Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: Effect of GATA4 on odontoblasts polarization, cell proliferation and secretion of root dentin matrix. ( A ) H&E-stained sections of the mandibular first molars showed that the odontoblasts have shorter height and flattened morphology in Wnt1-Cre;GATA4 fl/fl mice at P14 and P21 (Bar: 50 μm). ( B-E ) Immunohistochemistry staining images showing expression levels of DSPP (B), COL-1 (C), DCN (D), and PCNA (E) in root of Wnt1-Cre;GATA4 fl/fl mice at P14. ( F ) The height of odontoblasts was measured. Quantitative assessment of the molar root odontoblasts height from (A) at P14 and P21. ( G-J ) Percentages of DSPP (G), COL-1 (H), DCN (I), and PCNA-positive (J) cells in the control and mutant groups were calculated. ( K ) Double-labeled fluorescent immunostaining of DAPI-stained cell nuclei (blue), GFP (green), and merged images in tooth root at P17 after the injetion in vivo (Bar: 50 μm). ( L ) H&E-stained sections of the mandibular first molars showed that root dentin thickness was increased in GATA4 OE group mice at P17 (Bar: 50 μm). ( M ) Quantitative assessment of the molar root dentin thickness at P17. ( N ) Expression of GATA4 after lentivirus injected at P17 (Bar: 100 μm). ( O ) Percentages of GATA4-positive cells in the two groups were calculated. ( P ) Immunohistochemistry staining images showing expression levels of DSPP, COL-1, and DCN in root of mice at P17. ( Q ) Percentages of DSPP, COL-1, and DCN-positive cells in the two groups were calculated. Data expressed as the mean ± standard deviation, n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Staining, Immunohistochemistry, Expressing, Control, Mutagenesis, Labeling, Immunostaining, In Vivo, Injection, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: Characterization of DPSCs and expression of GATA4 in DPSCs. ( A ) Flow chart explaining cells isolation, culture and collection for FCM analyses. ( B, C ) Flow cytometry demonstrated that DPSCs expressed mesenchymal markers (CD44 and CD90) at a high level and generated the hematopoietic makers (CD14 and CD45) at a low level. ( D ) After mineralization for 3, 7, and 14 days, GATA4 protein expression was assessed at the indicated time points by western blotting. ( E ) DPSCs infected with lentivirus as assessed by fluorescence microscopy (Bar: 50 μm). ( F ) Efficiency of GATA4 knockdown after infection with lentivirus was analysed by western blotting. ( G ) Quantitative analysis of western blotting bands from (D) is shown as the ratio of GATA4 to GAPDH. ( H ) Quantitative analysis of western blotting bands from (F) is shown as the ratio of GATA4 to GAPDH. Data expressed as the mean ± standard deviation, n = 3. ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Isolation, Flow Cytometry, Generated, Western Blot, Infection, Fluorescence, Microscopy, Knockdown, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: Effect of GATA4 on migration, proliferation and odonto/osteogenic differentiation of DPSCs. ( A ) Effect of GATA4 knockdown on cell migration was assessed by wound scratch assays (Bar: 100 μm). ( B ) Effect of GATA4 knockdown on cell migration was assessed by transwell assay (Bar: 100 μm). ( C ) ALP staining observed after 7 days of mineralization (Bar: 100 μm). ( D ) After mineralization for 14 days, alizarin red staining was performed and observed with an image scanner (upper) and under a microscope (lower) (Bar: 100 μm). ( E ) The CCK8 assay was used to analyse the proliferation of DPSCs after infection with GATA4 lentivirus. ( F ) Quantitative assessment of ALP-positive areas. ( G ) Semi-quantitative estimation of calcium. ( H ) Expression levels of odonto/osteogenic-related genes (DSPP, BMP4, RUNX2, OSX, OPN, and OCN) were assessed by western blotting. ( I ) Quantitative analysis of western blotting bands from (H). ( J ) Expressions of odonto/osteogenic markers (Dspp, Dmp1, Col1a1, Bmp4, Runx2, Osx, Ocn, and Alp) were assessed by qRT-PCR. (Bar: 100 μm). Data expressed as the mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Migration, Knockdown, Transwell Assay, Staining, Microscopy, CCK-8 Assay, Infection, Expressing, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: Overexpression of GATA4 in DPSCs increased the odonto/osteogenic ability. ( A ) DPSCs infected with lentivirus control and pcDNA-GATA4 were observed under a fluorescence microscope (Bar: 50 μm). ( B ) Protein expression of GATA4 in the DPSCs was tested by western blotting after overexpression of GATA4. ( C ) Quantitative analysis of western blotting bands from (B). ( D ) ALP staining was observed after 7 days of mineralization. ( E ) ARS staining was performed 14 days after mineralization (Bar: 100 μm). ( F ) Quantitative assessment of ALP-positive areas after 7 days of osteogenic induction. ( G ) Semi-quantitative estimation of calcium. Data expressed as the mean ± standard deviation; n = 3. ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Over Expression, Infection, Control, Fluorescence, Microscopy, Expressing, Western Blot, Staining, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: GATA Binding Protein 4 Regulates Tooth Root Dentin Development via FBP1

doi: 10.7150/ijbs.36567

Figure Lengend Snippet: GATA4 enhanced glycolysis by negatively regulating FBP1 in DPSCs. ( A ) Co-immunoprecipitated proteins were then separated using SDS-PAGE and stained. ( B ) Mass spectrometry followed by peptide sequencing identified the two proteins as fructose-1,6-bisphosphatase 1 (FBP1) and isoform CRA_d. ( C ) Immunofluorescence staining revealed that GATA4 co-localizes with FBP1 in the nucleus in DPSCs (Bar: 100 μm). ( D ) Expression pattern of GATA4 during tooth development at embryonic day 13.5 (E13.5), E14.5, E15.5, P1, and P14 (Bar: 50 μm). ( E ) FBP1 expression was tested by western blotting after infection with GATA4 lentivirus in DPSCs. ( F ) Quantitative analysis of western blotting bands from (E). ( G ) FBP1 expression was tested by western blotting after GATA4 overexpression in DPSCs. ( H ) Quantitative analysis of western blotting bands from (G). ( I, J ) knockdown of GATA4 resulted in decreased glucose consumption and lactate production. ( K, L ) Overexpression of GATA4 resulted in increased glucose consumption and lactate production. Data expressed as the mean ± standard deviation; n = 3. ** P < 0.01.

Article Snippet: Recombinant lentivirus of shRNA target GATA4 (shGATA4; 5′-GAATAAATCTAAGACACCA-3′), control lentivirus (shCTRL; 5′-TTCTCCGAACGTGTCACGT-3′), lentivirus to overexpress GATA4 (pcDNA-GATA4) and blank lentivirus were purchased from GenePharma (Shanghai, China).

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Sequencing, Immunofluorescence, Expressing, Western Blot, Infection, Over Expression, Knockdown, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: CCDC137 knockdown suppresses bladder cancer progression by downregulating SCD

doi: 10.1186/s12967-025-07033-w

Figure Lengend Snippet: Exploration of potential downstream mechanism and upstream regulators of CCDC137. A Heatmap shows DEGs identified by RNA sequencing in T24-shCtrl and T24-shCCDC137-1 cells. B Bar plot displays GO and KEGG functional enrichment results of downregulated DEGs from RNA sequencing. C GSVA-Hallmark pathway enrichment analysis displayed differences between CCDC137 positive (CCDC137 +) and CCDC137 negative (CCDC137 −) cells based on single-cell sequencing data. D mRNA and protein expression of stearoyl-CoA desaturase (SCD) were detected by qRT-PCR and Western blot. E DecoupleR was used to analyze differences in transcription factor activity between CCDC137 + and CCDC137 − epithelial cells in single-cell sequencing data. F TF-Target Finder was employed to identify potential upstream transcription factors of CCDC137. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, no statistical significance

Article Snippet: The shCCDC137 lentiviral particles and corresponding control lentiviral particles (shCtrl) were purchased from Genechem Co., Ltd (Shanghai, China).

Techniques: RNA Sequencing, Functional Assay, Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Journal of Translational Medicine

Article Title: CCDC137 knockdown suppresses bladder cancer progression by downregulating SCD

doi: 10.1186/s12967-025-07033-w

Figure Lengend Snippet: In vivo validation of CCDC137 regulating tumor growth. A Subcutaneous xenograft models were established in nude mice to validate the regulatory role of CCDC137 in tumor growth. B Tumor volume growth curves were plotted over 28 days after tumor cell inoculation. C Tumor weights of T24-shCtrl and T24-shCCDC137-1 groups were measured on day 28. D The Graphical Abstract described the key results in this study. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, no statistical significance

Article Snippet: The shCCDC137 lentiviral particles and corresponding control lentiviral particles (shCtrl) were purchased from Genechem Co., Ltd (Shanghai, China).

Techniques: In Vivo, Biomarker Discovery

Journal: bioRxiv

Article Title: DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.1101/865626

Figure Lengend Snippet: (A) Schematic of DECR1 function in fatty acid (FA) β-oxidation. In order to translocate FAs into the mitochondria, CPT1 converts long-chain acyl-CoA species to their corresponding long-chain acylcarnitine species. This is followed by a dehydrogenation step mediated by acyl CoA dehydrogenase (ACAD) to generate trans-2-enoyl-CoA, the only intermediate that can be processed by downstream enzymes in the β-oxidation process. Many FAs have unsaturated bonds either on an odd-numbered carbon or in the cis-configuration, resulting in the generation of enoyl-CoA intermediates that cannot be directly processed via the downstream β-oxidation enzymes. These FAs require the activity of 3 auxiliary enzymes, ECI1, ECH1 and DECR1 in order to form trans-2-enoyl-CoA before undergoing β-oxidation. DECR1 catalyzes the conversion of either 2-trans,4-cis-dienoyl or 2-trans,4-trans-dienoyl-CoA to 3-trans-enoyl-CoA. A complete cycle of β-oxidation results in the release of the first two carbon units as acetyl-CoA, and a fatty-acyl-CoA minus two carbons. The acetyl-CoA enters the TCA cycle to produce energy (ATP). The shortened fatty-acyl-CoA is processed again starting with the ACADs to form trans-2-enoyl-CoA either directly or with the aid of the auxiliary enzymes depending on the presence of double bonds. This process continues until all carbons in the fatty acid chain are turned into acetyl-CoA. (B) DECR1 protein expression after 72 hours or 96 hours siRNA transfection. Densitometry quantification of relative DECR1 protein expression was normalized to the HSP90 internal control. (C) Linoleic acid level in LNCaP cells quantified in following 96 hours DECR1 knockdown using GC QQQ targeted metabolomics. (D) Relative quantities of the C10:2 acylcarnitine species in LNCaP cell conditioned medium (left) or cell lysates (right) (n=3). (E) Quantification of ATP levels in LNCaP cell lysates. LNCaP cells were transfected with DECR1 siRNAs for 48 hours and then starved in no-glucose medium and treated with the lipolysis inhibitor DEUP (100µM) in the presence (BSA-LA) or absence (BSA) of the PUFA linoleic acid for 48 hours before measuring ATP levels. ( F) Oxygen consumption rate (OCR) was assessed in LNCaP cells supplemented with the PUFA linoleic acid (LA) or (G) the saturated fatty acid palmitic acid (PA). Each data point represents an OCR measurement. ATP production, maximal mitochondrial respiration and mitochondrial spare capacity were assessed. (H) Extracellular acidification rate (ECAR) was assessed in LNCaP cells. Each data point represents an ECAR measurement. For experiments (F-H) LNCaP cells were transfected with DECR1 siRNAs for 72 hours, then starved in substrate limited medium for 24 hours; the assay was run in FAO assay medium. (I & J) Metabolites were quantified in LNCaP cells following 96 hours DECR1 knockdown using GC QQQ targeted metabolomics. Data in bar graphs are represented as the mean ± s.e.m (n=3). Statistical analysis was performed using two-tailed Student’s t -test: * p <0.05, ** p <0.01 and **** p <0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc. (San Diego, CA, USA) according to the manufacturer’s protocol.

Techniques: Activity Assay, Expressing, Transfection, Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.1101/865626

Figure Lengend Snippet: (A) Cell viability after DECR1 knockdown in non-malignant PNT1 prostate cells; hormone-responsive PCa cell lines (LNCaP and VCaP); castrate-resistant V16D and 22RV1 cell lines and enzalutamide-resistant MR94F cells cultured in full serum media. (B) Cell viability of stable DECR1-overexpressed LNCaP cells cultured in full serum media. Cell viability and cell death were measured using trypan blue exclusion following 96 hours DECR1 knockdown. Percentages are represented relative to the control siRNA; n = 3 independent experiments per cell line. (C) Clonogenic cell survival of LNCaP cells were assessed using colony formation assay. Stable DECR1-overexpressed cells or (D) stable DECR1 knockdown was achieved using two different short hairpin (sh) vectors and DECR1 expression was confirmed using western blot. Cells were cultured for 2 weeks, washed with PBS, fixed with paraformaldehyde and stained with 1% crystal violet for 30 minutes. Colonies with more than 50 cells were counted manually; data shown is representative of n = 2 independent experiments. (E) LNCaP and 22RV1 cell growth in 3D spheres. Spheroids were prepared using the hang drop assay following 48 hours DECR1 knockdown. Spheroid volumes were determined after five days of culturing the cells in 20 µl drops; at least 25 spheres per cell line were assessed using the ReViSP software, n = 3 independent experiments per cell line. (F) LNCaP, 22RV1 and MR49F cell migration and (G) 22RV1 cell invasion were assessed using transwell migration/invasion assay. Cells were transfected with DECR1 siRNA or control siRNA for 48 hours. Equal number of cells were transferred to the upper inserts in serum free medium; lower chambers were filled with medium containing 5% serum as a chemoattractant. Plates were incubated for a further 48 hours. Migrated/invaded cells on the lower face of the inserts were washed with PBS, fixed with paraformaldehyde, stained with 1% crystal violet for 30 minutes, and counted manually; data shown is representative of n = 3 independent experiments. (H) Violin plots of mKi67 and DECR1 mRNA expression in LNCaP tumors (n = 5 mice, shControl; n = 4 mice, shDECR1). (I) Representative KI67 IHC staining of LNCaP tumors. Scale bar, 100µm. Data in bar graphs are represented as the mean ± s.e.m. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test: * p <0.05, ** p <0.01, *** p <0.001 and **** p <0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc. (San Diego, CA, USA) according to the manufacturer’s protocol.

Techniques: Knockdown, Cell Culture, Control, Colony Assay, Expressing, Western Blot, Staining, Software, Migration, Invasion Assay, Transfection, Incubation, Immunohistochemistry

Journal: Stem Cell Research & Therapy

Article Title: CD90 serves as differential modulator of subcutaneous and visceral adipose-derived stem cells by regulating AKT activation that influences adipose tissue and metabolic homeostasis

doi: 10.1186/s13287-019-1459-7

Figure Lengend Snippet: CD90 silencing inhibits mitotic clonal expansion of S-ADSCs to influence adipocyte differentiation. S-ADSCs from mice ( n = 10) were infected with shCD90 or shControl lentivirus for 72 h and then were subjected to adipogenic induction. a The mRNA level of CD90 was detected by qPCR before adipogenic induction. b , c Mitotic clonal expansion was determined by EdU incorporation assay after 16 h of adipogenic induction. Fluorescence signals were detected by fluorescence microscope ( b ). Scale bar 100 μm. Growth indices as specified in Fig. are shown ( c ). d , e After 18 days of adipogenic induction, lipid contents were visualized using Oil Red O staining ( d ) and quantified by eluting Oil Red O ( e ). Scale bar 50 μm. f The mRNA levels of white adipocyte markers were measured by qPCR at days 0, 4, 8, and 12 during the induction. Data are presented as mean ± SEM. n = 4–6 per group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: In some experiments, S-ADSCs infected with shRNA CD90 (shCD90) or control shRNA (shControl) recombinant lentivirus (GenePharma, shanghai, China) were subjected to adipogenic induction.

Techniques: Infection, Fluorescence, Microscopy, Staining

Journal: Stem Cell Research & Therapy

Article Title: CD90 serves as differential modulator of subcutaneous and visceral adipose-derived stem cells by regulating AKT activation that influences adipose tissue and metabolic homeostasis

doi: 10.1186/s13287-019-1459-7

Figure Lengend Snippet: CD90 silencing causes glucose intolerance and adipocyte hypertrophy in mice. a , b S-ADSCs were infected with GFP-tagged shControl or shCD90 lentivirus (Genechem). The infection efficiency was determined by GFP signals under fluorescence microscope ( a ). BF, bright field. Scale bar = 100 μm. The mRNA level of CD90 was detected by qPCR ( b ). c GFP-tagged shControl lentivirus was injected into bilateral inguinal fat pads (7.0 × 10 6 TU per point, 3 points per fat pad) of male mice at the age of 8 weeks. After 2 or 4 weeks, ADSCs were isolated from inguinal SAT to examine GFP signals under fluorescence microscope. Scale bar = 50 μm. NS, normal saline. d – l Mice ( n = 10 per group) at the age of 8 weeks were injected with shCD90 (or shControl) lentivirus as mentioned above. The body weight ( d ) was recorded during 4 weeks of treatment. After 3 weeks of treatment, GTT was performed in mice with overnight fast; glucose levels ( e ) were determined at different time points after glucose injection. Areas under the curve ( f ) for GTT are shown. The inguinal SAT section was stained with H&E ( g ), and the areas of adipocytes were measured as pixies using Image-Pro Plus 6.0 ( h ). Scale bar = 50 μm. The mRNA level of Leptin in inguinal SAT was detected by qPCR ( i ). S-ADSCs from inguinal SAT were examined for the mRNA level of CD90 by qPCR ( j ) and proliferation by EDU incorporation assay. Fluorescence signals ( k ) detected by fluorescence microscope and growth indices ( l ) as specified in Fig. are shown. Scale bar = 100 μm. Bars represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: In some experiments, S-ADSCs infected with shRNA CD90 (shCD90) or control shRNA (shControl) recombinant lentivirus (GenePharma, shanghai, China) were subjected to adipogenic induction.

Techniques: Infection, Fluorescence, Microscopy, Injection, Isolation, Saline, Staining

Journal: American Journal of Cancer Research

Article Title: Isocitrate dehydrogenase 3A, a rate-limiting enzyme of the TCA cycle, promotes hepatocellular carcinoma migration and invasion through regulation of MTA1, a core component of the NuRD complex

doi:

Figure Lengend Snippet: IDH3A promotes EMT and cell migration as well as invasion in HCC. A and B. IDH3A was overexpressed or knocked down in HepG2 cells. The expression of IDH3A and EMT markers were determined by RT-qPCR and western blotting analyses (*P < 0.05). C. IDH3A was overexpressed or knocked down in MHCC-97H cells. The expression of MTA1 was determined by RT-qPCR and western blotting analyses (*P < 0.05). D. MHCC-97H cells were transfected with IDH3A siRNA and/or IDH3A for the wound healing assay (*P < 0.05). E. MHCC-97H cells were transfected with IDH3A siRNA and/or IDH3A for the transwell invasion assay (*P < 0.05).

Article Snippet: Lentiviruses carrying control shRNA (shControl) and IDH3A shRNA (shIDH3A) were purchased from Genepharma Inc.

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Wound Healing Assay, Transwell Invasion Assay

Journal: American Journal of Cancer Research

Article Title: Isocitrate dehydrogenase 3A, a rate-limiting enzyme of the TCA cycle, promotes hepatocellular carcinoma migration and invasion through regulation of MTA1, a core component of the NuRD complex

doi:

Figure Lengend Snippet: IDH3A regulates MTA1 expression in HCC. A. Heatmap of 3933 differentially expressed genes in HepG2 cells expressing IDH3A siRNA to siRNA control from three independent biological replicates. B. Bioinformatics analysis of the different gene expression events that were identified in IDH3A-depleted HepG2 cells using the DAVID Functional Annotation Tool (DAVID, https://david.ncifcrf.gov/). C. IDH3A was knocked down in HepG2 cells, RT-qPCR was performed to detect the roles of IDH3A (*P < 0.05). D. HepG2 cells were transfected with IDH3A siRNA and/or IDH3A. The expression of MTA1 was determined by RT-qPCR and western blotting analyses (*P < 0.05).

Article Snippet: Lentiviruses carrying control shRNA (shControl) and IDH3A shRNA (shIDH3A) were purchased from Genepharma Inc.

Techniques: Expressing, Control, Gene Expression, Functional Assay, Quantitative RT-PCR, Transfection, Western Blot

Journal: American Journal of Cancer Research

Article Title: Isocitrate dehydrogenase 3A, a rate-limiting enzyme of the TCA cycle, promotes hepatocellular carcinoma migration and invasion through regulation of MTA1, a core component of the NuRD complex

doi:

Figure Lengend Snippet: IDH3A promotes cell migration and invasion through regulating MTA1 expression in HCC. A. HepG2 cells were transfected with MTA1, IDH3A and/or MTA1 siRNA, MTA1 siRNA, IDH3A siRNA and/or MTA1. The expression of MTA1 and EMT markers were determined by RT-qPCR and western blotting analyses (*P < 0.05). B. MHCC-97H cells were transfected with MTA1, IDH3A and/or MTA1 siRNA, MTA1 siRNA, IDH3A siRNA and/or MTA1. The expression of MTA1 was determined by RT-qPCR and western blotting analyses (*P < 0.05). C. MHCC-97H cells were transfected with MTA1, IDH3A and/or MTA1 siRNA, MTA1 siRNA, IDH3A siRNA and/or MTA1 for wound-healing assay (*P < 0.05). D. MHCC-97H cells were transfected with MTA1, IDH3A and/or MTA1 siRNA, siMTA1, IDH3A siRNA and/or MTA1 for transwell invasion assay (*P < 0.05). E. MHCC-97H cells that were infected with lentiviruses carrying control shRNA, IDH3A shRNA and/or infected with retroviruses carrying MTA1 were injected intravenously through the tail vein of SCID mice (n=6). Lung metastasis was quantified by bioluminescence imaging.

Article Snippet: Lentiviruses carrying control shRNA (shControl) and IDH3A shRNA (shIDH3A) were purchased from Genepharma Inc.

Techniques: Migration, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Wound Healing Assay, Transwell Invasion Assay, Infection, Control, shRNA, Injection, Imaging

Journal: PLoS ONE

Article Title: CYB5D2 Requires Heme-Binding to Regulate HeLa Cell Growth and Confer Survival from Chemotherapeutic Agents

doi: 10.1371/journal.pone.0086435

Figure Lengend Snippet: A) Western blot analysis of HeLa cells following CYB5D2 shRNA-mediated knockdown (shCYB5D2) compared to shRNA control (shCTRL) HeLa cells. B) Cell proliferation of HeLa cells following CYB5D2 knockdown. Experiments were conducted in quadruplicate, with triplicate replicates in each experiment. Cell numbers determined at each time point are presented as mean ± SD. ** p <0.01 (two-tailed Student’s t -test). C) Anchorage-independent growth of shCYB5D2 and shCTRL HeLa cells. Representative images of soft agar plates (top panels) and phase contrast images of colonies at 50X magnification (bottom panels). Scale bar is equal to 200 µm. D) Mean number (left panel) and mean diameter (right panel) of colonies in 10 random fields were evaluated and presented as mean ± S.E.M. of three independent experiments. ** p <0.01 (two-tailed Student’s t -test).

Article Snippet: CYB5D2 shRNA (shCYB5D2) and non-specific shRNA control (shCTRL) lentiviral pool plasmids were purchased from Santa Cruz Biotechnology Inc., and packaged in 293T cells by co-transfecting each shRNA plasmid pool (10 μg) with plasmids (10 μg each) necessary for third-generation lentiviral production (pRSV-REV, pCMV-VSV-G, pMDLg/pRRE) , using the calcium phosphate transfection method.

Techniques: Western Blot, shRNA, Knockdown, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: CYB5D2 Requires Heme-Binding to Regulate HeLa Cell Growth and Confer Survival from Chemotherapeutic Agents

doi: 10.1371/journal.pone.0086435

Figure Lengend Snippet: A) Dual immunofluorescence of FLAG-tagged CYB5D2 and CYPOR-YFP. Stable CYB5D2-expressing HeLa cells were transiently transfected with CYPOR-YFP plasmid. Images were taken at 1000X magnification. Scale bar is equal to 5 μm. B) Western blot analysis of CYP51A1 protein levels following CYB5D2 shRNA-mediated knockdown (shCYB5D2) in HeLa cells compared to shRNA control (shCTRL) cells. C) Mevalonate treatment results in reduced survival of shCYB5D2 cells compared to shCTRL cells. D) CYP3A4 activity is reduced in shCYB5D2 cells compared to shCTRL cells, which is further evident following transiently transfecting CYPOR-YFP plasmid (+CYPOR) for 24 hours (h). E) Relative survival of HeLa cells expressing ectopic CYB5D2 or CYB5D2(D86G) compared to empty vector (EV) control cells (left panels), or shRNA control (shCTRL) and CYB5D2 shRNA-mediated knockdown (shCYB5D2) HeLa cells (right panels), following treatment with either paclitaxel (400 ng/ml), cisplatin (400 ng/ml) or doxorubicin (10 µM) for 24 and 48 h. At each time point, cell survival values were normalized to a dimethylsufloxide (DMSO)-treated control for each stable cell line. Relative cell survival values are presented as mean ± S.E.M. of three independent experiments (three replicates in each experiment). * p <0.05; ** p <0.01 (two-tailed Student’s t -test).

Article Snippet: CYB5D2 shRNA (shCYB5D2) and non-specific shRNA control (shCTRL) lentiviral pool plasmids were purchased from Santa Cruz Biotechnology Inc., and packaged in 293T cells by co-transfecting each shRNA plasmid pool (10 μg) with plasmids (10 μg each) necessary for third-generation lentiviral production (pRSV-REV, pCMV-VSV-G, pMDLg/pRRE) , using the calcium phosphate transfection method.

Techniques: Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Western Blot, shRNA, Knockdown, Control, Activity Assay, Stable Transfection, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Receptor Tyrosine Kinase TIE2/ Tek Pathway by NRF2 Activation in Neurovascular Endothelial Cells

doi: 10.3390/ijms27020770

Figure Lengend Snippet: Reduced TIE2/ Tek levels with SFN are dependent on NRF2 activity. ( A ) bEnd.3 cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h). Expression levels of 84 endothelial genes were analyzed by quantitative real-time PCR (qRT-PCR) and normalized to the geometric mean of Actb , Gapdh , B2m , Gusb , and Hsp90ab1 levels. Volcano plot comparing gene expression of vehicle- versus SFN-treated cells. A fold of change greater than 1.5 is represented by yellow (increased expression when compared to vehicle-treated cells) and blue (decreased expression when compared to vehicle-treated cells) dots. Data are the mean of n = 4. Statistical analysis was performed with the GeneGlobe Data Analysis Center from Qiagen. A p -value < than 0.05 was considered significant and is represented by a line. A red square highlights the Tek gene. ( B ) Representative immunoblots of NRF2 (arrowhead), TIE2, CLDN5, HO-1, and VCL, and GAPDH as a loading control from bEnd.3 cells maintained under low-serum conditions (16 h, 1% FBS) and treated with SFN (5 µM, 16 h). ( C ) Densitometric quantification of NRF2, HO-1, TIE2, and CLDN5 protein levels from representative immunoblots of B expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05 and *** p < 0.001 vs. vehicle according to Student’s t -test. ( D ) bEnd.3 cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h). Transcript levels of Hmox1 , Nqo1 , Slc7a11 , Tek , Cldn5 , Cdh5 , Ocln , and Tjp1 were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). * p < 0.05 and *** p < 0.001 vs. vehicle according to Student’s t -test. ( E ) Confocal analysis of double immunofluorescence with anti-TIE2 (green) and anti-CLDN5 (red) antibodies in vehicle (Veh.) or SFN-treated bEnd.3 cells. ( F ) Quantification of TIE2 and CLDN5 intensity signals in E. Values are mean ± SD (n = 3, with >50 cells counted per field). * p < 0.05 vs. VEH-treated cells according to Student’s t -test. ( G ) bEnd.3 cells were transduced with lentiviral vectors carrying short hairpin RNA against NRF2 (shNRF2) or a scramble sequence (shCTRL). Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h). Representative immunoblots of NRF2 (arrowhead), TIE2, CLDN5, HO-1, and LMNB, and GAPDH as a loading control. ( H ) Densitometric quantification of NRF2, HO-1, TIE2, and CLDN5 protein levels from representative immunoblots of G expressed as a ratio of LMNB and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05 and ** p < 0.01 vs. vehicle, or # p < 0.05 vs. shCTRL according to Student’s t -test. ( I ) Transcript levels of Nfe2l2 , Hmox1 , Nqo1 , and Slc7a11 and ( J ) of Tek , Cldn5 , Cdh5 , Ocln , and Tjp1 from bEnd.3 cells transduced with shNRF2 or shCTRL maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h) were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle, or # p < 0.05 and ### p < 0.001 vs. shCTRL according to Student’s t -test.

Article Snippet: Lentiviral vector pLKO.1 shRNA control (shCtrl) (Addgene Plasmid #10879) was purchased from Addgene; pLKO-puro shNRF2 (NM_010902) was purchased from Sigma-Aldrich.

Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Western Blot, Control, Immunofluorescence, Transduction, shRNA, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Receptor Tyrosine Kinase TIE2/ Tek Pathway by NRF2 Activation in Neurovascular Endothelial Cells

doi: 10.3390/ijms27020770

Figure Lengend Snippet: Overexpression of NRF2 alters TIE2/ Tek levels. ( A ) bEnd.3 cells were transduced with lentiviral vectors carrying overexpressed NRF2 insensitive to KEAP1 degradation (NRF2 ∆ETGE ) or a lentivirus empty vector. Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS). Representative immunoblots of NRF2 (arrowhead), TIE2, CLDN5, HO-1, and VCL, and GAPDH as a loading control. ( B ) Densitometric quantification of NRF2, HO-1, TIE2, and CLDN5 protein levels from representative immunoblots of A expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05 and *** p < 0.001 vs. empty vector according to Student’s t -test. ( C ) Transcript levels of Hmox1 , Nqo1 , and Slc7a11 and ( D ) of Tek , Cldn5 , Cdh5 , Ocln , and Tjp1 from bEnd.3 cells transduced with NRF2 ∆ETGE or empty vector maintained under low-serum conditions (16 h, 1% FBS) were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). ** p < 0.01 and *** p < 0.001 vs. empty vector according to Student’s t -test. ( E ) bEnd.3 cells were transduced with lentiviral vectors NRF2 ∆ETGE or an empty vector. Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS) and were subjected to ANGPT1 (400 ng/mL) purified from supernatant HEK293T-stable expression (CMP-ANGPT1) to the indicated time points. Representative immunoblots of NRF2 (arrowhead), TIE2, CLDN5, HO-1, and VCL, and GAPDH as a loading control. ( F ) Densitometric quantification of NRF2, TIE2, and CLDN5 protein levels from representative immunoblots of E expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. time 0 according to a one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Lentiviral vector pLKO.1 shRNA control (shCtrl) (Addgene Plasmid #10879) was purchased from Addgene; pLKO-puro shNRF2 (NM_010902) was purchased from Sigma-Aldrich.

Techniques: Over Expression, Transduction, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR, Purification, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Receptor Tyrosine Kinase TIE2/ Tek Pathway by NRF2 Activation in Neurovascular Endothelial Cells

doi: 10.3390/ijms27020770

Figure Lengend Snippet: Repression of TIE2/ Tek levels by NRF2 is not dependent on BACH1. ( A ) Subcellular fraction from bEnd.3 maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM) and hemin (10 µM) treatments for 16 h. Representative immunoblots of NRF2 (arrowhead), BACH1, TIE2, CLDN5, HO-1, and LMNB, and GAPDH as a loading control. Densitometric quantification of BACH1 ( B ), NRF2 nuclear ( C ), TIE2 cytosol ( D ), and HO-1 cytosol ( E ) protein levels from representative immunoblots of A expressed as a ratio of LMNB for nuclear fraction and GAPDH for cytosol fraction, respectively. Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle according to Student’s t -test. ( F , G ) bEnd.3 cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM) and hemin (10 µM) treatments for 16 h. Transcript levels of Hmox1 , Nqo1 , and Slc7a11 ( F ) and Tek , Bach1 , and Cldn5 ( G ) were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.05 and *** p < 0.001 vs. vehicle according to Student’s t -test. ( H ) bEnd.3 cells were transduced with lentiviral vectors carrying short hairpin RNA against BACH1 (shBACH1) or a scramble sequence (shCTRL). Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h). Representative immunoblots of NRF2 (arrowhead), BACH1, TIE2, CLDN5, HO-1, and VCL, and GAPDH as a loading control. ( I ) Densitometric quantification of NRF2, BACH1, HO-1, TIE2, and CLDN5 protein levels from representative immunoblots of H expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle, or ### p < 0.001 vs. shCTRL according to Student’s t -test. ( J ) Transcript levels of Hmox1 , Nqo1 , Bach1 , Tek , and Cldn5 from bEnd.3 cells transduced with shBACH1 or shCTRL maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h) were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle, or # p < 0.05 and ### p < 0.001 vs. shCTRL according to Student’s t -test. ns indicates not significant.

Article Snippet: Lentiviral vector pLKO.1 shRNA control (shCtrl) (Addgene Plasmid #10879) was purchased from Addgene; pLKO-puro shNRF2 (NM_010902) was purchased from Sigma-Aldrich.

Techniques: Western Blot, Control, Quantitative RT-PCR, Transduction, shRNA, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Receptor Tyrosine Kinase TIE2/ Tek Pathway by NRF2 Activation in Neurovascular Endothelial Cells

doi: 10.3390/ijms27020770

Figure Lengend Snippet: NRF2 does not modify Tek mRNA stability and does not bind its promoter. ( A ) bEnd.3 were maintained under low-serum conditions (16 h, 1% FBS), pre-treated with SFN (5 µM, 6 h), and subsequently subjected to actinomycin D (5 µg/mL) or sustained SFN (5 µM) at the indicated time points. The graph depicts the natural logarithm of the relative levels of the Tek mRNA as a function of actinomycin D or actinomycin D/SFN incubation time, normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . The mRNA half-life was determined using the linear part of the degradation curve. Data are mean ± S.D. (n = 3). No statistically significant differences ( p ≤ 0.05) were detected between vehicle- and SFN-treated groups at any of the analyzed time points according to a two-way ANOVA followed by a Bonferroni post hoc test. ( B ) Representative immunoblots of NRF2 (arrowhead) and TIE2, as well as VCL as a loading control, from bEnd.3 were maintained under low-serum conditions (16 h, 1% FBS), pre-treated with SFN (5 µM, 6 h), and subsequently subjected to CHX (100 µM) or sustained SFN (5 µM) at the indicated time points. ( C ) The graph depicts the natural logarithm of the relative levels of the TIE2 protein as a function of CHX or CHX/SFN incubation time from representative immunoblots of A normalized to VCL. The protein half-life was determined using the linear part of the degradation curve. Data are mean ± S.D. (n = 3). No statistically significant differences ( p ≤ 0.05) were detected between vehicle- and SFN-treated groups at any of the analyzed time points according to a two-way ANOVA followed by a Bonferroni post hoc test. ( D ) bEnd.3 cells were transduced with lentiviral vectors carrying overexpressed NRF2 insensitive to KEAP1 degradation (NRF2 ∆ETGE ) or a lentivirus empty vector. Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS). Chromatin immunoprecipitation (ChIP) analysis was performed with anti-IgG or anti-PolII antibodies, and the potential AREs in Tek with the highest were analyzed by qRT-PCR. The figure shows representative data normalized as the fold of enrichment with the anti-PolII antibody vs. the IgG antibody. The presence of already known AREs in Hmox1 (ARE1), Hmox1 (ARE2), and Nqo1 was analyzed as a positive control, and Actb was amplified as a negative control.

Article Snippet: Lentiviral vector pLKO.1 shRNA control (shCtrl) (Addgene Plasmid #10879) was purchased from Addgene; pLKO-puro shNRF2 (NM_010902) was purchased from Sigma-Aldrich.

Techniques: Incubation, Western Blot, Control, Transduction, Plasmid Preparation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Positive Control, Amplification, Negative Control