Journal: PLoS ONE

Article Title: Anti-Inflammatory and Anti-Fibrotic Profile of Fish Oil Emulsions Used in Parenteral Nutrition-Associated Liver Disease

doi: 10.1371/journal.pone.0115404

Figure Lengend Snippet: Human liver epithelial cell line THLE-3 was incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at 1/100 dilutions for 72 hours. Representative visible morphology and immunofluorescences for alpha smooth muscle actin (αSMA) and collagen type I (col type I) are showed.

Article Snippet: The reverse transcription was performed in 300 ng of total RNA with TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for α-SMA (Hs00559403_m1), α 1 (I)-collagen ( Col type I ; cat. n°: Hs00164004_m1), vimentin (cat. n°: Hs 00958116_m1), E-cadherin (cat. n°: Hs01023894_m1), zona occludens-1 ( ZO-1 ; cat. n°: Hs01551861_m1), Slug (cat. n°: Hs00950344_m1), Snail (cat. n°: Hs00195591_m1) and GAPDH (pre-designed by Applied Biosystems, cat. n°: 4310884E) as a housekeeping in a 7900HT Fast Real-Time PCR System (Applied Biosystem) using Universal Master Mix (Applied Biosystems).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Anti-Inflammatory and Anti-Fibrotic Profile of Fish Oil Emulsions Used in Parenteral Nutrition-Associated Liver Disease

doi: 10.1371/journal.pone.0115404

Figure Lengend Snippet: Human liver epithelial cell line THLE-3 was incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by TGFβ1 5 ng/mL stimulation for additional 72 hours. (A) Visible morphology and immunofluorescence for alpha smooth muscle actin (αSMA) and collagen type I (col type I) distribution and expression. B) Expression of mRNA of αSMA and col type I . Scale bar: 10 µm. Results are expressed as means ± SEM of six independent experiments. * p <0.05 related to the control group. # p <0.05 related to the stimulus.

Article Snippet: The reverse transcription was performed in 300 ng of total RNA with TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for α-SMA (Hs00559403_m1), α 1 (I)-collagen ( Col type I ; cat. n°: Hs00164004_m1), vimentin (cat. n°: Hs 00958116_m1), E-cadherin (cat. n°: Hs01023894_m1), zona occludens-1 ( ZO-1 ; cat. n°: Hs01551861_m1), Slug (cat. n°: Hs00950344_m1), Snail (cat. n°: Hs00195591_m1) and GAPDH (pre-designed by Applied Biosystems, cat. n°: 4310884E) as a housekeeping in a 7900HT Fast Real-Time PCR System (Applied Biosystem) using Universal Master Mix (Applied Biosystems).

Techniques: Incubation, Immunofluorescence, Expressing

Journal: Cureus

Article Title: Investigation of the Use of Circulating Long Non-coding RNA HOXA Transcript at the Distal Tip (LncRNA HOTTIP) as a Biomarker in Breast Cancer

doi: 10.7759/cureus.50019

Figure Lengend Snippet: HOTTIP values (arbitrary units) In science and technology, an arbitrary unit or procedure defined unit is a relative unit of measurement that shows the ratio of amount of substance or other quantities to a predetermined reference measurement. HOTTIP: HOXA transcript at the distal tip, GAPDH: glyceraldehyde-3-phosphate dehydrogenase (used as an endogenous control for quantitative RT-PCR analysis because its expression is consistent at different time points and various experimental manipulations), CT: threshold cycle for each sample for each gene expression measured, ΔCT: CT HOTTIP - CT GAPDH, ΔΔCT: ΔCT sample - ΔCT pool, 2^(-ΔΔCT): standard strategy for qPCR data analysis based on the PCR efficiency

Article Snippet: The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were estimated in the same samples using the respective predesigned primers - TaqMan probe set (Hs03929097_g1, Thermo Fisher Scientific) - and served as a housekeeping gene.

Techniques: Control, Quantitative RT-PCR, Expressing, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: MUC16 is overexpressed in lung tissue from idiopathic pulmonary fibrosis (IPF) patients. Lung tissue was obtained from healthy controls ( n = 17) and IPF patients ( n = 20). ( A ) MUC16 mRNA expression was analysed by real-time polymerase chain reaction (PCR). ( B ) MUC16 protein expression levels were analysed by Western blotting ( n = 14). ( C ) Immunohistochemistry of MUC16. Top panel: healthy lung sections, bottom panel: IPF lung sections. Scale bar: 100 μm. ATII cells (arrows) and fibroblasts (arrowhead) show positivity for MUC16 immunostaining. Data are shown as the ratio compared to β-actin for protein expression and 2 −ΔCt for mRNA levels. Data are presented as a scatter dot plot with median and interquartile ranges. p values are based on the Mann–Whitney test.

Article Snippet: Reverse transcription was performed in 300 ng of total RNA with a TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for MUC16 (Hs01065189_m1), α-SMA (Hs00559403_m1), α 1 (I)-collagen (collagen type I; Hs00164004_m1), SNAIL (Hs00195591_m1) and SLUG (Hs00161904_m1) in a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Universal Master Mix (Applied Biosystems).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Immunostaining, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: TGF-β1 and MUC16 collaborate to induce the alveolar epithelial to mesenchymal transition. The A549 cell line transfected with control siRNA(−) or siRNA-MUC16 was stimulated for 48 h ( A – E ) or 72 h ( F ) with 5 ng/mL TGF-β1 to measure MUC16 ( A ), collagen type I ( B ), α-SMA ( C ), Slug ( D ), and Snail ( E ) mRNA expression by real-time PCR and collagen type I protein levels by Western blotting, quantification was performed by densitometry ( F ). Data are expressed as 2 −ΔCt for mRNA levels and relative to β-actin for protein levels. The results are expressed as means ± SE. Student t-test of three independent experiments performed in triplicate. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−).

Article Snippet: Reverse transcription was performed in 300 ng of total RNA with a TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for MUC16 (Hs01065189_m1), α-SMA (Hs00559403_m1), α 1 (I)-collagen (collagen type I; Hs00164004_m1), SNAIL (Hs00195591_m1) and SLUG (Hs00161904_m1) in a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Universal Master Mix (Applied Biosystems).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: TGF-β1 and MUC16 collaborate to induce the fibroblast to myofibroblast transition. The MRC5 cell line transfected with control siRNA(−) or siRNA-MUC16 was stimulated for 48 h ( A – D ) or 72 h ( E ) with 5 ng/mL TGF-β1 to measure collagen type I ( A ), α-SMA ( B ), Slug ( C ), and Snail ( D ) mRNA expression by real-time PCR and collagen type I protein levels by Western blotting, quantification was performed by densitometry ( E ). Data are expressed as 2 −ΔCt for mRNA levels and relative to β-actin for protein levels. The results are expressed as means ± SE. Student t-test of three independent experiments performed in triplicate. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−) siRNA(−) + TGF-β1.

Article Snippet: Reverse transcription was performed in 300 ng of total RNA with a TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for MUC16 (Hs01065189_m1), α-SMA (Hs00559403_m1), α 1 (I)-collagen (collagen type I; Hs00164004_m1), SNAIL (Hs00195591_m1) and SLUG (Hs00161904_m1) in a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Universal Master Mix (Applied Biosystems).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: MUC16 mediates TGF-β1-induced lung fibroblast proliferation. TGF-β1 10 ng/mL was added in siRNA-MUC16 or siRNA(-) lung MRC5 fibroblasts during 48 h, and proliferation was evaluated by the BrdU assay. The results are expressed as means ± SE. Student t-test of three independent experiments performed in triplicate. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−) + TGF-β1.

Article Snippet: Reverse transcription was performed in 300 ng of total RNA with a TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for MUC16 (Hs01065189_m1), α-SMA (Hs00559403_m1), α 1 (I)-collagen (collagen type I; Hs00164004_m1), SNAIL (Hs00195591_m1) and SLUG (Hs00161904_m1) in a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Universal Master Mix (Applied Biosystems).

Techniques: BrdU Staining

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: MUC16 modulates the effect of TGF-β1 on SMAD3 phosphorylation. ( A ) A549 and ( B ) MRC5 cell lines transfected with control siRNA(−) or siRNA-MUC16 were stimulated for 40 min with TGF-β1 10 ng/mL to measure p-Smad3 and for 48 h with TGF-β1 5 ng/mL to measure β-catenin by Western blot. A549 ( C ) and MRC5 ( D ) cells were stimulated with 10 ng/mL TGF-β1 for 40 min. Total protein was extracted and immunoprecipitated using MUC16 antibody and probed against p-Smad3 and MUC16 by Western blot (representative images are shown). ( E ) A549 cells were stimulated with 10 ng/mL TGF-β1 for 1 h. MUC16 and p-Smad3 co-localisation was analysed by confocal microscopy to generate a two-dimensional cytofluorogram that selected common localised points of both antibodies (white colour). Scale bars: 10 μm. ( F ) Smad Binding Element (SBE) measure following 10 ng/mL TGF-β1 stimulation during 18 h in A549 cells transfected with siRNA(−) or siRNA-MUC16. Data are expressed relative to Smad3 or β-actin protein level (A-D). The results are expressed as means ± SE of three independent experiments performed in triplicate. Student t-test was used. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−) + TGF-β1.

Article Snippet: Reverse transcription was performed in 300 ng of total RNA with a TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for MUC16 (Hs01065189_m1), α-SMA (Hs00559403_m1), α 1 (I)-collagen (collagen type I; Hs00164004_m1), SNAIL (Hs00195591_m1) and SLUG (Hs00161904_m1) in a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Universal Master Mix (Applied Biosystems).

Techniques: Transfection, Western Blot, Immunoprecipitation, Confocal Microscopy, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: Schematic illustration showing the collaboration of MUC16 with the TGF-β1 cellular pathway in idiopathic pulmonary fibrosis. After TGF-β1 stimulation, MUC16 forms a protein complex with pSmad3, allowing the proper phosphorylation and the consequent activation of the Smad Binding Element (SBE) to promote the expression of pro-fibrotic markers, cellular transformations such as epithelial to mesenchymal transition (EMT), fibroblast to mesenchymal transition (FMT), and cell proliferation.

Article Snippet: Reverse transcription was performed in 300 ng of total RNA with a TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for MUC16 (Hs01065189_m1), α-SMA (Hs00559403_m1), α 1 (I)-collagen (collagen type I; Hs00164004_m1), SNAIL (Hs00195591_m1) and SLUG (Hs00161904_m1) in a 7900HT Fast Real-Time PCR System (Applied Biosystems) using Universal Master Mix (Applied Biosystems).

Techniques: Activation Assay, Binding Assay, Expressing

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: Overexpression of CCAAT/enhancer‐binding protein delta (CEBPD) confers a pro‐proliferative phenotype in BFTC909 and TCCSUP cells. Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) and immunoblotting (A) showed that the transcript and protein levels of CEBPD were lower in the BFTC909 and TCCSUP cells among the four urothelial carcinoma (UC)‐derived cell lines. Hence, Exogenous CEBPD expression was performed in BFTC909 and TCCSUP cells to examine the biological impact of CEBPD on tumorigenesis. The mRNA (B) and protein (C) levels of CEBPD were significantly upregulated in BFTC909 and TCCSUP cells after successful exogenous expression of the CEBPD gene compared with the mock‐transfected cell lines. (D) Proliferation assay showed that overexpression of CEBPD in BFTC909 and TCCSUP significantly increased pro‐proliferative phenotype at 24–72 h after seeding compared to the mock‐transfected cells. All experiments were conducted in triplicate and results were represented as the mean ± SEM. For immunoblot assay, one representative image was shown and GAPDH was regarded as a loading control. Statistical significance: * p < .05

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Over Expression, Binding Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Expressing, Transfection, Proliferation Assay, Control

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: CCAAT/enhancer‐binding protein delta (CEBPD) overexpression confers mTORC1‐driven metabolic reprogramming and leads to glucose addiction. The potential relationship between CEBPD and proteins associated with the mTOR pathway was explored by immunoblotting analysis. (A) pMAPK3/1, pPI3K, pAKT1, pMTOR, pRPS6, pEIF4EBP1 and SKP2 were significantly upregulated, while cleaved CASP3 was notably decreased in CEBPD‐overexpressing BFTC909 and TCCSUP cells treated with culture medium containing glucose compared to mock‐expressing cells. (B) 2,3‐Bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl) 2H‐tetrazolium‐5‐carboxanilide (XTT) assay indicated that cell viability was notably inhibited in mock‐expressing BFTC909 and TCCSUP cells under glucose starvation (culture medium without glucose) for 72 h compared to mock‐expressing cells in a complete cell culture medium. Furthermore, CEBPD overexpression further exacerbated glucose withdrawal‐induced cell viability suppression in these two cell lines. (C) Immunoblotting data indicated that glucose deficiency suppresses the effect of CEBPD on the upregulation of pAKT1, pMTOR, pRPS6, pEIF4EBP1 and SKP2 protein and the downregulation of CASP3 in these two distinct UC‐derived cells. However, the protein levels of pMAPK3/1 and pPI3K were not comparable in CEBPD‐overexpressing BFTC909 and TCCSUP cells under glucose starvation conditions. Glucose uptake assay (D), lactate level analysis (E), Seahorse XFp Analyzer assessment (F–G), OCR, and MitoDsRed‐tagged fluorescent mitochondrial staining (H) were performed to evaluate the cellular glucose uptake, lactate production, oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and mitochondrial status. The data showed that stable overexpression of CEBPD notably increased glucose uptake (D), lactate production (E), the ECAR (F) and mitochondrial fragmentation/fission (H) but decreased the OCR (G) in BFTC909 and TCCSUP cells compared to mock‐expressing cells. To clarify the metabolic switch from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis under CEBPD regulation, the relationship between CEBPD and hexokinase 2 ( HK2 ) was estimated through analysis of the Gene Expression Omnibus (GEO) database (GSE13507 dataset, n = 188), indicating a significantly positive correlation between the mRNA levels of CEBPD and HK2 in bladder cancer specimens (I). The result was validated by quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) (J), promoter activity assay (K), and immunoblotting (L) and indicated that the transcript (J) and protein (L) levels of HK2 were markedly upregulated in CEBPD‐overexpressing BFTC909 and TCCSUP cells versus mock‐transfected cells via the upregulation of HK2 promoter activity (K). Additionally, CEBPD overexpression increased the protein level of SLC2A1 but not LDH A/C (L). All experiments were performed in triplicate and data was represented as the mean ± SE. For immunoblot assay and fluorescent image data, one representative image is displayed. GAPDH was served as a loading control for immunoblot assay. Statistical significance: * p < .05

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Binding Assay, Over Expression, Western Blot, Expressing, XTT Assay, Cell Culture, Derivative Assay, Staining, Phospho-proteomics, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Transfection, Control

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: Collaboration of CCAAT/enhancer‐binding protein delta (CEBPD) and MYC in the regulation of SLC2A1 and HK2. (A) To assess the crosstalk between the dosage/gene expression of CEBPD and MYC , we reassessed The Cancer Genome Atlas (TCGA) bladder cancer dataset and found that 32.2% (49/152) and 46.7% (71/152) of urothelial carcinomas (UCs) showed copy number gains (CNGs) involving CEBPD and MYC , respectively. Moreover, a notably positive correlation (45/152, r = 0.427, p < .001) between CEBPD and MYC dosage was observed. The Red dashed line indicated the log2 ratio of 0.2, which represent the cut‐off of copy number gain. (B) Assessment of the GEO database (GSE13507 dataset, n = 188) identified a markedly positive relation between the transcript levels of MYC and CEBPD ( r = 0.477, p < .001). Copy number variation (CNV) profiled by real‐time PCR using three distinct probes targeting three different regions of the MYC and CEBPD chromosomes indicated that the overexpression of MYC through viral delivery systems could induce CEBPD amplification in BFTC909 and TCCSUP (D) cells but not vice versa (C). (E) Immunohistochemistry (IHC; left) and chromogenic in situ hybridization (CISH; right) assays revealed an inconsistency between the expression and gene dosage of MYC in a representative case of UTUC with MYC amplification but a low MYC expression. (F) The effect of CEBPD overexpression on the promotion of MYC transcription was not comparable in BFTC909 and TCCSUP cells, while (G) CEBPD overexpression robustly increased the protein level of MYC in both cell lines, suggesting the possibility that CEBPD exerts posttranslational regulation of MYC expression. (H) Evaluation of protein stability by cycloheximide (CHX) chase assays coupled with immunoblotting shows that MYC protein expression was restored in CEBPD‐expressing BFTC909 and TCCSUP cells compared to mock cells. The statistical results were shown in a line graph (I). Moreover, the MYC protein levels were abundantly increased in the mock‐transfected BFTC909 and TCCSUP groups treated with MG132, a ubiquitin‐proteasome inhibitor, compared to the vehicle‐treated group. MYC protein had the same effect between MG132 or vehicle treatment in CEBPD‐overexpressing BFTC909 and TCCSUP cells (J). CEBPD overexpression notably inhibited the mRNA (K) and protein (L) levels of FBXW7 in BFTC909 and TCCSUP cells. (M) CEBPD overexpression significantly suppressed FBXW7 promoter activity in BFTC909 and TCCSUP cells, confirming that CEBPD upregulates MYC expression by depleting FBXW7‐mediated MYC degradation. All experiments were executed in triplicate and the results were represented as the mean ± SEM. For immunoblot assay, IHC and CISH data, one representative image was displayed. GAPDH was shown as a loading control for immunoblot assay. Statistical significance: * p < .05

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Binding Assay, Gene Expression, Real-time Polymerase Chain Reaction, Over Expression, Amplification, Immunohistochemistry, Chromogenic In Situ Hybridization, Expressing, Western Blot, Transfection, Ubiquitin Proteomics, Activity Assay, Control

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: Correlations between CCAAT/enhancer‐binding protein delta (CEBPD) and MYC amplification/expression and miR‐429 and HK2 expression and other important clinicopathological parameters in urinary bladder urothelial carcinoma (UBUC)

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Amplification, Expressing

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: Correlations between CCAAT/enhancer‐binding protein delta (CEBPD) and MYC amplification/expression and miR‐429 and HK2 expression and other important clinicopathological parameters in upper urinary tract urothelial cancer (UTUC)

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Amplification, Expressing

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: CCAAT/enhancer‐binding protein delta (CEBPD) promotes HK2 expression through the transcriptional suppression of hsa‐miR‐429. (A) Immunoblotting assays confirmed that MYC, SLC2A1 and HK2 expression was markedly increased by exogenous CEBPD expression in BFTC909 and TCCSUP cells. The increase in MYC, SLC2A1 and HK2 expression induced by CEBPD overexpression was partially attenuated by two distinct MYC siRNAs in BFTC909 and TCCSUP cells, confirming the critical role of MYC in CEBPD‐driven glycolysis. (B) A putative CEBPD binding site at the hsa‐miR‐429 promoter was predicted by the JASPAR database ( http://jaspar.genereg.net/ ). (C) Moreover, a predicted targeting site of hsa‐miR‐429 on the 3′‐untranslated region (3′‐UTR) of HK2 mRNA was identified. The red font indicated the seed sequence of miRNA to the mRNA. Lowercase letters mean unmatched position while capital letters showed the matched region between miRNA and mRNA. To evaluate the regulation of HK2 expression by hsa‐miR‐429, a mutant‐ HK2 mRNA‐3′‐UTR was constructed as indicated. We identified that treatment with the miR‐429 mimic did not affect CEBPD (D) or MYC (E) transcription in BFTC909 and TCCSUP cells. Exogenous CEBPD overexpression significantly inhibited the expression (F) and promoter transactivity (G) of hsa‐miR‐429 through quantitative RT‐PCR and luciferase reporter assays, respectively, in BFTC909 and TCCSUP cells. (H) Chromatin immunoprecipitation (ChIP) indicated that CEBPD is evidently recruited to the hsa‐miR‐429 promoter region in BFTC909 and TCCSUP cells, confirming the direct binding and transcriptional regulation of CEBPD on hsa‐miR‐429. (I) Interestingly, the transcription of HK2 was robustly diminished by treatment with the miR‐429 mimic but promoted after treatment with the hsa‐miR‐429 inhibitor in BFTC909 and TCCSUP cells. Moreover, miR‐429 mimic treatment significantly inhibited the relative luciferase activity of BFTC909 and TCCSUP cells transfected with pMIR‐WT‐ HK2 ‐3′‐UTR ‐Luc but not pMIR‐mutant‐ HK2 ‐3′‐UTR‐Luc (J). (K) The transcription of HK2 was increased in CEBPD‐overexpressing BFTC909 and TCCSUP cells compared to mock‐expressing cells. However, compared with the vehicle, the miR‐429 mimic robustly inhibited the increase in HK2 transcripts in CEBPD‐overexpressing BFTC909 and TCCSUP cells. The aforementioned evidence revealed that CEBPD promotes HK2 expression through the transcriptional suppression of hsa‐miR‐429. All experiments were performed in triplicate and the results were represented as the mean ± SEM. For immunoblot assay, one representative image was displayed. GAPDH was shown as a loading control for immunoblot assay. Statistical significance: * # p < .05

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Binding Assay, Expressing, Western Blot, Over Expression, Sequencing, Mutagenesis, Construct, Quantitative RT-PCR, Luciferase, Chromatin Immunoprecipitation, Activity Assay, Transfection, Control

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: High expression of MYC, CCAAT/enhancer‐binding protein delta (CEBPD) and HK2 along with low FBXW7 and hsa‐miR‐429 expression predicts adverse features and poor patient outcomes in urinary bladder urothelial carcinoma (UBUC) and upper urinary tract urothelial cancer (UTUC). (A) IHC and in situ hybridization showed that MYC amplification; high expression of MYC, CEBPD, and HK2; and low expression of FBXW7 and hsa‐miR‐429 were strongly relevant to UBUC patients ( n = 295) with high tumour stage. (B–C) Moreover, Kaplan‐Meier survival analysis indicated that both MYC amplification and high MYC expression are significant survival determinants in UBUC ( n = 295; B1 and B2) and UTUC ( n = 340; C1 and C2), while the survival impacts of MYC expression can be further enriched by the coexpression of CEBPD. (B3 and C3), suggesting potential synergistic effects of MYC and CEBPD in the promotion of UC progression. In addition, low expression of miR‐429 and high expression of HK2 also confer a poor prognosis in terms of disease‐specific survival in UBUC ( n = 295; B4 and B5) and UTUC ( n = 340; C4 and C5) patients. The UBUC and UTUC cohorts were taken from the biobank of Chi Mei Medical Center that collected specimens after an operation with curative intent between January 1996 and May 2004 as previously described. ²² This study was approved by the institutional review board of Chi Mei Medical Center (IRB10207‐001). For immunohistochemistry, one representative image was shown upper urinary tract urothelial cancer (UTUC)

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Expressing, Binding Assay, In Situ Hybridization, Amplification, Immunohistochemistry

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: Univariate log‐rank and multivariate analyses for disease‐specific and metastasis‐free survival in upper urinary tract urothelial cancer (UTUC)

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Amplification, Expressing

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: Univariate log‐rank and multivariate analyses for disease‐specific and metastasis‐free survival in urinary bladder urothelial carcinoma (UBUC)

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Amplification, Expressing

Journal: Clinical and Translational Medicine

Article Title: Biological significance of MYC and CEBPD coamplification in urothelial carcinoma: Multilayered genomic, transcriptional and posttranscriptional positive feedback loops enhance oncogenic glycolysis

doi: 10.1002/ctm2.674

Figure Lengend Snippet: Diabetes mellitus (DM) exacerbates CCAAT/enhancer‐binding protein delta (CEBPD)‐driven tumour aggressiveness. (A) Analysis of the National Health Insurance Research Database (NHIRD) showed that urinary bladder urothelial carcinoma (UBUC) (A1, n = 8436, p < .001) and upper urinary tract urothelial cancer (UTUC) (A5, n = 3232, p = .003) patients with concomitant DM had a higher death rate than non‐DM patients. However, unlike the results for the NHIRD, which includes a large number of patients, concomitant DM only displayed marginal significance for the prediction of poor disease‐specific survival (DSS) in UBUC (A2, n = 295, p = .0547) and UTUC (A6, n = 340, p = .0867) in our cohort, which includes few cases, indicating that the significance of DM in patients’ outcome might be mild. Interestingly, for those cases with high CEBPD expression in our cohort, the presence of DM conferred a significantly worse prognosis in UBUC (A3, n = 88, p = .0046) and UTUC (A7, n = 89, p = .0187). Conversely, tumours harbouring high CEBPD expression were even more aggressive in DM patients in both the UBUC (A4, n = 55, p = .0001) and UTUC (A8, n = 64, p < .0001) cohorts. The aforementioned evidence revealed that CEBPD‐mediated aggressiveness in UC can be exacerbated by concomitant hyperglycemic disorder, a symptom that characterizes diabetes, probably because high‐glucose conditions help to reinforce CEBPD‐mediated glycolysis in cancer cells. (B) Experiments on a BFTC909‐derived xenograft model ( n = 8 for each group) further reinforced that the effects of CEBPD on promoting tumour growth could be exacerbated by concomitant hyperglycemia. First, compared with the mock conditions, CEBPD overexpression promoted the growth of xenografted tumours in SCID/beige mice without high‐fat diet‐induced DM. Accelerated tumour growth driven by CEBPD overexpression was exacerbated in mice with high‐fat‐diet‐induced DM compared with those with CEBPD overexpression alone or induced DM alone (B1). The CEBPD‐induced and DM‐induced effects on tumour aggressiveness and the DM‐induced exacerbation of CEBPD‐driven tumour growth were all statistically significant (B2). (C) IHC analysis of xenografted samples at day 25 post‐injection showed that pAKT1, pMTOR, pRPS6, pEIF4EBP, MKI67, MYC and HK2 were highly expressed and that hsa‐miR‐429 were downregulated in the CEBPD‐overexpressing and/or DM‐induced groups compared to the control group. Of note, the upregulation of MYC and HK2 and downregulation of hsa‐miR‐429 were more prominent in the CEBPD‐overexpressing group than in the mock group, regardless of DM induction. This suggests that the CEBPD‐mediated effects on glycolysis might be regardless of hyperglycemia or normoglycemia conditions. For immunohistochemistry, one representative image was shown. Data are shown as the mean ± SEM. Statistical significance: * p < .05

Article Snippet: The mixture containing cDNA, predesigned TaqMan assay reagent (probe and primer set: CEBPD [Hs00270931_m1], MYC [Hs00153408_m1], FBXW7 [Hs00217794_m1] and HK2 [Hs01034055_g1]; Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) were subjected to quantitative RT‐PCR to determine the mRNA level via a StepOne Plus System (Applied Biosystems) at the indicated thermal cycling: 20 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The cycle threshold (Ct) value of the target gene was normalized to that of the reference gene POLR2A , which served as the ∆Ct value.

Techniques: Binding Assay, Expressing, Derivative Assay, Over Expression, Injection, Control, Immunohistochemistry

Journal: Hormones & cancer

Article Title: The Androgen Receptor supports tumor progression after the loss of ovarian function in a preclinical model of obesity and breast cancer

doi: 10.1007/s12672-017-0302-9

Figure Lengend Snippet: LC-mass spectrometry was used to measure (A) estradiol, and (B) testosterone in mammary/breast adipose tissue from postmenopausal women (N=3 women, 1-5 pieces of tissue each), or from lean or obese rats taken 3 weeks post OVX. For human samples, circles of the same color are from the same patient. (C) Q-PCR analysis of Cyp19a1 (aromatase) in rat ovary (N=4), rat mammary adipose (MG; N=29), and rat mammary tumor (N=37). Bars represent means.

Article Snippet: Rat Tissue Q-PCR Analysis Total RNA was isolated from rat ovary, mammary adipose (MG), or mammary tumor tissue using Trizol, followed by column clean-up (Qiagen). cDNA was prepared using the Verso kit (Thermo Scientific) and qPCR was performed using Taqman reagents, with commercially available predesigned Cyp19a1 primer/probe set (Rn00567222_m1; Life Technologies).

Techniques: Mass Spectrometry

Journal: Hormones & cancer

Article Title: The Androgen Receptor supports tumor progression after the loss of ovarian function in a preclinical model of obesity and breast cancer

doi: 10.1007/s12672-017-0302-9

Figure Lengend Snippet: (A) QPCR analysis of Fkbp5 expression in MCF7 cells treated with varying doses of testosterone in the presence or absence of IL-6. (B) QPCR analysis of Fkbp5 expression in MCF7 cells treated with low (0.1 nM) or high (10 nM) testosterone in the presence or absence of IL-6, and co-treated either with Fulvestrant (Fulv; 100 nM) or Enzalutamide (Enza; 10 μM). (C-D) Expression of Polr2a in cells treated with or without IL-6 and varying doses of testosterone (c), or in cells treated with IL-6, testosterone, or inhibitors (d) included as a control. (E-F) Expression of AR (e) and Fkbp5 (f) in primary breast tumor samples from women with high or low IL-6, as profiled in GSE54430. (G-H) Expression of Cyp19a1 (g) and Esr1 (h) in MCF7 cells treated with or without IL-6, and in human breast adipose tissue (HBA). T-tests determined statistical significance. * p<0.05; ** p<0.01; ***p<0.001.

Article Snippet: Rat Tissue Q-PCR Analysis Total RNA was isolated from rat ovary, mammary adipose (MG), or mammary tumor tissue using Trizol, followed by column clean-up (Qiagen). cDNA was prepared using the Verso kit (Thermo Scientific) and qPCR was performed using Taqman reagents, with commercially available predesigned Cyp19a1 primer/probe set (Rn00567222_m1; Life Technologies).

Techniques: Expressing, Control

Journal: Journal of Translational Medicine

Article Title: Type 2 diabetes is associated with decreased PGC1α expression in epicardial adipose tissue of patients with coronary artery disease

doi: 10.1186/s12967-016-0999-1

Figure Lengend Snippet: PGC1α, UCP1 and PRDM16 mRNA expression levels in EAT and SAT. TaqMan ® real time PCR for PGC1α, UCP1 and PRDM16 was performed on human EAT ( a – c ) and thoracic SAT ( d – f ) from coronary artery disease (CAD) patients with type 2 diabetes (CAD-DM2) (n = 22) and without it (CAD-NDM2) (n = 22) and non-CAD patients (NCAD) (n = 23). Results were expressed as the mean ± SEM of the completed experiment in duplicate. *P < 0.05 vs CAD-NDM2 and # P < 0.05 vs NCAD. PGC1α peroxisome proliferator-activated receptor gamma coactivator-1 alpha; UCP1 uncoupling protein 1; PRDM16 PR domain containing 16; EAT epicardial adipose tissue; SAT subcutaneous adipose tissue

Article Snippet: The gene expression levels in the adipose tissue were determined by real time quantitative polymerase chain reaction (PCR) using a predesigned and validated Taqman primer/probe sets [UCP1 (Hs00222453_m1, RefSeq NM_021833.4), PGC1α (Hs01016719_m1, RefSeq NM_013261.3), PRDM16 (Hs00922674_m1, RefSeq NM_022114.3) and cyclophilin A (Hs99999904_m1, RefSeq NM_021130.3)].

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Type 2 diabetes is associated with decreased PGC1α expression in epicardial adipose tissue of patients with coronary artery disease

doi: 10.1186/s12967-016-0999-1

Figure Lengend Snippet: Comparison of PGC1α, UCP1 and PRDM16 mRNAs in EAT and SAT. TaqMan ® real-time PCR analysis for mRNA expressions of PGC1α ( a ), UCP1 ( b ) and PRDM16 ( c ) in human adipose tissues (EAT and SAT) from coronary artery disease (CAD) patients. mRNA expression in thoracic SAT were compared as a fraction of epicardial fat, which was arbitrarily assigned the value of 1. Results were expressed as the mean ± SEM of the completed experiment in duplicate (n = 36). PGC1α peroxisome proliferator-activated receptor gamma coactivator-1 alpha; UCP1 uncoupling protein 1; PRDM16 PR domain containing 16; EAT epicardial adipose tissue; SAT subcutaneous adipose tissue

Article Snippet: The gene expression levels in the adipose tissue were determined by real time quantitative polymerase chain reaction (PCR) using a predesigned and validated Taqman primer/probe sets [UCP1 (Hs00222453_m1, RefSeq NM_021833.4), PGC1α (Hs01016719_m1, RefSeq NM_013261.3), PRDM16 (Hs00922674_m1, RefSeq NM_022114.3) and cyclophilin A (Hs99999904_m1, RefSeq NM_021130.3)].

Techniques: Comparison, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Translational Medicine

Article Title: Type 2 diabetes is associated with decreased PGC1α expression in epicardial adipose tissue of patients with coronary artery disease

doi: 10.1186/s12967-016-0999-1

Figure Lengend Snippet: Multiple regression analysis for prediction of EAT PGC1α mRNA levels

Article Snippet: The gene expression levels in the adipose tissue were determined by real time quantitative polymerase chain reaction (PCR) using a predesigned and validated Taqman primer/probe sets [UCP1 (Hs00222453_m1, RefSeq NM_021833.4), PGC1α (Hs01016719_m1, RefSeq NM_013261.3), PRDM16 (Hs00922674_m1, RefSeq NM_022114.3) and cyclophilin A (Hs99999904_m1, RefSeq NM_021130.3)].

Techniques: