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: 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: 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: iScience

Article Title: Balancing selection at the human salivary agglutinin gene ( DMBT1 ) driven by host-microbe interactions

doi: 10.1016/j.isci.2022.104189

Figure Lengend Snippet:

Article Snippet: For sequencing on a MinION (Oxford Nanopore), a sequencing library was prepared from total RNAusing the sequence-specific cDNA-PCR Sequencing kit (SQK-PCS-109, Oxford Nanopore Technologies, UK) following the manufacturer’s instructions.

Techniques: Virus, Recombinant, Sequencing, DNA Purification, Expressing, Positive Control, Software