Journal:

Article Title: hSK4, a member of a novel subfamily of calcium-activated potassium channels

doi:

Figure Lengend Snippet: Primary structure and tissue distribution of hSK4. (a) Amino acid sequence alignment of hSK4 and rSK1 with hSK1, rSK2, and rSK3. Sequences were aligned with the computer program pileup (GCG) using default parameters. Gaps are represented by dots. A dark line is drawn above putative transmembrane domains, denoted by the labels S1-S6, in addition to the P region. Shading denotes absolutely conserved residues. Consensus sites for phosphorylation by protein kinases A and C are marked by open squares and circles, respectively. Leucine heptad repeats are indicated by darkened boxes. The National Center for Biotechnology Information accession numbers for the nucleotide sequences of hSK4 and rSK1 are AF000972 and AF000973, respectively. (b) Kyte–Doolittle hydrophilicity analysis of hSK4 using a window of 20 residues. Numbers along the vertical axis refer to free energy of transfer to water. (c) Dendrogram based on the alignment in a. Horizontal branch lengths are inversely proportional to the similarity between sequences, whereas vertical branch lengths are for illustrative purposes only. (d) Northern blot analysis of hSK4 transcript using 3′ untranslated cDNA as probe. Molecular sizes are indicated in kilobases.

Article Snippet: This fragment then was labeled by random priming using a Prime-It II kit (Stratagene) and [ 32 P]dCTP to a specific activity of approximately 10 9 dpm/μg and used to probe a multiple tissue human Northern blot (CLONTECH).

Techniques: Sequencing, Northern Blot

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Mmu-miR-151-5p cleaves E2f6 in the absence of a seed match. (a) Genomic locus of mmu-miR-151 encoded by a LINE2 repeat element. (b) Schematic of the binding site of mmu-miR-151-5p to E2f6 3′UTR. (c) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (per 5p and mut 5p) in presence of miR-151-5p overexpression (sh-151-5p). (d) Western blot for E2f6in presence of sh-151-5p or a scrambled control (sh-scr). Actin serves as a loading control. Uncropped blot in . (e) E2f6 qPCR on miR-151-5p overexpression. Error bars, s.e.m. (n = 3 replicates). (f) Dual-luciferase reporter assay for E2f6 3′UTR or control ( Sox4 3′UTR) in presence of an increasing dosage of miR-151-5p inhibitor. (g) Dual-luciferase reporter assay in Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-5p and a functional copy of Ago2 or cleavage deficient Ago2 (D597A) or Ago1. (h) 5′-RACE of E2f6 . Arrowhead in the E2f6 3′UTR sequence (schematic) indicates the 5′ end of majority of E2f6 cleavage products in mouse lung tissue. Agarose gel showing E2f6 cleaved products (shown by an asterisk) is shown in the top gel (uncropped gel in ). The bottom gel serves as a RACE reaction control to detect the presence of E2f6 and ARHGDIA cDNAs. (c,f, g) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates), ns denotes not significant,*** P = 0.001 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Luciferase, Reporter Assay, Over Expression, Western Blot, Control, Functional Assay, Sequencing, Agarose Gel Electrophoresis, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: miR-151-3p suppresses E2f6 expression by binding to E2f6 3′UTR adjacent to where miR-151-5p binds. (a) Schematic of a putative binding site of the miR-151-3p to the E2f6 3′UTR region adjacent to where 5p strand binds, in both mice and humans. The seed regions (nucleotides 2-8) are indicated for both the 5p and 3p arms. (b) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (mut 3p and seed 3p as shown in the schematic) in presence of a miR-151-3p overexpression (sh-151-3p) or a scrambled control(sh-scr). A reporter construct with deletion of the entire miR-151-3p binding site in E2f6 3′UTR was also included. (c) Dual-luciferase reporter assay in wildtype MEF and Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-3p. For comparision, dual-luciferase reporter assay for E2f6 3′UTR in presence of sh-151-5p is also shown. (b,c) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Over Expression, Control, Construct

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Precursor miR-151 competes with the mature miR-151-5p for binding to E2f6 3′UTR. (a) Thermodynamics of pre-miR-151 binding to E2f6 . (b) Schematic of the stem-loop structure of the pre-miR-151 with the 5p arm (blue), 3p arm (purple) and two adenosines (green) substituted to guanosines (orange). (c) Northern analysis of miR-151 processing from pre-miR-151 overexpression plasmid (pEZX-151) or the double mutant form of pre-miR-151 (pEZX-DM). Let-7a serves as a loading control. (d) Dual-luciferase reporter assay for E2f6 3′UTR in presence of only the mature miR-151-5p (sh-miR-151-5p), or both the pre-miR-151 and mature miR-151-5p (pEZX-151 and pEZX-DM). (e) Schematic of the binding site of pre-miR-151 in E2f6 3′UTR and its modifications ( E2f6 3p del and E2f6 3p-5p swap). (f) In-vitro gel shift assay with radiolabed (denoted by an asterisk) synthetic pre-miR-151(I) or a control pre-miR-122 and increasing molar concentrations of wildtype E2f6 3′UTR (1, 10 and 100 nM) or its modified forms. (g) Dual-luciferase analysis for E2f6 3′UTR (wt), 3p del or 3p-5p swap reporters with pEZX-151 or pEZX-DM. (h) In-vitro gel shift assay of E2f6 3′UTR bound to miR-151-5p with increasing molar concentrations of a synthetic pre-miR-151 or a control pre-miR-122. Bands below the blue star and orange star represent radiolabeled pre-miR-151 and pre-miR-122 respectively. “*” denotes radiolabeled oligos. (d, g) For reporter assays, normalization was done with respect to a scrambled control (sh-scr). Error bars, s.e.m. (n = 3 biological replicates, each with 3 technical replicates). * P = 0.05, ** P = 0.01 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Northern Blot, Over Expression, Plasmid Preparation, Mutagenesis, Control, Luciferase, Reporter Assay, In Vitro, Gel Shift, Modification, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Pre-miR-151 binds to E2f6 in vivo and may protect the E2f6 transcript in quiescent tissues. (a) Schematic of the ChIRP method used to pull-down E2f6 mRNA from mouse brain. Quantitative PCR of (b) E2f6 mRNA and a control Ctdnep1 mRNA, (c) pre-miR-151 and a control pre-miR-124, (d) mature miR-151-5p and a control miR-124, pulled down by the biotinylated tiling oligonucleotides against the E2f6 3′UTR or a control lacZ mRNA. Quantitative PCR of (e) E2f6 mRNA, (f) pri-miR-151, (g) mature miR-151-5p in quiescent and non-quiescent tissues. In each case ( e — g ), the data are presented as fold induction after normalization to the liver sample (value = 1). (h) Northern analysis of miR-151 processing in various tissues. The blot on the left was probed with a LNA probe against mature miR-151-5p as shown by the schematic above the blot. The primary or intermediate product in the miR-151 biogenesis pathway is indicated by an arrowhead (→) and the mature miR-151-5p is indicated by a circle (○). U6 serves as a loading control. The blot on the right was probed (sequence is provided in ) for a region just outside the annotated stem loop structure of mmu-miR-151 (as shown by the schematic above the blot). (i) Quantitative PCR analyses of E2f6 , pri-miR-151 and miR-151-5pduring differentiation of muscle cells (C2C12) ( b — g, i ) For qPCR data, error bars, s.e.m. (n = 2 biological replicates, each with 3 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Control, Northern Blot, Sequencing

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Demonstration of the deletion in region 22q11.2. A. FISH technique. B. MLPA technique.

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Congenital heart diseases in 47 patients with the 22q11.2 deletion syndrome and the surgical corrections performed

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Phenotypic characteristics of 60 patients with the 22q11.2 deletion syndrome

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Main phenotypic characteristics of patients with the 22q11.2 deletion syndrome. A) Narrow palpebral fissure. B) Elongated face and/or nose. C) Thin lips.

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Pictures showing evolving features of patients with the 22q11.2 deletion syndrome at different ages. A) Newborn with thin lips and dysplasic ears. These phenotypic features become more characteristic at school age. B) Newborn with facial dysmorphism (elongated face and nose, narrow palpebral fissure, thin lips). C) Infant with elongated face and nose more evident during development

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Distribution of values of total lymphocytes, CD4 + , CD8 + and CD19 + in patients with the 22q11.2 deletion syndrome. A) Number of total lymphocytes. B) CD4 + count. C) CD8 + count. D) CD19 + count. Each dot ( • ) corresponds to an individual patient. Max: Maximum; Min: Mimum

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: p130/E2F4 Binds to and Represses the cdc2 Promoter in Response to p53

doi: 10.1074/jbc.m005101200

Figure Lengend Snippet: FIG. 1. Repression of cdc2/prolactin promoter chimeras by p53. A, lucifer- ase activity after G2 arrest induced by p53 overexpression. Stable pools of cells con- taining the promoter chimeras shown in B and C were released from a mimosine block in the presence or absence of tetra- cycline (TET). Removal of tetracycline in- duces the expression of high levels of p53, which causes cell cycle arrest mainly in G2. Luciferase activity was measured 48 h after removal of mimosine. Rlu: relative light units. Standard errors are shown by the bars. B, schematic diagrams of chime- ras and average fold repression from two independent experiments (data shown in A). C, sequences of chimeric promoter constructs in the region of the R box.

Article Snippet: Membranes were probed with monoclonal antibodies specific for p53 (DO-1), actin (C-2), and rabbit polyclonal antibodies specific for Cdc2 (C-19), p130 (C-20), Rb (C-15), p107 (C-18), or E2F4 (C108), all from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Over Expression, Blocking Assay, Expressing, Luciferase, Construct

Journal: Journal of Biological Chemistry

Article Title: p130/E2F4 Binds to and Represses the cdc2 Promoter in Response to p53

doi: 10.1074/jbc.m005101200

Figure Lengend Snippet: FIG. 2. Effects of mutations in the CDE and CHR elements on repression of the cdc2 promoter by p53. A, fold repression of wild- type and mutant promoters. Mutations were generated in a cdc2 pro- moter fragment extending to 294 relative to the start of transcription. Wild-type and mutant reporter constructs were stably transfected into TR9-7 cells. Asynchronously growing pools of cells were incubated in the presence or absence of tetracycline for 72 h, followed by measure- ment of luciferase activity. Standard errors were less that 10% of the mean in all experiments. mCDE, mutant CDE; mCHR, mutant CHR. B, details of mutations in the cdc2 promoter and comparison with the relevant regions of the chimeric promoters shown in Fig. 1.

Article Snippet: Membranes were probed with monoclonal antibodies specific for p53 (DO-1), actin (C-2), and rabbit polyclonal antibodies specific for Cdc2 (C-19), p130 (C-20), Rb (C-15), p107 (C-18), or E2F4 (C108), all from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Mutagenesis, Generated, Construct, Stable Transfection, Transfection, Incubation, Luciferase, Activity Assay, Comparison

Journal: Journal of Biological Chemistry

Article Title: p130/E2F4 Binds to and Represses the cdc2 Promoter in Response to p53

doi: 10.1074/jbc.m005101200

Figure Lengend Snippet: FIG. 4. Role of p21/waf1 in repression of the cdc2 promoter by p53. A, HCT116 cells in which p21/waf1 was inactivated by gene tar- geting were transfected transiently with various amounts of a p53 expression plasmid and either the cdc2 promoter (CC) or prolactin chimeric (PP) reporter constructs described in Fig. 1. Luciferase activity measured 48 h after transfection was corrected for transfection effi- ciency by using a cotransfected b-galactosidase construct. A represent- ative experiment is shown. B, role of p21/waf1 in repression of the cdc2 promoter in response to DNA damage. HCT116 cells with or without p21/waf1 were transfected stably with the CC or PP reporter constructs. Stable pools of cells were treated with adriamycin to induce DNA damage, followed by measurement of luciferase activity 48 h later. Standard errors were less than 15% of the mean in all experiments. C, effects of overexpression of p21/waf1 on the cdc2 promoter. A pool of TR9-7 cells stably transfected with a reporter construct extending up to 294 of the cdc2 promoter was infected at the indicated multiplicities with an adenovirus to overproduce p21/waf1. Luciferase activity was measured 72 h after infection. Standard errors are shown by the bars.

Article Snippet: Membranes were probed with monoclonal antibodies specific for p53 (DO-1), actin (C-2), and rabbit polyclonal antibodies specific for Cdc2 (C-19), p130 (C-20), Rb (C-15), p107 (C-18), or E2F4 (C108), all from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Luciferase, Activity Assay, Stable Transfection, Over Expression, Infection

Journal: Journal of Biological Chemistry

Article Title: p130/E2F4 Binds to and Represses the cdc2 Promoter in Response to p53

doi: 10.1074/jbc.m005101200

Figure Lengend Snippet: FIG. 6. Detection of E2F4 and Rb family members in TR9-7 cells. A, Western analysis of TR9-7 cells with an- tibodies to p53, Rb, p107, p130, and E2F4 before and after induction of p53. Asyn- chronously growing TR9-7 cells were in- cubated for 48 h in the presence or ab- sence of tetracycline (TET) to induce p53 expression. Cells incubated for 48 h in 0.25% serum and then stimulated for 24 h with 10% serum are also shown. B, gel mobility shift analysis using a consensus binding site for E2F-containing com- plexes. TR9-7 cells were released from a mimosine block in the presence or ab- sence of tetracycline to induce p53- dependent G2 arrest. Nuclear lysates were incubated with a radioactively la- beled probe in the presence or absence of antibodies to p130 or E2F4.

Article Snippet: Membranes were probed with monoclonal antibodies specific for p53 (DO-1), actin (C-2), and rabbit polyclonal antibodies specific for Cdc2 (C-19), p130 (C-20), Rb (C-15), p107 (C-18), or E2F4 (C108), all from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Incubation, Mobility Shift, Binding Assay, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: p130/E2F4 Binds to and Represses the cdc2 Promoter in Response to p53

doi: 10.1074/jbc.m005101200

Figure Lengend Snippet: FIG. 7. Gel mobility shift analysis of the cdc2 promoter. The experiments were carried out with a radioactively la- beled probe derived from the R box of the human cdc2 promoter. TR9-7 cells re- leased from a mimosine block and incu- bated in the presence or absence of tetra- cycline (TET) for 72 h were lysed and analyzed. A, effect of p53 expression on complexes bound to the R box region of the human cdc2 promoter. The analysis was carried out in the presence or absence of an antibody to p130. B, supershift anal- ysis of a p53-induced complex with anti- bodies to E2F1, E2F4, and Rb family members. Lysates from cells arrested in G2 by overexpression of p53 were used for analyses in the presence or absence of the indicated antibodies. C, competition anal- ysis of a p53-induced complex. Binding reactions were carried out in the absence or presence of the indicated competitor probes, added at a 100-fold molar excess over the labeled probe. Competition was carried out with probes with a wild-type sequence or with mutations in either the CDE or CHR, as shown. Mutated bases are shown in lowercase letters. The muta- tions are the same as used in Fig. 2.

Article Snippet: Membranes were probed with monoclonal antibodies specific for p53 (DO-1), actin (C-2), and rabbit polyclonal antibodies specific for Cdc2 (C-19), p130 (C-20), Rb (C-15), p107 (C-18), or E2F4 (C108), all from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Mobility Shift, Derivative Assay, Blocking Assay, Expressing, Over Expression, Binding Assay, Labeling, Sequencing

Journal: eBioMedicine

Article Title: Metabolic role of the hepatic valine/3-hydroxyisobutyrate (3-HIB) pathway in fatty liver disease

doi: 10.1016/j.ebiom.2023.104569

Figure Lengend Snippet: Hepatic HIBCH mRNA expression and plasma 3-HIB concentrations are associated with fatty liver . A: Graphical representation of Spearman correlations for hepatic HIBCH mRNA and different variables in 66 liver donors with different degrees of liver fat content and known NAFLD/NASH status ranging in BMI from 23 to 46 kg/m 2 (Liver cohort). Correlations are significant for p < 0.05 (indicated by red outline for the analysis without adjustment for multiple testing). B: Hepatic HIBCH mRNA expression in people from the Liver cohort. Participants were stratified into different groups based on NAFLD/NASH status, BMI, T2D status and SAT and VAT adiposity. C: Graphical representation of Spearman correlations for plasma 3-HIB and different variables in 192 participants with abdominal obesity (BMI ≥ 30 kg/m 2 ) and/or WC ≥ 102 cm (for males) and WC ≥ 88 cm (for females) (CARBFUNC cohort). Liver density was measured by CT imaging and calculated as HU units and divided by spleen density. Because increased liver density reflects lower fat content, the correlation coefficient in the figure was inverted to a positive value (i.e., reflecting more liver fat). Abd. SAT, abdominal subcutaneous adipose tissue; Clamp GIR, glucose infusion rate from euglycemic hyperinsulinemic clamp; FFA, free fatty acids; HDL-C, high-density lipoprotein cholesterol; IL-6, interleukin-6; LDL-C, low-density lipoprotein cholesterol; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; TAG, triacylglycerols; WC, waist circumference; WHR; Waist-Hip-Ratio.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (One-way ANOVA–Sidak's test, Kruskal–Wallis—Dunn's test or Mann–Whitney test).

Article Snippet: Gene expression in liver samples (1 μg RNA input) was analyzed by qPCR using the TaqMan assay as described previously with target-specific probes ( HIBCH : Hs00961835_g1; PDK4 : Hs01037712_m1; HPRT1 : Hs01003267_m1).

Techniques: Expressing, Imaging, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Metabolic role of the hepatic valine/3-hydroxyisobutyrate (3-HIB) pathway in fatty liver disease

doi: 10.1016/j.ebiom.2023.104569

Figure Lengend Snippet: Lipid storage in hepatocytes is accompanied by increased HIBCH expression and release of 3-HIB . Huh7 liver cells were treated with and without free FAs (1:1 M ratio of 50 μM PA and 50 μM OA) for 24 h before analyses. Gene expression was measured by RNA-sequencing. A: HIBCH expression in control and FA-treated cells, shown as RPKM (n = 6). B: Representative images of Oil-Red-O lipid-stained hepatocytes (left) and quantification of lipid accumulation (right) in Huh7 cells (n = 6). C: Relative FA uptake in Huh7 cells treated with or without FA for 24 h, followed by addition of fluorescent dodecanoic acid whose uptake was detected spectrophotometrically after 1 h (n = 9–12). D: Average net medium appearance per hour of 3-HIB during a 24 h period (n = 6). E: A linear mixed model accounting for the group structure was used to examine the correlation between HIBCH mRNA expression (RPKM) and extracellular 3-HIB concentrations (GC–MS/MS analysis of cell culture medium) across the control and FA-treated samples. F: GSEA showing up- and down-regulated pathways by HIBCH expression (HIBCH correlation) in Huh7 (HALLMARK pathway analysis) (n = 6). Gene sets are ordered by normalized enrichment score (NES) and significant adjusted p-value for each pathway are shown. Ctrl, control; FA, fatty acids; GSEA, gene set enrichment analysis; RPKM, reads per kilobase per million mapped reads.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Unpaired t-test or Mann–Whitney test).

Article Snippet: Gene expression in liver samples (1 μg RNA input) was analyzed by qPCR using the TaqMan assay as described previously with target-specific probes ( HIBCH : Hs00961835_g1; PDK4 : Hs01037712_m1; HPRT1 : Hs01003267_m1).

Techniques: Expressing, RNA Sequencing Assay, Control, Staining, Gas Chromatography-Mass Spectrometry, Cell Culture, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Metabolic role of the hepatic valine/3-hydroxyisobutyrate (3-HIB) pathway in fatty liver disease

doi: 10.1016/j.ebiom.2023.104569

Figure Lengend Snippet: HIBCH overexpression in hepatocytes affects cellular pathways related to FA metabolism and oxidative phosphorylation . Huh7 liver cells were transfected with pCMV6-HIBCH or control (pCMV6-empty vector) plasmid (0.2 μg per well in a 24-well plate) diluted in Opti-MEM® Reduced Serum Media and TransIT-X2® transfection reagent (Mirus). 24 h after transfection, the cells were treated with and without free FAs (1:1 M ratio of 50 μM PA and 50 μM OA) for 24 h before analyses. A: HIBCH expression in Huh7 cells measured by qPCR (n = 6). B: The quantitative values of HIBCH relative to α-VINCULIN in Huh7 cells (n = 3). C: Average net medium appearance per hour of 3-HIB and valine during a 24 h period (n = 6). D: Relative FA uptake in Huh7 cells treated with or without FA for 24 h, followed by addition of fluorescent dodecanoic acid whose uptake was detected spectrophotometrically after 1 h (n = 9–12). E: GSEA showing up-and down-regulated pathways by HIBCH overexpression relative to control expression (without and with FAs) in Huh7 (HALLMARK pathway analysis of RNA sequencing data) (n = 24). Gene sets are ordered by normalized enrichment score (NES) and significant adjusted p-values for each pathway are shown. F: Number of up-and down-regulated genes by HIBCH overexpression with/without FA treatment in Huh7 cells shown as a Venn diagram (adjusted p-value cutoff <0.1). FA, fatty acid treatment; NES, normalized enrichment score.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (–unpaired t-test or Mann–Whitney test).

Article Snippet: Gene expression in liver samples (1 μg RNA input) was analyzed by qPCR using the TaqMan assay as described previously with target-specific probes ( HIBCH : Hs00961835_g1; PDK4 : Hs01037712_m1; HPRT1 : Hs01003267_m1).

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Expressing, RNA Sequencing Assay, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Metabolic role of the hepatic valine/3-hydroxyisobutyrate (3-HIB) pathway in fatty liver disease

doi: 10.1016/j.ebiom.2023.104569

Figure Lengend Snippet: HIBCH knockdown and FA treatment in hepatocytes have both similar and distinct metabolic effects . Huh7 liver cells were treated with and without free Fas (1:1 M ratio of 50 μM PA and 50 μM OA) combined with siRNA-mediated knockdown of HIBCH and siRNA non-targeting control for 24 h before analyses. A: HIBCH expression in Huh7 cells measured by mRNA sequencing (n = 6). B: Average net medium appearance per hour of 3-HIB and valine during a 24 h period (n = 6). C: Relative FA uptake in Huh7 cells treated with or without FA for 24 h, followed by addition of fluorescent dodecanoic acid whose uptake was detected spectrophotometrically after 1 h (n = 9–12). D: Principal component (PC) analysis of samples in all four treatment groups in the experiment are shown. E: Representative functional categories and genes in the four different gene expression patterns responding to FA treatment and HIBCH knockdown. FA, fatty acid treatment; KD, knockdown.∗p < 0.05, ∗∗P < 0.01, ∗∗∗p < 0.001 (Ordinary one-way ANOVA–Sidak's test).

Article Snippet: Gene expression in liver samples (1 μg RNA input) was analyzed by qPCR using the TaqMan assay as described previously with target-specific probes ( HIBCH : Hs00961835_g1; PDK4 : Hs01037712_m1; HPRT1 : Hs01003267_m1).

Techniques: Knockdown, Control, Expressing, Sequencing, Functional Assay

Journal: eBioMedicine

Article Title: Metabolic role of the hepatic valine/3-hydroxyisobutyrate (3-HIB) pathway in fatty liver disease

doi: 10.1016/j.ebiom.2023.104569

Figure Lengend Snippet: HIBCH and 3-HIB are responsive to PDK4 inhibition . A: Graphical representation of Spearman correlations for hepatic PDK4 mRNA and different variables in 66 liver donors with different degrees of liver fat content and known NAFLD/NASH status ranging in BMI from 23 to 46 kg/m 2 (Liver cohort). Correlations are significant for p < 0.05 (indicated by black outline for the analysis without adjustment for multiple testing). B: Hepatic PDK4 mRNA expression in participants from the Liver cohort. Participants were stratified based on NAFLD/NASH status, BMI, T2D status and SAT and VAT adiposity. C–E: Huh7 liver cells were treated with and without free FAs (1:1 M ratio of 50 μM PA and 50 μM OA) combined with PDK4 inhibitor (final concentration of 6 or 12 μM PS10) (DMSO was used as control) for 24 h before analyses. C: HIBCH mRNA expression in Huh7 cells measured by qPCR, calculated relative to the reference gene HPRT (n = 6). D: Effect of PDK4 inhibitor on relative FA uptake in Huh7 cells treated with or without FA for 24 h, followed by addition of fluorescent dodecanoic acid whose uptake was detected spectrophotometrically after 1 h (n = 9–12). E: Average net medium appearance per hour of 3-HIB and valine in Huh7 cells during a 24 h period (n = 6) in response to PDK4 inhibitor. Clamp GIR, glucose infusion rate from euglycemic hyperinsulinemic clamp; FA, fatty acid treatment; FFA, free fatty acids; HDL-C, high-density lipoprotein cholesterol; IL-6, interleukin-6; LDL-C, low-density lipoprotein cholesterol; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; TAG, triacylglycerols.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Ordinary one-way ANOVA–Sidak's test, unpaired t-test, Kruskal–Wallis—Dunn's test or Mann–Whitney test).

Article Snippet: Gene expression in liver samples (1 μg RNA input) was analyzed by qPCR using the TaqMan assay as described previously with target-specific probes ( HIBCH : Hs00961835_g1; PDK4 : Hs01037712_m1; HPRT1 : Hs01003267_m1).

Techniques: Inhibition, Expressing, Concentration Assay, Control, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Metabolic role of the hepatic valine/3-hydroxyisobutyrate (3-HIB) pathway in fatty liver disease

doi: 10.1016/j.ebiom.2023.104569

Figure Lengend Snippet: Altered HIBCH expression affects mitochondrial respiration, ROS production and extracellular metabolite concentrations . Huh7 liver cells were transfected with siRNA-mediated knockdown of HIBCH and siRNA non-targeting control or with pCMV6-HIBCH or control (pCMV6-empty vector) plasmid (0.2 μg per well in a 24-well plate) diluted in Opti-MEM® Reduced Serum Media and TransIT-X2® transfection reagent (Mirus). The cells were treated with and without free FAs (1:1 M ratio of 50 μM PA and 50 μM OA) for 24 h, before analyses. A: Seahorse Cell Mito Stress Assay (OCR measurements) was performed using the Seahorse XFe96 Analyzer to assess the mitochondrial respiration in Huh7 cells (n = 10–12) Basal levels (the three first OCR measurements) were obtained, before adding oligomycin, CCCP and rotenone/antimycin A. Basal respiration, ATP production, maximal respiration, spare capacity and uncoupling were calculated for each well based on the OCR measurements. B: Effect of HIBCH knockdown on ROS production in Huh7 cells treated with or without FA for 24 h, followed by addition of fluorescent probe whose uptake was detected spectrophotometrically after 1 h (n = 11). C: Average net medium appearance per hour of the metabolites during a 24 h period (n = 6). FA, fatty acid; KD, knockdown; OCR, oxygen consumption rate.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (unpaired t-test).

Article Snippet: Gene expression in liver samples (1 μg RNA input) was analyzed by qPCR using the TaqMan assay as described previously with target-specific probes ( HIBCH : Hs00961835_g1; PDK4 : Hs01037712_m1; HPRT1 : Hs01003267_m1).

Techniques: Expressing, Transfection, Knockdown, Control, Plasmid Preparation

Journal: eBioMedicine

Article Title: Metabolic role of the hepatic valine/3-hydroxyisobutyrate (3-HIB) pathway in fatty liver disease

doi: 10.1016/j.ebiom.2023.104569

Figure Lengend Snippet: Overview of BCAA and propionate metabolism and effects of HIBCH knockdown in cultured Huh7 hepatocytes . Down- and upregulated genes (from RNA-seq) are marked in blue and red colour, respectively. Increased and decreased net medium appearance of metabolites based on extracellular measurements are marked with dotted lines in blue and red colour, respectively. Genes and metabolites that are unchanged by HIBCH knockdown are marked in grey colour and black dotted lines, respectively.

Article Snippet: Gene expression in liver samples (1 μg RNA input) was analyzed by qPCR using the TaqMan assay as described previously with target-specific probes ( HIBCH : Hs00961835_g1; PDK4 : Hs01037712_m1; HPRT1 : Hs01003267_m1).

Techniques: Knockdown, Cell Culture, RNA Sequencing Assay

Journal: eBioMedicine

Article Title: Metabolic role of the hepatic valine/3-hydroxyisobutyrate (3-HIB) pathway in fatty liver disease

doi: 10.1016/j.ebiom.2023.104569

Figure Lengend Snippet: 3-HIB supplementation to human hepatocytes alters key metabolic functions . Huh7 liver cells were treated with and without free FAs (1:1 M ratio of 50 μM PA and 50 μM OA) combined with and without 3-HIB supplementation (final concentration of 25 μM) (water was used as control), for 24 h before analyses. A: HIBCH mRNA expression in Huh7 cells measured by qPCR, calculated relative to the reference gene HPRT (n = 6). B: Relative FA uptake in Huh7 cells treated with or without FA for 24 h, followed by addition of fluorescent dodecanoic acid whose uptake was detected spectrophotometrically after 1 h (n = 9–12). C: Seahorse Cell Mito Stress Assay (OCR measurements) was performed using the Seahorse XFe96 Analyzer to assess the mitochondrial respiration in Huh7 (n = 10–12) 24 h after treatment. Basal levels (the three first OCR measurements) were obtained, before adding oligomycin, CCCP and rotenone/antimycin A, as indicated at the top in the upper left figure. Basal respiration, ATP production, maximal respiration, spare capacity and uncoupling were calculated for each well based on the OCR measurements. D: Effect of 3-HIB supplementation on ROS generation in Huh7 cells treated with or without FA for 24 h, followed by addition of fluorescent probe whose uptake was detected spectrophotometrically after 1 h (n = 11). FA, fatty acid treatment.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (unpaired t-test).

Article Snippet: Gene expression in liver samples (1 μg RNA input) was analyzed by qPCR using the TaqMan assay as described previously with target-specific probes ( HIBCH : Hs00961835_g1; PDK4 : Hs01037712_m1; HPRT1 : Hs01003267_m1).

Techniques: Concentration Assay, Control, 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: Blood

Article Title: MicroRNA regulation of STAT4 protein expression: rapid and sensitive modulation of IL-12 signaling in human natural killer cells.

doi: 10.1182/blood-2011-05-356162

Figure Lengend Snippet: Figure 1. miRNAs directly target sequences in the STAT4 3UTR. (A) Human STAT4 might be the molecular target of miR-132, miR-212, and miR-200a. This diagram represents a sequence alignment of miR-132, -212, and -200a and their target sites in STAT4 3UTR, and relative mutated versions. (B) miRNAs reduced luciferase activity in cells transfected with wild-type reporter (WT-3UTR), but not in cells transfected with mutated-type reporter (132/212 or 200a MT-3UTR). HEK293T cells were cotransfected with wild (or mutated) type STAT4 3UTR firefly luciferase reporter plasmids, pTK-Renilla-luciferase plasmids, together with control (ctrl), miR-132, miR-212, miR-200a mimics, or equimolar mixtures of the 3 indicated miRNAs (final concentration as indicated). After 48 hours, firefly luciferase activity was measured and normalized by renilla luciferase activity. Representative data from 3 independent experiments are shown, and each bar indicates the mean value SEM (n 3). *P .05, **P .01 versus cells transfected control mimics.

Article Snippet: After the blocking step, the blots were probed with specific anti-human STAT4 antibodies (Santa Cruz Biotechnology), and then visualized with appropriate HRP-conjugated secondary antibody (Pierce) and an ECL detection system (Pierce).

Techniques: Sequencing, Luciferase, Activity Assay, Transfection, Control, Concentration Assay

Journal: Blood

Article Title: MicroRNA regulation of STAT4 protein expression: rapid and sensitive modulation of IL-12 signaling in human natural killer cells.

doi: 10.1182/blood-2011-05-356162

Figure Lengend Snippet: Figure 2. Dose- and time-dependent expression of IFN- and STAT4 in IL-12-treated NK cells. (A) NK cells were treated with 0, 0.1, 1, or 10 ng/mL IL-12 and incubated for the times indicated in the presence of IL-2. Culture supernatants were collected at the times indicated points from 2 to 32 hours. The IFN- levels in the medium were quantified by ELISA. Bars at 2, 4, and 8 hours indicate cumulative IFN- levels in culture supernatants; Bars at 16, 24 and 32 hours indicate IFN- production in each preceding 8 hours in culture supernatants. (B) NK cells were treated with 0 or 10 ng/mL IL-12 and incubated for indicated times in presence of IL-2. Total RNA was purified from the respective cell pellets and analyzed by qRT-PCR for the expression of IFN- mRNA. (C) NK cells were cultured as in panel A. Cells were collected at the times indicated and whole-cell extracts were prepared. Western blots were performed with anti-STAT4. (D) NK cells were treated with 0 or 10 ng/mL IL-12 and incubated for indicated times in presence of IL-2. Total RNA was purified from the respective cell pellets and analyzed by qRT-PCR for the expression of STAT4 mRNA. “2 12 / 2” represents the fold change in mRNA levels calculated by comparing the value of IL-2 IL-12–cultured cells (2 12) to that of IL-2-cultured samples2 in parallel. The results are mean SEM of representative experiments with NK cells from 3 different donors. *P .05, **P .01 vs 16 hours (A) or 8 hours (B) same concentration of IL-12 treated cells. #P .05, ##P .01 versus 0 hours untreated cells.

Article Snippet: After the blocking step, the blots were probed with specific anti-human STAT4 antibodies (Santa Cruz Biotechnology), and then visualized with appropriate HRP-conjugated secondary antibody (Pierce) and an ECL detection system (Pierce).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Cell Culture, Western Blot, Concentration Assay

Journal: Blood

Article Title: MicroRNA regulation of STAT4 protein expression: rapid and sensitive modulation of IL-12 signaling in human natural killer cells.

doi: 10.1182/blood-2011-05-356162

Figure Lengend Snippet: Figure 6. miR-132, miR-212, and miR-200a negatively regulate STAT4 expression and IL-12–induced IFN- production in NK cells. (A) Human NK cells (2 105) were transfected with control mimics (miR ctrl), miR-491-5p, miR-132, miR-212, miR-200a, or an equimolar mixture of miR-132, miR-212, and miR-200a (miR mix) mimics as indicated at an aggregate final concentration of 50nM. After 48 hours culture in the presence of IL-2, the expression of STAT4 was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. (B) Summary of data from panel A, mean of STAT4 levels in indicated miRNA overexpression cells were analyzed to determine the relative effects of different miRNAon STAT4 protein levels. The results are STAT4 MFI mean SEM of representative experiment with NK cells from 3 different donors. *P .05 versus miR ctrl. (C) Cells treated as in panel A (but not harvested) were washed with complete growth medium and cultured with or without 10 ng/mL of IL-12 for another 12 hours in the presence of IL-2. Cellular expression of phosphorylated STAT4 was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. (D) Summary of data from panel C. Mean of p-STAT4 levels in indicated miRNA overexpression cells was analyzed to determine the relative effects of different miRNA on IL-12–induced STAT4 phosphorylation. The results are p-STAT4 MFI mean SEM of representative experiment with NK cells from 3 different donors. *P .05 versus miR ctrl. (E) Cells treated as in panel C. Cellular expression of IFN- was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. (F) Summary of data from panel E. Mean of IL-12–induced IFN- expression in indicated miRNA overexpression cells (IFN- MFI with IL-12 – IFN- MFI without IL-12) was calculated to determine the relative effects of different miRNA on IL-12–induced IFN- expression. The results are increased IFN- MFI mean SEM of representative experiment with NK cells from 3 different donors. *P .05, **P .01 versus miR ctrl. The gray dotted histogram represents the isotype control staining of NK cells.

Article Snippet: After the blocking step, the blots were probed with specific anti-human STAT4 antibodies (Santa Cruz Biotechnology), and then visualized with appropriate HRP-conjugated secondary antibody (Pierce) and an ECL detection system (Pierce).

Techniques: Expressing, Transfection, Control, Concentration Assay, Cytometry, Over Expression, Cell Culture, Phospho-proteomics, Staining

Journal: Blood

Article Title: MicroRNA regulation of STAT4 protein expression: rapid and sensitive modulation of IL-12 signaling in human natural killer cells.

doi: 10.1182/blood-2011-05-356162

Figure Lengend Snippet: Figure 7. Inhibition of IL-12–induced miR-132, miR-212, or miR-200a blocks the reduction of STAT4 induced by IL-12 and increases the IFN- production induced by further IL-12 stimulation. (A) Human NK cells (2 105) were transfected with control inhibitor (anti-miR ctrl), anti–miR-132, anti–miR-212, anti–miR-200a, or an equimolar mixture of anti–miR-132, miR-212, and miR-200a inhibitor (anti-miR mix) as indicated at an aggregate final concentration of 50nM. These transfected cells were treated with or without IL-12 (10 ng/mL) for 48 hours in the presence of IL-2, and the expression of STAT4 was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. The gray dotted line represents the isotype control staining of NK cells. (B) Summary of data from panel A, mean of IL-12–induced STAT4 reduction in cells transfected with anti-miR as indicated (STAT4 MFI without IL-12 – STAT4 MFI with IL-12) was calculated to determine the relative inhibitory effects of different miRNA inhibitors on IL-12–induced STAT4 reduction. The results are STAT4 MFI decreased mean SEM of representative experiment with NK cells from 3 different donors. *P .05, **P .01 versus anti-miR ctrl. (C) Cells treated with IL-12 and the indicated mir inhibitors as in panel A (but not harvested) were washed with complete growth medium and secondarily stimulated with 10 ng/mL of IL-12 for 12 hours in the presence of IL-2. Cellular expression of IFN- was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. The gray dotted line represents the isotype control staining of NK cells. (D) Summary of data from panel C, mean of the inhibitory effect of IL-12 pretreatment on secondary IL-12–induced IFN- production in indicated anti-miR–transfected cells (IFN- MFI without IL-12 priming – IFN- MFI with IL-12 priming) was calculated to determine the relative inhibitory effects of different miRNA inhibitors on the inhibitory effect of IL-12 pretreatment on secondary IL-12–induced IFN- production. The results are decreased IFN- MFI mean SEM of representative experiment with NK cells from 3 different donors. *P .05 versus anti-miR ctrl.

Article Snippet: After the blocking step, the blots were probed with specific anti-human STAT4 antibodies (Santa Cruz Biotechnology), and then visualized with appropriate HRP-conjugated secondary antibody (Pierce) and an ECL detection system (Pierce).

Techniques: Inhibition, Transfection, Control, Concentration Assay, Expressing, Cytometry, Staining