Journal: PLoS ONE

Article Title: Transcriptional and Epigenetic Regulation of KIF14 Overexpression in Ovarian Cancer

doi: 10.1371/journal.pone.0091540

Figure Lengend Snippet: Promoter deletion constructs fused to Luciferase were tested for activity in HeLa, SKOV3 and OvCa429 cells. pRSV-Luc, positive control, 0, empty vector control. Numbers are base pairs relative to the transcriptional start site (0). Bioinformatic analysis (Genomatix) of the most active region within the KIF14 promoter (−2366 to −2245) identified putative binding sites for transcription factors YY1, HSF1 and Sp1. Specific transcription factor recognition sites are underlined, and space within sequence denotes location of deletion constructs. N = 3, * Significance at P <0.05, unpaired t-test. P = 0.01 (HeLa); P = 0.01 (SKOV3); P = 0.03 (OvCa429).

Article Snippet: 1 μL of synthesized cDNA was added to 1X TaqMan PCR master mix (ABI), and 1X TaqMan Gene Expression Assay primer-probe mix for KIF14 (Hs00978216_m1), Sp1 (Hs00916521_m1), YY1 (Hs00231533_m1), and HSF1 (Hs01027608_g1).

Techniques: Construct, Luciferase, Activity Assay, Positive Control, Plasmid Preparation, Control, Binding Assay, Sequencing

Journal: PLoS ONE

Article Title: Transcriptional and Epigenetic Regulation of KIF14 Overexpression in Ovarian Cancer

doi: 10.1371/journal.pone.0091540

Figure Lengend Snippet: siRNA knockdown of endogenous Sp1 (orange), HSF1 (green), and YY1 (red) transcription factors, and measurement of their mRNA expression along with corresponding KIF14 mRNA levels (blue) via real-time PCR in A SKOV3 and B OvCa429 cells. Y-axes: normalized mRNA expression relative to MOCK. GL2, control siRNA; N = 3, * Significance at P <0.05, unpaired t-test. Three different siRNA molecules (A, B, C) were used to knock down each gene. P values for panel A (SKOV3 cells): P = 0.02 for Sp1 expression (orange) with Sp1 siRNAs A, B, and C; P = 0.03 (siRNA-A), P = 0.047 (siRNA–B), P = 0.04 (siRNA–C) for KIF14 expression (blue). P = 0.001 for HSF1 expression (green) with HSF1 siRNAs A, B, and C; P = 0.23 (siRNA-A), P = 0.12 (siRNA-B), P = 0.4 (siRNA-C) for KIF14 expression (blue). P = 0.01 for YY1 expression (red) with YY1 siRNAs A, B and C; P = 0.006 for KIF14 expression (blue) with YY1 siRNAs A, B, and C. P values for panel B (OvCa429 cells): P = 0.03 for Sp1 expression (orange) with Sp1 siRNAs A, B, and C; P = 0.05 (siRNA-A), P = 0.04 (siRNA-B), P = 0.045 (siRNA-C) for KIF14 expression (blue). P = 0.003 for HSF1 expression (green) with HSF1 siRNAs A, B, and C; P = 0.31 (siRNA-A), P = 0.45 (siRNA-B), P = 0.39 (siRNA-C) for KIF14 expression (blue). P = 0.02 (siRNA-A), P = 0.01 (siRNA-B), P = 0.01 for YY1 expression (red); P = 0.02 (siRNA-A), P = 0.001 (siRNA-B), P = 0.006 (siRNA-C) for KIF14 expression (blue).

Article Snippet: 1 μL of synthesized cDNA was added to 1X TaqMan PCR master mix (ABI), and 1X TaqMan Gene Expression Assay primer-probe mix for KIF14 (Hs00978216_m1), Sp1 (Hs00916521_m1), YY1 (Hs00231533_m1), and HSF1 (Hs01027608_g1).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: Transcriptional and Epigenetic Regulation of KIF14 Overexpression in Ovarian Cancer

doi: 10.1371/journal.pone.0091540

Figure Lengend Snippet: A siRNA knockdown of endogenous Sp1 (orange), HSF1 (green), and YY1 (red) transcription factors, and measurement of their protein expression along with corresponding KIF14 protein levels (blue) via immunoblot in SKOV3 cells. x-axis: normalized protein expression relative to MOCK. B Representative immunoblot of KIF14 and transcription factor expression. Numbers represent normalized expression values for the experiment shown. Similar results were seen with OvCa429 cells. GL2, control siRNA; N = 3; *, P <0.05 for transcription factor expression; #, P <0.05 for KIF14 expression, unpaired t-test. P values for panel A (SKOV3 cells): P = 0.009 (siRNA-A), P = 0.003 (siRNA-B), P = 0.006 (siRNA-C) for Sp1 expression (orange); P = 0.005 (siRNA-A), P = 0.007 (siRNA-B), P = 0.004 (siRNA-C) for KIF14 expression (blue). P = 0.01 for HSF1 expression (green) with HSF1 siRNAs A, B, and C; P = 0.54 (siRNA-A), P = 0.65 (siRNA-B), P = 0.41 (siRNA-C) for KIF14 expression (blue). P = 0.001 for YY1 expression (red) with YY1 siRNAs A, B and C; P = 0.01 (siRNA-A), P = 0.02 (siRNA-B), P = 0.005 (siRNA-C) for KIF14 expression (blue).

Article Snippet: 1 μL of synthesized cDNA was added to 1X TaqMan PCR master mix (ABI), and 1X TaqMan Gene Expression Assay primer-probe mix for KIF14 (Hs00978216_m1), Sp1 (Hs00916521_m1), YY1 (Hs00231533_m1), and HSF1 (Hs01027608_g1).

Techniques: Knockdown, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Transcriptional and Epigenetic Regulation of KIF14 Overexpression in Ovarian Cancer

doi: 10.1371/journal.pone.0091540

Figure Lengend Snippet: ChIP assays of endogenous YY1, Sp1 and HSF1 followed by real time PCR with the KIF14 promoter region (−2300 to −2133) in cell lines SKOV3, OvCa429, and HeLa compared to IgG (negative control). Values represent average quantity of promoter region product relative to IgG control. Error bars represent standard deviation of triplicate assays.

Article Snippet: 1 μL of synthesized cDNA was added to 1X TaqMan PCR master mix (ABI), and 1X TaqMan Gene Expression Assay primer-probe mix for KIF14 (Hs00978216_m1), Sp1 (Hs00916521_m1), YY1 (Hs00231533_m1), and HSF1 (Hs01027608_g1).

Techniques: Real-time Polymerase Chain Reaction, Negative Control, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Transcriptional and Epigenetic Regulation of KIF14 Overexpression in Ovarian Cancer

doi: 10.1371/journal.pone.0091540

Figure Lengend Snippet: Quantitative mRNA expression analysis of primary OvCa tumors with KIF14 HIGH (red) and KIF14 LOW (green) mRNA expression (but no KIF14 genomic gain) for Sp1 (circle), YY1 (triangle), and HSF1 (diamond) normalized to normal ovary expression (set as 1, black dashed line). Mean, black line. Individual tumors represented by symbols. * Significance at P <0.05, paired t-test. P = 0.03 (Sp1), P = 0.01 (YY1), P = 0.32 (HSF1).

Article Snippet: 1 μL of synthesized cDNA was added to 1X TaqMan PCR master mix (ABI), and 1X TaqMan Gene Expression Assay primer-probe mix for KIF14 (Hs00978216_m1), Sp1 (Hs00916521_m1), YY1 (Hs00231533_m1), and HSF1 (Hs01027608_g1).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Transcriptional and Epigenetic Regulation of KIF14 Overexpression in Ovarian Cancer

doi: 10.1371/journal.pone.0091540

Figure Lengend Snippet: Pearson correlation between KIF14 gain/no gain tumors and Sp1 , HSF1 and YY1 mRNA.

Article Snippet: 1 μL of synthesized cDNA was added to 1X TaqMan PCR master mix (ABI), and 1X TaqMan Gene Expression Assay primer-probe mix for KIF14 (Hs00978216_m1), Sp1 (Hs00916521_m1), YY1 (Hs00231533_m1), and HSF1 (Hs01027608_g1).

Techniques:

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 1. Mutant C1413S COUP-TFI Dimerizes with wtCOUP-TFI and Inhibits Its DNA Binding Activity A, mutCOUP-TFI interacts with wtCOUP-TFI in a yeast two-hybrid system. Y190 yeast cells were transformed with the different vectors as shown. The resulting b-galactosidase activities were assayed and are shown as Miller units. Each bar represent the mean 6 SEM of four values obtained in two independent experiments. B, mutCOUP-TFI is retained on a GST-wtCOUP-TFI matrix. 4 ml of 35S-labeled in vitro translated mutCOUP-TFI were allowed to interact either with GST alone (lane 2) or a GST-DCOUP-TFI

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Mutagenesis, Binding Assay, Activity Assay, Transformation Assay, Labeling, In Vitro

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 2. Stable Expression of COUP-TFI in Transfected P19 EC Cells A, RT-PCR analysis of transgene expression. Control cells (pcDNA), wild-type (wt), or mutant (mut) COUP-TF expressing cells were grown for 24 h as a monolayer in the presence or absence of cAMP as indicated before RNA extraction and RT-PCR analysis of COUP-TFI and the invariant PO gene expression. Results show the ethidium bromide staining of a 2% agarose gel. B and C, EMSAs of control (pcDNA) and wtCOUP-TFI cells treated as aggregates for 48 h with cAMP. An asterisk indicates the putative Ear2/DNA complex (see text), an arrow shows the position of the COUP-TFI/DNA complex, and an open circle shows the position of the supershifted complex in the presence of COUP-TFI antibodies (Ab). The oligonucleotides indicated in panel C were added as competitors at a 20-fold molar excess. D, DR-1 binding activity in P19 cell aggregates treated with 1026 M RA for 48 h. E, DR-1 binding activities in the different cell lines cultured as aggregates for 48 h and treated as indicated with all-trans-retinoic acid (RA) and/or cAMP. Symbols are the same as in Fig. 1B. Note that the mutant COUP-TFI inhibits endogenous COUP-TFs binding to the probe.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Control, Mutagenesis, RNA Extraction, Gene Expression, Staining, Agarose Gel Electrophoresis, Binding Assay, Activity Assay, Cell Culture

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 4. RT-PCR Analysis of the Expression of Selected Ret- inoid-Responsive Genes RT-PCR analysis was run with RNA extracted from aggre- gates of pcDNA, wt, and mutant COUP-TFI cells treated as indicated for 48 h. Bmp-4, Bone morphogenetic protein-4; E-cad, E-cadherin.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 5. P19 Cell Endogenous COUP-TFs Control Axogenesis Aggregated cells were plated after a 3-day RA treatment and further cultured for 5 days in the presence or not of 1 mM cAMP before anti-NF200 immunohistochemistry (magnification, 3400). Note the absence of neurites in mutCOUP-TFI cells treated with cAMP, despite the presence of neurofilament (NF200)-positive cell bodies (dark staining). Photographs show details of the different cultures and are representative of the content of the entire plates.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Control, Cell Culture, Immunohistochemistry, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 6. COUP-TFI Promotes Neuronal Migration A, Aggregates of cells treated for 3 days with 1 mM RA were plated onto tissue culture-grade plastic and further cultured for 24 h before the RGDS and SDGRG peptides were added at the indicated concentrations. After an additional day in culture, cells were fixed and stained for the presence of NF200 by immunohistochemistry (magnification, 3220). Neurons and neurites were intensely stained and either spread on the top of astrocytes [wtCOUP-TFI (no peptide)], or packed together [mutCOUP-TFI (no peptide), or wtCOUP-TFI RGDS 0.1 mg/ml]. B, The percentage of aggregates presenting either migrating neurons out of the core of the aggregates or a completely spread organization was determined and is shown as the percentage of aggregates with neuron outgrowth in function of the cell lines, in the absence of peptide (control) or in the presence of SDGRG and RGDS peptides at 0.1 mg/ml. Results are shown as the mean 6 SEM for 150 aggregates of each cell line scored in three independent experiments.

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Migration, Cell Culture, Staining, Immunohistochemistry, Control

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells.

doi: 10.1210/mend.14.12.0562

Figure Lengend Snippet: Fig. 8. COUP-TFI Regulates the Synthesis of Vitronectin A, RT-PCR analysis was run with RNA extracted from aggregates (agg.) or monolayer cell cultures (mon.) of pcDNA, wt, and mutant COUP-TFI cells treated as indicated for 48 h. B, Alignment of the human and mouse proximal promoter regions of the vitronectin gene (black dots indicate identical nucleotides between mouse and human, and dashes represent gaps that were introduced to maximize sequence homology). The conserved nuclear receptor (NR) half-binding sites are boxed as well as putative binding sites for other transcription factors. C, The mouse vitronectin promoter is activated by COUP-TFI in transient transfection assays. P19 cells were cotransfected with a mouse vitronectin promoter fragment (2528/147) linked to a luciferase coding sequence and the indicated amounts of wt or mutCOUP-TFI expression vectors. Results are shown as the mean 6 SEM (n 5 3) of the relative luciferase activities (raw luciferase activities divided by b-galactosidase activities).

Article Snippet: Briefly, 4 mg of WCEs were preincubated with 1 mg poly(dI-dC), and eventually with competitors, in 20 ml of binding buffer (20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 2.5 mM MgCl2) at room temperature for 15 min. For supershifting experiments, 1 ml of antibodies against COUP-TFI or COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 30 min with WCEs before addition of binding buffer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Sequencing, Binding Assay, Transfection, Luciferase, Expressing

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: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 1 The expression of DEF6 is increased in hypertrophic hearts and cardiomyocytes. A mRNA levels of DEF6 in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 5). B Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 4). C mRNA levels of DEF6 in NRCMs administrated with PBS or 24 h of PE (50 μM) (n = 5). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in NRCMs administrated with PBS or 24 h of PE (n = 4). *P < 0.05, ***P < 0.001 vs. sham or PBS. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Student’s t test (A, C) or Mann–Whitney U test (B, D).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 2 Ablation of DEF6 mitigates TAC-induced cardiac hypertrophy. A Strategy to construct KO mice and the sequencing results of WT and KO mice. B Protein levels of cardiac DEF6 in WT and KO mice (n = 5). C Comparisons of HW, HW/BW, LW/BW, and HW/TL in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). D Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area from groups (n = 6). E RT-PCR analyses of the hypertrophic markers in the indicated groups (n = 4). F–H Comparisons of the LVEDd, LVESd, LVPWd, FS, and EF values in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). I Left, PSR-stained LV sections in WT and KO mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). J RT-PCR analysis of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by One-way ANOVA (C, D, F–I) or Kruskal–Wallis test (E, J).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Construct, Sequencing, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 3 Overexpression of DEF6 aggravates TAC-induced cardiac hypertrophy. A mmunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the hearts of mice injected with AAV9-vector or AAV9-DEF6 (n = 4). B Comparisons of HW, HW/BW, LW/ BW, and HW/TL in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). C Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area between groups (n = 6). D RT-PCR analyses of the hypertrophic markers in each groups (n = 4). E–G Comparisons of LVEDd, LVESd, LVPWd, FS, and EF in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). H Left, PSR-stained LV sections in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). I RT-PCR analyses of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AAV9- vector or AAV9-vector sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AAV9-vector TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by Mann–Whitney U test (A) or One-way ANOVA (B, C, E–H) or Kruskal–Wallis test (D, I).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Over Expression, Expressing, Injection, Plasmid Preparation, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 4 DEF6 exacerbates PE-induced cardiomyocyte hypertrophy. A Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCMs infected with AdshRNA or AdshDEF6 (n = 3). B Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with AdshRNA or AdshDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). C RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCVs infected with Advector or AdDEF6 (n = 3). E Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with Advector or AdDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). F RT-PCR analyses of the hypertrophic markers in cultured NRCVs of each groups (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AdshRNA or AdshRNA PBS or Advector or Advector PBS, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AdshRNA PE or Advector PE. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Mann–Whitney U test (A, D) or One-way ANOVA (B, E) or Kruskal–Wallis test (C, F).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Western Blot, Expressing, Cell Culture, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 6 Prohypertrophic effect of DEF6 depends on Rac1-MEK-ERK signaling. A Co-IP of DEF6 was performed with anti-Flag and probed by Western blots with anti-HA (left); Co-IP of Rac1 was performed with anti-HA and probed by Western blots with anti-Flag (right). B In vitro GST pulldown assays for the interaction of purified Flag-DEF6 and GST-HA-Rac1 (left), as well as Flag-Rac1 and GST-HA-DEF6 (right). C The activity of Rac1 changes in the same direction as the DEF6 expression. D Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with Advector or AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM) (n = 3). E Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with Advector and AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM). (n ≥48 cells per group). F RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). G Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with AdshRNA or AdshDEF6 and with Adcontrol or AdRac1(G12V) under PE 24 h of PE treatment (50 μM) (n = 3). H Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with the indicated adenovirus and administrated with 24 h of PE (50 μM) (n ≥48 cells per group). I RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). •P < 0.05, P < 0.01,•P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, ###P < 0.001 vs. AdDEF6 PBS PE or AdshDEF6 Adcontrol PE, and n.s. indicates no significance. Data are displayed as mean ± SD. Statistical analysis were conducted by Kruskal–Wallis test.

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Activity Assay, Expressing, Cell Culture, Infection, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: Environmental research

Article Title: Infant sex-specific placental cadmium and DNA methylation associations

doi: 10.1016/j.envres.2015.02.004

Figure Lengend Snippet: Cadmium-high/low status and DNA methylation, stratified by infant sex.

Article Snippet: Little is known about the function of this gene. table ft1 table-wrap mode="anchored" t5 caption a7 Gene/name Gene ontology (molecular function) CpG Site Female Infants Male Infants β a p-value β a p-value HOPX/HOP homeobox Sequence-specific DNA binding and transcription factor activity cg04528060 −1.84 0.04 0.10 0.95 KIAA1211/KIAA121 cg04528060 −5.86 0.28 −9.65 0.04 CPB2/Carboxypeptidase B2 Metallocarboxypeptidase activity, zinc ion binding cg00613224 0.58 0.05 −1.28 0.09 GPR160/G protein-coupled receptor 160 G-protein coupled receptor activity cg15958576 −4.88 0.02 −0.86 0.74 cg10903116 −5.38 0.03 −0.10 0.97 SEC62/SEC62 (S. cerevisiae) Protein transporter & receptor activity cg10903116 3.52 0.05 5.16 0.07 Open in a separate window a β = log 2 fold change in gene expression for every two-fold increase in methylation DNA methylation and gene expression, stratified by infant sex.

Techniques: DNA Methylation Assay, Ubiquitin Proteomics

Journal: Environmental research

Article Title: Infant sex-specific placental cadmium and DNA methylation associations

doi: 10.1016/j.envres.2015.02.004

Figure Lengend Snippet: Cadmium-related differential DNA methylation and infant birth weight, stratified by infant sex.

Article Snippet: Little is known about the function of this gene. table ft1 table-wrap mode="anchored" t5 caption a7 Gene/name Gene ontology (molecular function) CpG Site Female Infants Male Infants β a p-value β a p-value HOPX/HOP homeobox Sequence-specific DNA binding and transcription factor activity cg04528060 −1.84 0.04 0.10 0.95 KIAA1211/KIAA121 cg04528060 −5.86 0.28 −9.65 0.04 CPB2/Carboxypeptidase B2 Metallocarboxypeptidase activity, zinc ion binding cg00613224 0.58 0.05 −1.28 0.09 GPR160/G protein-coupled receptor 160 G-protein coupled receptor activity cg15958576 −4.88 0.02 −0.86 0.74 cg10903116 −5.38 0.03 −0.10 0.97 SEC62/SEC62 (S. cerevisiae) Protein transporter & receptor activity cg10903116 3.52 0.05 5.16 0.07 Open in a separate window a β = log 2 fold change in gene expression for every two-fold increase in methylation DNA methylation and gene expression, stratified by infant sex.

Techniques: DNA Methylation Assay, Methylation

Journal: Environmental research

Article Title: Infant sex-specific placental cadmium and DNA methylation associations

doi: 10.1016/j.envres.2015.02.004

Figure Lengend Snippet: DNA methylation and gene expression, stratified by infant sex.

Article Snippet: Little is known about the function of this gene. table ft1 table-wrap mode="anchored" t5 caption a7 Gene/name Gene ontology (molecular function) CpG Site Female Infants Male Infants β a p-value β a p-value HOPX/HOP homeobox Sequence-specific DNA binding and transcription factor activity cg04528060 −1.84 0.04 0.10 0.95 KIAA1211/KIAA121 cg04528060 −5.86 0.28 −9.65 0.04 CPB2/Carboxypeptidase B2 Metallocarboxypeptidase activity, zinc ion binding cg00613224 0.58 0.05 −1.28 0.09 GPR160/G protein-coupled receptor 160 G-protein coupled receptor activity cg15958576 −4.88 0.02 −0.86 0.74 cg10903116 −5.38 0.03 −0.10 0.97 SEC62/SEC62 (S. cerevisiae) Protein transporter & receptor activity cg10903116 3.52 0.05 5.16 0.07 Open in a separate window a β = log 2 fold change in gene expression for every two-fold increase in methylation DNA methylation and gene expression, stratified by infant sex.

Techniques: DNA Methylation Assay, Gene Expression, Sequencing, Binding Assay, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: Variant identification pipeline for WES in P1 and P2 (see also Appendix Tables S1 and ). Unrelated familial trees showing affected children in black and healthy individuals in white. Sanger sequencing of the region corresponding to mutations in ALPI in both families. Location of ALPI on chromosome 2 and diagrams featuring ALPI gene with its nine exons and ALPI protein with its N‐terminal signal peptide, phosphatase domain and C‐terminal recognition signal for the transamidase complex (GPI‐anchor attachment site), which removes the GPI signal sequence and replaces it by a preformed GPI precursor glycolipid. Arrows point to mutations identified in P1 and P2. Numbers indicate amino acid position.

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Variant Assay, Sequencing, Recognition Signal

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: Confirmatory Sanger sequencing for siblings in Family 2. Multiple alignments of ALPI orthologs from different species using the Clustal Omega software. Residues altered by mutations in P1 and P2 are boxed in red. Conserved residues are indicated as follow: full identity (*), similar characteristics (:) (> 0.5 in the Gonnet PAM 250 matrix), weak similarities (.) (< 0.5 in the Gonnet PAM 250 matrix).

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Sequencing, Software

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: A, B Analysis with ALPI antibody of HEK293T cells (lacking endogenous ALPI expression) after mock transduction or transduction with lentiviral particles encoding indicated mutants. (A) Western blot of whole‐cell lysates. (B) Flow cytometry analysis of surface‐labelled cells. C–E 3D modelling of ALPI calculated on the basis of the crystal structure of ALPP. ALPI is represented with one monomer in yellow, and the second in blue. Alanine 350, alanine 360 and catalytic Ser111 are represented in stick in violet, cyan and red, respectively. Deletion of the C‐terminal from Gln439 is shown in pink. Top and bottom views shown in (D) correspond to a 90° rotation. F pNPP phosphatase activity of WT and ALPI mutants measured by the OD405 nm of the reaction supernatants. n = 5, error bars indicate SD. ** P = 0.0079, nonparametric, unpaired two‐tailed Mann–Whitney test. G IL‐8 inhibition of LPS‐induced transcription in THP1 cells by WT and ALPI mutants. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; EV, empty vector. n = 3, error bars indicate SD. ** P = 0.022, nonparametric, unpaired two‐tailed Mann–Whitney test. Source data are available online for this figure.

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Expressing, Transduction, Western Blot, Flow Cytometry, Activity Assay, Two Tailed Test, MANN-WHITNEY, Inhibition, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: WT ALPI and mutant expression by qRT‐PCR. Data are representative of three experiments and are expressed as mean ± SD.

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: A, B Immunofluorescence microscopy of duodenum (A) and ileum (B) sections from Patient 1 (A) and Patient 2 (B) compared to normal or IBD controls. Sections were stained with DAPI (4′,6‐diamidino‐2‐phenylindole) for DNA (blue) and antibodies against ALPI alone (second column, red) or ALPI and E‐cadherin (green) (first column, merge staining). Magnification 20× (scale bar = 100 μm). C Effect of AP inhibitors (L‐phe, L‐arg) on AP activity in stools of non‐inflamed controls ( n = 20). Data are expressed as mean ± SD. **** P < 0.0001, nonparametric, unpaired two‐tailed Mann–Whitney test. D ALPI activity in stools of non‐inflamed ( n = 20) or IBD controls ( n = 14) and of P1. AP values are expressed as units of ALPI/g stool and as medians with interquartiles; * P = 0.0122, nonparametric, unpaired two‐tailed Mann–Whitney test.

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Immunofluorescence, Microscopy, Staining, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis

doi: 10.15252/emmm.201708483

Figure Lengend Snippet: A, B Immunofluorescence microscopy of duodenum (A) and ileum (B) sections from P1 (A) and P2 (B) compared with normal or IBD controls. Sections were counterstained with blue RedDot2 dye for DNA and antibodies against ALPI alone (red), TNAP alone (green) or TNAP and ALPI (first column, merge staining). Magnification 20× (Scale bars: 100 μm).

Article Snippet: Total RNA (5 μg) was reverse transcribed using M‐MLV reverse transcriptase (Invitrogen). qRT–PCR was performed with an Assays‐on‐Demand probe (Applied Biosystems, Thermo Fisher Scientific) specific for ALPI‐FAM (Hs00357579_g1) and RPLPO‐FAM (Large Ribosomal Protein; Hs99999902_m1), which was used for normalization. qPCR was performed with a ABI PRISM 7900 (Applied Biosystems).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: RNA sequencing reveals that tomatidine modulates ATF4-dependent ER stress genes in pancreatic cancer cells Human and murine PDAC cell lines were treated for 40 h with 6.4 μg/mL tomatidine, RNA was isolated, and RNA sequencing was performed to analyze differences in gene regulation. N = 3 biological separate experiments. (A–D) (A) Heatmap of all genes and how they change in tomatidine-treated vs. untreated cells for Panc1 cells (FDR<0.05). Volcano plot highlighting ER stress-related genes in (B) Panc1 and (C) MT5 cells. (Upregulated genes on the right of the central axis and vice versa with higher fold change as we go away from the origin on y axis) (D) IPA upstream analysis of ATF4-related genes in Panc1 cells. (E) Heatmap elucidating targeting of ATF4-related genes in treated vs. untreated Panc1 cells. (Fold change: +2.5 to −1.5; FDR<0.03). (F) Top 10 pathways focusing on UPR, ATF4, and ER stress via Reactome analysis of the RNA-sequencing data of treated vs. untreated samples for Panc1 cells. (FDR< 0.03).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: RNA Sequencing, Isolation

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: ATF4 expression in PDAC (A) KM plotter was utilized to examine ATF4 expression in human PDAC specimens examining high vs. low expression compared to overall survival. (B) Data distribution elucidating significance of the overall survival curve prepared by KM Plotter. (C and D) (C) scRNA-seq datasets of pancreatic cancer tissue from metastatic patients were obtained from NIH dbGAP (accession phs002045.v1.p1) and (D) analyzed for ATF4 expression. (See also Table S1 ). (E) Pancreatic tumor tissue from KPC mice were stained by multiplex IF and imaged using Akoya Vectra Polaris and the Phenochart software to analyze ATF4 in multiple cellular compartments. Markers used to determine ATF4 (white), epithelial cells (CK19; red), stroma (αSMA; orange and PDGFRβ; Green), CD8 T+ cells (yellow), and macrophages (F4/80; turquoise). Scale bar = 100 μm.

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Staining, Multiplex Assay, Software

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine inhibits ATF4-dependent signaling in PDAC (A and B) (A) MiaPaca-2 tumor cells were treated with tomatidine for 72 h and cell lysates were immunoblotted for ATF4, 4EBP1, and phospho-4EBP1(p-4EBP1) protein expression and (B) p-4EBP1/4EBP1 levels quantified by densitometry. (C) Immunofluorescence (IF) was performed on vehicle (DMSO) or tomatidine-treated Panc1 cells to track ATF4 (FITC-Green) translocation from nucleus (DAPI-Blue) to cytoplasm. Scale bar = 50 μm. (D–G) (D) Nuclear to cytoplasmic translocation was quantified. Panc1 cells were treated with vehicle (DMSO) or tomatidine for 40 h and ATF4 transcriptional activity was analyzed by chromatin immunoprecipitation (ChIP) qPCR evaluating binding of ATF4 to the downstream promoter regions of (E) eIF4EBP1 (F) CHOP- B site and (G) ASNS. Data are reported as the means + SEMs. n = 3 or more independent biological replicates (4B, One-way ANOVA with Tukey’s test for pairwise comparisons was used to analyze the data; 4D-G, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Immunofluorescence, Translocation Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: In vivo tomatidine treatment inhibits pancreatic tumor growth (A–C) (A) MT5 tumor-bearing C57BL/6 mice (5 mice/group) were treated with 5 mg/kg daily i.p. injections of tomatidine or vehicle control (40% HPBCD) and monitored for tumor growth. (n = 5/group). RNA isolated from the tumor tissues of MT5 tumor-bearing C57BL/6 mice treated with vehicle or 5 mg/kg daily i.p. injections of tomatidine were assessed for (B) ATF4 and (C) eIF4EBP1 expression via qPCR. Data are reported as the means + SEMs. n = 5 mice per group. (5A, mixed between-within subjects ANOVA shows a significant interaction between days of treatment and group; 5B-C, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: In Vivo, Control, Isolation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine mediated inhibition of ATF4 signaling can increase sensitivity to ferroptotic cell death in PDAC (A) Panc-1 tumor cells were treated with tomatidine (6.4 μg/mL) and analyzed by RNA sequencing. Ingenuity pathway analysis of the regulated genes suggested ferroptosis as a top hit for tomatidine-treated cells. (B–D) (B) Pancreatic cancer cells were treated with vehicle (DMSO), erastin (to induce ferroptosis), ferrostatin-1 (to inhibit ferroptosis), tomatidine, or in different combinations and lipid peroxidation of (C) Panc-1 and (D) MiaPaca-2 was analyzed by flow cytometry using Bodipy-11. (E) Panc-1 and MiaPaca-2 cells treated with vehicle (DMSO) or tomatidine and lysates collected after 24 h were immunoblot for GPX4 expression. (F) Panc1 cells were plated overnight and treated with tomatidine for 6 h and then assayed by Seahorse assay to analyze mitochondrial fitness. (G) Schematic showing how tomatidine can regulate ATF4-dependent signaling to induce ferroptosis in pancreatic cancer. Data are reported as the means + SEMs. n = 3 or more independent biological replicates. (7C-D, Two-tailed independent student’s test was used to analyze the data; 7F, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05, ∗∗p < 0.007).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Inhibition, RNA Sequencing, Flow Cytometry, Western Blot, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet:

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Control, Recombinant, Viability Assay, Luciferase, ChIP-qPCR, Gene Expression, Software

Journal: Journal of Neuroscience

Article Title: SOCS3-Mediated Blockade of JAK/STAT3 Signaling Pathway Reveals Its Major Contribution to Spinal Cord Neuroinflammation and Mechanical Allodynia after Peripheral Nerve Injury

doi: 10.1523/jneurosci.5007-09.2010

Figure Lengend Snippet: Figure 5. Effects of transducing primary glial cells or BV2 microglia with LV–SOCS3t on JAK/STAT3 pathway activity and inflammatory state markers. A, Stimulation of primary glia with IL-6 (50 ng/ml) resulted in rapid (15 min) pSTAT3 accumulation (i.e., JAK/STAT3 activation, Western blot). This effect was prevented in cells transduced 48 h earlier with LV–SOCS3t (350 or 35 ng/ml p24). Data are shown as mean SEM of three independent experiments. #p 0.001, IL-6-treated versus untreated cell cultures;*p 0.001,IL-6-treatedLV–SOCS3t-transducedcellsversusIL-6-treateduninfectedcells.B,C,Inbothprimaryglialcells (B) and BV2 microglial cell line (C), IL-6-induced production (after 3 h incubation with IL-6) of inflammatory markers (IL-6, CCL2, TNF) was efficiently inhibited in cells transduced 48 h before with LV–SOCS3t. IL-6 can also induce ATF3 production in BV2 microglia, this effect being significantly prevented in LV–SOCS3t-transduced cells. Each bar is the mean SEM (n 4 for each group). #p 0.05, IL-6-treated cells versus control cell cultures; *p 0.05, IL-6-treated LV–SOCS3t-infected cells versus IL-6- treated uninfected cells. R.Q., Relative quantification; A.U., arbitrary unit.

Article Snippet: Assay-onDemand Gene TaqMan PCR probes (Applied Biosystems) were used for target genes: SOCS3 (GenBank accession number Rn00585674_s1), integrin alpha M (ITGAM) (GenBank accession number Rn00709342_ m1), GFAP (GenBank accession number Rn01460868_m1), IL-6 (Rn00561420_m1), IL-1 (GenBank accession number Rn00580432_ m1), ATF3 (GenBank accession number Rn00563784_m1), CCL2 (GenBank accession number Rn00580555_m1), tumor necrosis factor- (TNF ) (GenBank accession number Rn99999017_m1), GAPDH (GenBank accession number Rn99999916_s1), and ribosomal subunit 18S (RS 18S) (GenBank accession number Hs 99999901_s1).

Techniques: Activity Assay, Activation Assay, Western Blot, Incubation, Control, Infection, Quantitative Proteomics