippet: LC3B-knockdown impairs the epithelial-mesenchymal transition process in nasopharyngeal carcinoma 6–10B cells. (A) Western blot analysis showed that the basal level of LC3B was higher in 6–10B cells than in 5–8F cells. (B) Western blot analysis indicated that the expression of LC3B was effectively inhibited by siRNA in 6–10B cells. (C) Reverse transcription-quantitative PCR revealed that LC3B-knockdown upregulated E-cadherin mRNA expression and downregulated vimentin, Snail and Slug mRNA expression levels. mRNA levels were normalized to β-actin (*P<0.05 vs. siR-NT group). siR-NT, control small interfering RNA nucleotides; LC3B, microtubule-associated protein 1 light chain 3B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: LC3B small interfering RNA (siRNA; catalogue no. sc-43390) and control siRNA (catalogue no. sc-37007) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Knockdown, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Small Interfering RNA

Journal: PeerJ

Article Title: Titanium dioxide dental implants surfaces related oxidative stress in bone remodeling: a systematic review

doi: 10.7717/peerj.12951

Figure Lengend Snippet: A descriptive summary of the findings are tabulated based on the focused question.

Article Snippet: 5 , ( ) ) China , Study on potential toxic of titanium oxide nanoparticles on osteoblasts , Cell culture study , Osteoblast cells , Titanium oxide nanoparticles (TiO2-NPs) , less than 25 nm. , * The cellular and molecular cross talk in bone remodeling were not identified. * The effect of TiO 2 NPs induced oxidative stress on the osteogenesis-angiogenesis coupling in bone remodeling were not identified. , MTS reagent kit for cytoactive detection. LDH reagent kit for cytotoxicity detection. ROS detection reagent kit. RIPA cell lysis. Transmission electron microscopy (TEM). Zetasizer particle size analyzer Malvern Instrument, Ultrasonic oscillation instrument. Inverted phase contrast microscope. , ND , Osteogenesis , TiO 2 -NPs can cause the decrease of the survival rate of the osteoblast and increase of the content of the LDH released by the cell, and with dosage dependency effect , 71.66 Strong Evidence.

Techniques: In Vivo, In Vitro, Titanium Dioxide, Cell Culture, Lactate Dehydrogenase Assay, Flow Cytometry, Membrane, Permeability, RNA Extraction, Quantitative RT-PCR, Clinical Proteomics, Mass Spectrometry, Peroxidation Assay, Activity Assay, Glutathione Assay, FRAP Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Micro-CT, Staining, Immunohistochemical staining, Immunohistochemistry, Expressing, Electrophoresis, Colorimetric Assay, Lysis, Transmission Assay, Electron Microscopy, Microscopy, WST-1 Assay, Nitration, Immunoenzymatic Assay, Immunoprecipitation, Biomarker Discovery, Real-time Polymerase Chain Reaction, MTT Assay, Tube Formation Assay, Wound Healing Assay, ROS Assay, ATP Assay, X-ray Diffraction, BIA-KA, Confocal Laser Scanning Microscopy, Laser-Scanning Microscopy, Viability Assay, Immunofluorescence, Control, Inhibition, CCK-8 Assay, ALP Assay, Modification, Protein-Protein interactions

Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: DDX17 plays an important role in maintaining mitochondrial morphology and function in cardiomyocytes. a Transmission electron microscopy (TEM) of LVs from control (Con) and Ddx17 -cardiomyocyte-specific knockout (cKO) mice (scale bars: low-1 μm, medium-500 nm, high-200 nm) ( n = 5). b , c Statistical analysis of mitochondrial length and mitochondrial area in control (Con) and Ddx17 -cKO mice ( n = 48). d , e Mitochondrial membrane potential (ΔΨm) analyzed by JC-1 red/green fluorescence intensity in control (Con) and Ddx17 -overexpressing ( Ddx17 -OE) HL-1 cells treated with normoxic and hypoxic conditions ( n = 3); scale bar: 50 μm. f , g Mitochondrial permeability transition pore (mPTP) analyzed by calcein-AM fluorescence intensity in control (Con) and Ddx17 -overexpressing ( Ddx17 -OE) HL-1 cells ( n = 4); scale bar: 90 μm. h , i NMVMs were infected with Ddx17 -overexpressing adenovirus ( Ddx17 -OE) or its control (Con) for 24 h and then treated with PBS or Dox for 24 h. Mitochondria of cardiomyocytes were stained with Mito-Tracker Red and nuclei were stained with DAPI, and the rate of mitochondrial fission was analyzed by confocal microscopy ( n = 4); scale bar corresponds to 20 μm. j Cellular ATP concentration in the NMVMs of control (Con) and Ddx17 -cKO mice treated with PBS or Dox ( n = 4) . k Cellular ATP concentration in the NMVMs of control (Con) and Ddx17 -Tg mice treated with PBS or Dox ( n = 4) . l , m 8-OHdG and MDA levels in NMVMs from each group ( n = 3). n – r HL-1 cardiomyocytes were infected with Ddx17 overexpressing adenovirus ( Ddx17 -OE) or its control (Con) for 24 h and then treated with PBS or 0.5 μM Dox for 24 h. Based on the measured mitochondrial OCR of HL-l cells in response to 1 μM oligomycin, 1 μM FCCP and 0.5 μM rotenone/antimycin A, the basal respiration, maximal respiration, ATP production and spare respiratory capacity were measured using a Seahorse flux analyser ( n = 4). s Western blot of DRP1, MFN1 and MFN2 in the left ventricle of mice in the control + saline (Con + Saline), Ddx17 -cKO + saline (Ddx17 -cKO + Saline), control + doxorubicin (Con + Dox) and Ddx17 -cKO + doxorubicin ( Ddx17 -cKO + Dox) groups ( n = 6). t Western blot of DRP1, MFN1 and MFN2 in LVs from mice in the control + saline (Con + Saline), Ddx17 -transgene + saline ( Ddx17 -Tg + Saline), control + doxorubicin (Con + Dox) and Ddx17 -transgene+doxorubicin ( Ddx17 -Tg + Dox) groups ( n = 6). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Transmission Assay, Electron Microscopy, Control, Knock-Out, Membrane, Fluorescence, Permeability, Infection, Staining, Confocal Microscopy, Concentration Assay, Western Blot, Saline

Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: DDX17 coordinates with BCL6 in the transcriptional repression of the Drp1 gene in cardiomyocytes. a , b GO terms responding to promoters with higher ( a ) and lower ( b ) methylation levels in NMVMs from Ddx17 -Tg mice compared to controls (Con) ( n = 3). Representative genes are indicated below. c Motif enrichment analysis of proximal NDRs in Ddx17 -Tg and control (Con) NMVMs ( n = 3). d , e Co-IP experiments were performed with DDX17 ( d ) and BCL6 ( e ) antibodies to analyze the interaction of DDX17 and BCL6 in control (Con) and Ddx17 -cKO NMVMs ( n = 4). f Immunofluorescence staining of DDX17 (red), BCL6 (green) and nuclei (DAPI, blue) in cultured wild-type NMVMs; scale bar, 20 μm ( n = 3). g To investigate the regulatory effects of DDX17 and BCL6 overexpression on Drp1 promoter activity, HEK293A cells were transfected with Ddx17 (pcDNA- Ddx17 ) and/or Bcl6 (pcDNA- Bcl6 ) expression plasmids and simultaneously cotransfected with the Drp1 0.8-kb wild-type promoter (pGL3- Drp1 -WT) luciferase reporter plasmid, and Drp1 promoter activity was analyzed by luciferase assay ( n = 3). h HEK293A cells were transfected with Ddx17 (pcDNA- Ddx17 ) and/or Bcl6 (pcDNA- Bcl6 ) expression plasmids and simultaneously cotransfected with the 0.8 kb Drp1 promoter mutation plasmid (pGL3- Drp1 -MUT) with mutated BCL6 binding sites using the pGL3-basic plasmid ( n = 3). i HEK293A cells were cotransfected with the Drp1 promoter plasmid (pGL3- Drp1 -WT) and different concentrations of the Bcl6 expression plasmid (pcDNA- Bcl6 ) to detect Drp1 promoter activity ( n = 3). j ChIP analysis of NMVMs revealed the recruitment of BCL6 to regions containing BCL6 binding sites within the promoter region of Drp1 by quantitative real-time PCR ( n = 3). k – n HL-1 cells were transfected with Ddx17 siRNA and/or Drp1 siRNA for 24 h and then treated with doxorubicin for 24 h. Expression of DDX17, DRP1 and c-CASP-3 was analyzed by western blot, and GAPDH was used as a protein loading control ( n = 3). In all co-transfection experiments, pcDNA3.1 was used as the equilibrium plasmid in the different transfection mixtures to balance the total amount of DNA, and NCi was used as the equilibrium RNA in the different transfection mixtures to balance the total amount of RNA ( n = 3). o – q HL-1 cardiomyocytes were transfected with NCi (Con) or Ddx17 siRNA ( Ddx17 -KD) for 24 h and then treated with PBS or 0.5 μM Dox for 24 h. Cardiomyocyte mitochondria and cytoplasm were isolated and the expression of mitochondrial DRP1 (Mito-DRP1) and cytoplasmic DRP1 (Cyto-DRP1) was analyzed by western blot. COX IV and β-tubulin were used as protein loading controls for mitochondria and cytoplasm, respectively ( n = 4). r HL-1 cells were transfected with NCi (Con) and Ddx17 siRNA ( Ddx17 -KD) for 24 h, then treated with PBS or 0.5 μM Dox for 24 h and divided into Con, Ddx17 -KD, Con + Dox and Ddx17 -KD + Dox groups. Cardiomyocyte mitochondria and cytoplasm were extracted separately using a mitochondrial isolation kit. Cytochrome c levels in mitochondria and cytoplasm were analyzed by western blot. COX IV was used as a protein loading control for mitochondria and β-tubulin as a protein loading control for cytoplasmic proteins ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Methylation, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Cell Culture, Over Expression, Activity Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Cotransfection, Isolation

Journal: Signal Transduction and Targeted Therapy

Article Title: DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure

doi: 10.1038/s41392-024-01831-2

Figure Lengend Snippet: DDX17 expression levels are positively correlated with cardiac function in patients with different stages of heart failure. a H&E staining of myocardial tissue from the control (Con) and heart failure (HF) patients ( n = 3); scale bar, 100 μm. b Wheat germ agglutinin staining of tissue samples isolated from Con and HF patients ( n = 3); scale bar, 100 μm. c Transmission electron microscopy images of myocardial tissue from Con and HF patients ( n = 3); scale bar, 2 μm. d Correlation of the DDX17 mRNA levels with left ventricular EF in myocardial tissue from heart failure patients. e Correlation of DRP1 mRNA levels with left ventricular EF in myocardial tissue from heart failure patients. f Correlation of DDX17 with DRP1 mRNA levels in the myocardium of heart failure patients. g Graphic summary of DDX17 protecting cardiac function by promoting mitochondrial homeostasis through the BCL6-DRP1 pathway in heart failure

Article Snippet: Negative control (NCi, Cat. No. sc-37007, Santa Cruz, USA), mouse Ddx17 siRNA (Cat. No. sc-142922, Santa Cruz, USA) and Drp1 siRNA (Cat. No. sc-45953, Santa Cruz, USA) were purchased from Santa Cruz Co.

Techniques: Expressing, Staining, Control, Isolation, Transmission Assay, Electron Microscopy

Journal: JHEP Reports

Article Title: XBP1-mediated activation of the STING signalling pathway in macrophages contributes to liver fibrosis progression

doi: 10.1016/j.jhepr.2022.100555

Figure Lengend Snippet: XBP1 regulates macrophage activation in a STING-NLRP3-dependent manner. (A) Western blot was performed to analyse the levels of NLRP3 in liver macrophages isolated from Xbp1 FL/FL and Xbp1 M-KO mice treated with CCl 4 , BDL, or MCD. Statistical analysis was carried out using 1-way ANOVA. (B) Western blot was performed to analyse the levels of TLR4, p-IRE1α, XBP1, NLRP3, cleaved caspase-1, pro-caspase-1, cleaved IL-1β, and pro-IL-1β in Xbp1 FL/FL and Xbp1 M-KO BMDMs treated with LPS or LPS + ATP. Statistical analysis was carried out using 1-way ANOVA. (C) Western blot was performed to analyse the levels of STING in liver macrophages isolated from Xbp1 FL/FL and Xbp1 M-KO mice treated with CCl 4 , BDL, or MCD. Statistical analysis was carried out using 1-way ANOVA. (D) Western blot was performed to analyse the levels of cGAS, STING, p-TBK1, TBK1, p-IRF3, and IRF3 in Xbp1 FL/FL and Xbp1 M-KO BMDMs treated with LPS. Statistical analysis was carried out using 1-way ANOVA. (E) Immunofluorescence staining showing STING (green) and NLRP3 (red) colocalization in Xbp1 FL/FL and Xbp1 M-KO BMDMs stimulated with LPS. (F) Immunofluorescence was used to detect the expression of NLRP3 in LPS-stimulated Xbp1 M-KO BMDMs transfected with LV- Tmem173 or Sh- Irf3 . (G) The gene levels of Tnfa , Il6 , Il1b, and Cxcl10 in BMDMs; n = 3/group; 1-way ANOVA. (H) Schematic diagram of the putative IRF3 binding site within the Nlrp3 promoter. The BMDMs were subjected to ChIP assay with anti-IRF3 or IgG antibody. The representative results from 3 independent experiments are shown. (I) Relative Bnip3 mRNA expression in Xbp1 FL/FL BMDMs treated with Sh- Irf3 or KIN1148 with or without LPS stimulation were determined using qPCR; n = 3 biological replicates/group; 1-way ANOVA. (J) Western blot was performed to analyse the levels of p-IRF3 in liver macrophages isolated from Xbp1 FL/FL and Xbp1 M-KO mice treated with CCl 4 , BDL, or MCD. Statistical analysis was carried out using 1-way ANOVA. (K) Western blot was performed to analyse the levels of p-IRF3 in human normal or fibrotic liver tissue; Student t test. The values are shown as the mean ± SD. Statistical significance was assessed by Student t test or ANOVA. ∗∗ p <0.01; ∗ p <0.05. BDL, bile duct ligation; BMDMs, bone marrow-derived macrophages; BNIP3, BCL2/adenovirus E1B interacting protein 3; CCl 4 , carbon tetrachloride; ChIP, chromatin immunoprecipitation; cGAS, cyclic GMP-AMP synthase; IRE1α, inositol-requiring enzyme-1α; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; MCD, methionine/choline-deficient diet; NLRP3, nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3; STING, stimulator of interferon genes; TBK1, TANK binding kinase 1; TLR, toll-like receptor; TNF-α, tumour necrosis factor alpha; XBP1, X-box binding protein 1.

Article Snippet: In the CCl 4 group, the BCL2/adenovirus E1B interacting protein 3 ( Bnip3 ) siRNA or scramble siRNA (Santa Cruz, CA, USA) was mixed with mannose-conjugated polymers (Polyplus-transfection, Illkirch, France) according to the manufacturer’s instructions, and injected into the mice via the tail vein (2 mg/kg) every 3 days beginning 2 weeks after CCl 4 injections.

Techniques: Activation Assay, Western Blot, Isolation, Immunofluorescence, Staining, Expressing, Transfection, Binding Assay, Ligation, Derivative Assay, Chromatin Immunoprecipitation

Journal: JHEP Reports

Article Title: XBP1-mediated activation of the STING signalling pathway in macrophages contributes to liver fibrosis progression

doi: 10.1016/j.jhepr.2022.100555

Figure Lengend Snippet: XBP1 deficiency promotes mitophagy activation in macrophages. (A) KEGG pathway enrichment analysis of the differentially expressed pathways in LPS-stimulated Xbp1 FL/FL and Xbp1 M-KO BMDMs. (B) Heat map showing the clustering of 25 upregulated genes that affect mitophagy in response to LPS according to genotype. (C) Relative Bnip3 mRNA expression in LPS-stimulated Xbp1 FL/FL and Xbp1 M-KO BMDMs were determined using qPCR; n = 3 biological replicates/group; Student t test. (D) Autophagic microstructures in BMDM mitochondria were examined by transmission electron microscopy, 5,000 × magnification; scale bars, 2 μm. Arrowheads, mitophagy. Representative of 3 experiments. (E) Western blot was performed to determine intracellular LC3B, p62, PINK1, Parkin, and BNIP3 protein levels in LPS-stimulated Xbp1 FL/FL and Xbp1 M-KO BMDMs. Statistical analysis was carried out using 1-way ANOVA. (F) Relative Bnip3 mRNA expression in LPS-stimulated Xbp1 FL/FL BMDMs treated with or without toyocamycin or tunicamycin were determined using qPCR; n = 3 biological replicates/group. Statistical analysis was carried out using 1-way ANOVA. (G) Schematic diagram of the putative XBP1 binding site within the Bnip3 promoter. The BMDMs were subjected to ChIP assay with anti-XBP1 or IgG antibody. Representative results from 3 independent experiments are shown. (H) Luciferase activity in BMDMs co-transfected with Bnip3 -promoter luciferase reporter plasmid and with the increasing multiplicity of infection of Ad- sXbp1 or Ad-CON. n = 3/group. Statistical analysis was carried out using 1-way ANOVA. (I) The expression levels of Bnip3 in liver tissue from mice with CCl 4 -, BDL-, and MCD-induced liver fibrosis were examined using quantitative real-time PCR; n = 6 mice/group; 1-way ANOVA. (J) The correlation between XBP1 levels and BNIP3 expression in human fibrotic liver tissue was assessed using Pearson’s correlation analysis; n = 54. The values are shown as the mean ± SD. Statistical significance was assessed by the Student t test or ANOVA. ∗∗ p <0.01; ∗ p <0.05. BDL, bile duct ligation; BMDMs, bone marrow-derived macrophages; BNIP3, BCL2/adenovirus E1B interacting protein 3; CCl 4 , carbon tetrachloride; ChIP, chromatin immunoprecipitation; KEGG, Kyoto Encyclopedia of Genes and Genomes; LPS, lipopolysaccharide; MCD, methionine/choline-deficient diet; XBP1, X-box binding protein 1.

Article Snippet: In the CCl 4 group, the BCL2/adenovirus E1B interacting protein 3 ( Bnip3 ) siRNA or scramble siRNA (Santa Cruz, CA, USA) was mixed with mannose-conjugated polymers (Polyplus-transfection, Illkirch, France) according to the manufacturer’s instructions, and injected into the mice via the tail vein (2 mg/kg) every 3 days beginning 2 weeks after CCl 4 injections.

Techniques: Activation Assay, Expressing, Transmission Assay, Electron Microscopy, Western Blot, Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Ligation, Derivative Assay, Chromatin Immunoprecipitation

Journal: JHEP Reports

Article Title: XBP1-mediated activation of the STING signalling pathway in macrophages contributes to liver fibrosis progression

doi: 10.1016/j.jhepr.2022.100555

Figure Lengend Snippet: BNIP3-mediated mitophagy activation decreases mtDNA release and STING-NLRP3 activation in Xbp1 -deficient macrophages. (A) Western blot was performed to examine intracellular BNIP3, LC3B, and p62 protein levels in LPS-stimulated Xbp1 M-KO BMDMs transfected with SCR-siRNA or Bnip3 -siRNA. Statistical analysis was carried out using the Student t test. (B) Autophagic microstructures in mitochondria in LPS-stimulated Xbp1 M-KO BMDMs transfected with SCR-siRNA or Bnip3 -siRNA were examined by transmission electron microscopy, 5,000 × magnification; scale bars, 2 μm. Arrowheads, mitophagy. (C) The levels of ROS in LPS-stimulated Xbp1 M-KO BMDMs transfected with SCR-siRNA or Bnip3 -siRNA were examined by DCFH-DA (original magnification 200 × ). (D) Relative total mtDNA amounts in LPS-stimulated Xbp1 M-KO BMDMs transfected with SCR-siRNA or Bnip3 -siRNA were determined using qPCR with primers specific for mtDNA ( D-loop ) and nuclear DNA ( Tert ); n = 3 biological replicates/group; Student t test. (E) mtDNA released from mitochondria in Xbp1 M-KO BMDMs transfected with SCR-siRNA or Bnip3 -siRNA, as shown by confocal microscopy. Arrowheads, mtDNA released into cytoplasm. (F) The mitochondrial membrane potential of Xbp1 M-KO BMDMs transfected with SCR-siRNA or Bnip3 -siRNA was determined by TMRM staining. (G) The levels of ROS in LPS-stimulated Xbp1 FL/FL BMDMs treated with PBS or MitoTEMPO were examined using DCFH-DA (original magnification 200 × ). (H) Relative amounts of total cytosolic mtDNA in LPS-stimulated Xbp1 FL/FL BMDMs treated with PBS or MitoTEMPO were determined using qPCR with primers specific for mtDNA ( D-loop ) and nuclear DNA ( Tert ); n = 3 biological replicates/group; Student t test. (I) Schematic showing the administration protocol for mannose-conjugated SCR-siRNA and Bnip3 -siRNA in CCl 4 -, BDL-, and MCD-induced murine liver fibrosis models for the experiments shown in (J). (J) Xbp1 M-KO male mice were subjected to CCl 4 -, BDL-, or MCD-induced experimental fibrosis and injected with SCR-siRNA or Bnip3 -siRNA via the tail vein, and the collected livers were subjected to H&E and Sirius Red staining and α-SMA immunohistochemical analysis; scale bar=100 μm. Representative of 6 mice/group. (K) The proportions of the Sirius Red- and α-SMA-positive areas were quantified; n = 6 mice/group; 1-way ANOVA. The values are shown as mean ± SD. Statistical significance was assessed by Student t test or ANOVA. ∗∗ p <0.01. BMDMs, bone marrow-derived macrophages; BNIP3, BCL2/adenovirus E1B interacting protein 3; LC3B, microtubule-associated protein 1 light chain 3 beta; LPS, lipopolysaccharide; NLRP3, nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3; p62, sequestosome 1; ROS, reactive oxygen species; STING, stimulator of interferon genes; XBP1, X-box binding protein 1.

Article Snippet: In the CCl 4 group, the BCL2/adenovirus E1B interacting protein 3 ( Bnip3 ) siRNA or scramble siRNA (Santa Cruz, CA, USA) was mixed with mannose-conjugated polymers (Polyplus-transfection, Illkirch, France) according to the manufacturer’s instructions, and injected into the mice via the tail vein (2 mg/kg) every 3 days beginning 2 weeks after CCl 4 injections.

Techniques: Activation Assay, Western Blot, Transfection, Transmission Assay, Electron Microscopy, Confocal Microscopy, Membrane, Staining, Injection, Immunohistochemical staining, Derivative Assay, Binding Assay

Journal: Nucleic acids research

Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

doi: 10.1093/nar/gkw1208

Figure Lengend Snippet: Figure 1. Establishing MTX concentrations that induce DNA breaks and the effect of MTX on DNA. (A) Analysis of the cell cycle in mESCs with different doses of MTX after 24 h of treatment, by flow cytometry. The results were graded according to the cell cycle phase. (B) Rate of apoptosis in mESCs at different doses of MTX after 24 h of treatment, by flow cytometry. (C) Comet assays in mESCs at different doses of MTX after 24 h of treatment. The length of the DNA tail and tail %DNA for each dose of MTX are shown. Values are means ± SD (standard deviation) derived from three independent experiments. ** Indicates a statistically significant difference compared with the control (P < 0.01) based on Student’s two-tailed t-test. (D) Representative images of nuclei with gH2AX foci in mESCs following 0.12 M MTX for 24 h, by immunofluorescent staining gH2AX (panel 2). Nuclear DNA was stained by DAPI (panel 1). Merged images (panel 3) are shown. One representative nucleus with associated merged images (panel 4) are shown. Red: gH2AX; blue: DAPI staining. Bar, 5 M.

Article Snippet: The bands were detected using an enhanced chemiluminescence technique (Amersham Biosciences). siRNA transfection mESCs were transfected with UBF siRNA (sc-29515, Santa Cruz) targeting mouse UBF1/2.

Techniques: Flow Cytometry, Standard Deviation, Derivative Assay, Control, Two Tailed Test, Staining

Journal: Nucleic acids research

Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

doi: 10.1093/nar/gkw1208

Figure Lengend Snippet: Figure 2. DSBs enrichment workflow and specificity of the DNA DSBs (A) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. (B) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). (C) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. (D–F) Capillary electrophoresis to detect DSB enrichment products after SbfI (D), PmeI (E) and HindIII (F) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). (G, H) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium (G) and cultured in complete medium with 0.12 M MTX (H). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).

Article Snippet: The bands were detected using an enhanced chemiluminescence technique (Amersham Biosciences). siRNA transfection mESCs were transfected with UBF siRNA (sc-29515, Santa Cruz) targeting mouse UBF1/2.

Techniques: Control, Amplification, Sequencing, In Situ, Electrophoresis, Agarose Gel Electrophoresis, Fluorescence, Labeling, Ligation, Cell Culture

Journal: Nucleic acids research

Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

doi: 10.1093/nar/gkw1208

Figure Lengend Snippet: Figure 3. Genome-wide hot spots of DSBs in rRNA genes units in MTX-treated mESCs (A) The relative proportion of reads of DSBs in chromosomes of mESCs from the MTX and control obtained by Illumina sequencing. (B) Genome-wide distribution of DSB sites induced by MTX. (C) Genome-wide MTX sensitivity landscape of DSBs in each chromosome in mESCs by hypergeometric test and visualized using Circos. (D) Significantly enriched DSBs in rRNA genes in mESCs compared to the rest of the genome calculated the fold-change in the number of reads. (E) Distribution of DSBs peak in the IGS regions of rRNA genes in mESCs detected with F-seq in complete medium and in complete medium with 0.12 M MTX for 24 h. The control is in black and the treated is in red. X-axis represents the rRNA gene base number, Y-axis represents the read counts.

Article Snippet: The bands were detected using an enhanced chemiluminescence technique (Amersham Biosciences). siRNA transfection mESCs were transfected with UBF siRNA (sc-29515, Santa Cruz) targeting mouse UBF1/2.

Techniques: Genome Wide, Control, Illumina Sequencing

Journal: Nucleic acids research

Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

doi: 10.1093/nar/gkw1208

Figure Lengend Snippet: Figure 4. Verification of hot spots of DSBs in rRNA genes units and the H3K4me1 modifications correlated with them (A) Schematic representation of a mouse rRNA genes repeat unit. Black squares represent regions that do not break and white squares represent hot spots of DSBs. Primer pairs (solid bars) and their approximate positions relative to the transcription start site are indicated. (B) Enrichment of gH2AX in rRNA genes breakage sites obtained with anti-gH2AX. The occupancy was determined by chromatin cross-linking and immunoprecipitation (ChIP) analysis using chromatin prepared from mESCs cultured in complete medium and complete medium with 0.12 M MTX for 24 h. Chromatin DNA was quantitated by real-time PCR with primer sets, as indicated in (A). Values are means ± SD (standard deviation) derived from three independent experiments. *P < 0.05; **P < 0.01. (C) Binding of four histone modifications (H3K27me3, H3K9me3, H3K4me3, and H3K4me1) to the DSBs in rRNA genes after 0.12 M MTX treatment compared with the control, determined by ChIP-qPCR analysis. *P < 0.05; **P < 0.01. (D) Expression changes of histone methyltransferases (MLL3, MLL4) and demethylases (KDM5b, KDM5c) in mESCs cultured in complete medium and complete medium with 0.12 M MTX for 24 h, by RT-qPCR. Assays were performed in triplicate and the mean ± SD was calculated. *P < 0.05; **P < 0.01. (E) Comparison of H3K4me1 binding and DSBs peak in rRNA genes units detected with F-seq. The raw data of H3K4me1 in mESCs were from SRR002255 ES H3K4me1 ChIP-Seq (UCSC Genome Browser). Thin red lines show the position of overlapped DSBs and H3K4me1 inside the IGS regions. X-axis represents the rRNA gene base number, Y-axis represents the read counts. (F) Correlation heatmap of pairwise comparisons using GenometriCorr R package between median signals for DSBs, H3K27me3, H3K9me3, H3K4me3 and H3K4me1 in the entire rRNA genes unit detected with F-seq.

Article Snippet: The bands were detected using an enhanced chemiluminescence technique (Amersham Biosciences). siRNA transfection mESCs were transfected with UBF siRNA (sc-29515, Santa Cruz) targeting mouse UBF1/2.

Techniques: Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation, Derivative Assay, Binding Assay, Control, ChIP-qPCR, Expressing, Quantitative RT-PCR, Comparison, ChIP-sequencing

Journal: Nucleic acids research

Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

doi: 10.1093/nar/gkw1208

Figure Lengend Snippet: Figure 5. MTX promotes rRNA transcription and UBF specifically binds to DSB regions of rRNA genes (A) Binding of UBF to the DSBs in rRNA genes unit after 0.12 M MTX treatment compared with the control, determined by ChIP-qPCR analysis. *P <0.05; **P <0.01. (B) Detection of UBF expression in mESCs cultured in complete medium and complete medium with 0.12 M MTX treatment, by western blotting. -Tubulin was used as a loading control. Positions of molecular weight markers are indicated. *P <0.05. (C) Expression levels of 45S pre-rRNA in mESCs cultured in complete medium with 0.12 M MTX for 3, 6, 12 or 24 h, determined by RT-qPCR. The assays were performed in triplicate and the mean ± SD was calculated. **P <0.01. (D) Detection of ongoing rRNA synthesis by FUrd incorporation assays on mESCs subjected to 0.12 M MTX for 24 h. Transcription was monitored by a FUrd pulse to observe incorporation into nascent nucleolar transcripts. FUrd detection with BrdU antibody was by confocal microscopy (panel 2). Nuclear DNA was stained by DAPI (panel 1). Merged images (panel 3) are shown. One representative nucleus with associated merged images (panel 4) are shown. Green: FUrd; blue: DAPI staining. Bar, 5 M. Relative intensity of FUrd foci and the number of nucleoli per cell with FUrd foci were analyzed. The histogram shows the average number of nucleoli per cell based on an analysis of 93 cells from two independent experiments. (E) Co- localization between nucleolar proteins and rRNA transcripts by double-stained preparations of nucleolar markers (panel 2, Pol-I largest subunit A194) and FUrd pulse (panel 3, BrdU antibody).Nuclear DNA was stained by DAPI(panel 1).Merged images (panel 4) are shown. Green: A194(Pol-I); red: FUrd; blue: DAPI staining. Bar, 5 M. (F) Co-localization between rRNA transcripts and UBF by double-stained preparations of FUrd pulse (panel 2, BrdU antibody) and UBF(panel3, upstream binding factor antibody).Nuclear DNA was stained by DAPI (panel 1). Merged images (panel 4) are shown. Green: UBF; red: FUrd; blue: DAPI staining. Bar, 5 M. (G) Efficiency of siRNA mediated UBF depletion on mRNA level. The levels of the mRNAs UBF were analysed in mESCs with either a non-targeting siRNA (control siRNA) or siRNA directed against UBF(UBF siRNA). The assays were performed in triplicate and the mean ± SD was calculated. (H) Efficiency of siRNA mediated UBF depletion on protein level. mESCs were transfected with either control siRNA or UBF siRNA and UBF1/2 were determined 72 h post-transfection by Western-blotting. -Tubulin was used as a loading control. Positions of molecular weight markers are indicated. (I) Expression levels of 45S pre-rRNA in mESCs with control siRNA or UBF siRNA cultured in complete medium with 0.12 M MTX for 24 h, determined by RT-qPCR. The assays were performed in triplicate and the mean ± SD was calculated. **P <0.01.

Article Snippet: The bands were detected using an enhanced chemiluminescence technique (Amersham Biosciences). siRNA transfection mESCs were transfected with UBF siRNA (sc-29515, Santa Cruz) targeting mouse UBF1/2.

Techniques: Binding Assay, Control, ChIP-qPCR, Expressing, Cell Culture, Western Blot, Molecular Weight, Quantitative RT-PCR, Confocal Microscopy, Staining, Transfection

Journal: Nucleic acids research

Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

doi: 10.1093/nar/gkw1208

Figure Lengend Snippet: Figure 6. Effects of supplementary folinic acid on cell cycle and DSBs induced by MTX (A) mESC colony formation was partially reversed by folinic acid. mESCs were cultured in complete medium, complete medium with 0.12 M MTX, and supplementary folinic acid (50 mg/L) for 24 h. The colonies were photographed at 100× resolution. Representative fields are shown. (B) Comet assay in mESCs by 0.12 M MTX treatment and after supplementary folinic acid treatment. The length of DNA tail and tail %DNA are shown for each treatment. Values are means ± (standard deviation) derived from three independent experiments. ** Indicates statistically significant difference from control (P < 0.01) based on Student’s two-tailed t-test. (C) Fraction of gH2AX positive cells in the indicated phase of the cell cycle following 0.12 M MTX for 24 h or supplementary folinic acid. Data are from three independent experiments. (D) H3K4 methylation states at representative sites of DSBs in rRNA genes unit after supplementary folinic acid compared with MTX treatment alone, as determined by ChIP-qPCR. Values are means ± SD derived from three independent experiments. *P < 0.05; **P < 0.01. (E) Binding of UBF binding to the DSBs in rRNA genes unit after supplementary folinic acid compared with the MTX treatment alone, by ChIP-qPCR analysis. *P < 0.05; **P < 0.01. (F) Expression levels of 45S pre-rRNA determined by RT-qPCR in mESCs cultured in complete medium with 0.12 M MTX for 24 h and supplementary folinic acid. The assays were performed in triplicate and the mean ± SD was calculated. **P < 0.01.

Article Snippet: The bands were detected using an enhanced chemiluminescence technique (Amersham Biosciences). siRNA transfection mESCs were transfected with UBF siRNA (sc-29515, Santa Cruz) targeting mouse UBF1/2.

Techniques: Cell Culture, Single Cell Gel Electrophoresis, Standard Deviation, Derivative Assay, Control, Two Tailed Test, Methylation, ChIP-qPCR, Binding Assay, Expressing, Quantitative RT-PCR