Journal: Molecular Therapy. Nucleic Acids

Article Title: Effective Small Interfering RNA Therapy to Treat CLCN7 -dependent Autosomal Dominant Osteopetrosis Type 2

doi: 10.1038/mtna.2015.21

Figure Lengend Snippet: In vitro and in vivo tests of CLCN7 mutant -specific siRNAs . ( a ) Cartoon depicting the pEGFP-C1 vector used in the study. ( b ) HEK293 cells stably transfected with the pEGFP-C1 vector carrying the indicated mutations. Expression of the CLCN7 gene was quantified by real-time RT-PCR on RNA extracted from mutant transfectants, against cells transfected with the empty vector, which did not express CLCN7 mRNA (first bar from left). ( c–e ) HEK293 cells transfected with the indicated vectors, were treated with the CLCN7 mutant -specific siRNA listed in as the most effective per each mutation. Concentration-dependent regulation of CLCN7 assessed by real-time RT-PCR, normalized with GAPDH . ( f ) RT-PCR using primer pairs specific for the Clcn7 G213R mRNA showing transcript amplification only in heterozygous ( Clcn7 G213R/WT ) and homozygous ( Clcn7 G213R/G213R ) osteoclasts, while in wild-type osteoclasts ( Clcn7 WT/WT ) no transcript was amplified. ( g ) Direct DNA sequencing of the amplified transcript shown in f for the Clcn7 G213R/WT osteoclasts, demonstrating only the mutant sequence. ( h ) Osteoclasts generated from the bone marrow mononuclear cells of Clcn7 WT/WT and Clcn7 G213R/WT mice were treated with the indicated concentration of scrambled (SCR) or Clcn7 G213R -specific siRNA. Real-time RT-PCR was performed using the primer pairs specific for the mutant transcript validated in ( f ) and ( g ). ( i ) Osteoclasts were generated from the bone marrow mononuclear cells of Clcn7 WT/WT and Clcn7 G213R/WT mice onto bone slices and treated with the indicated concentration of SCR and Clcn7 G213R -specific siRNA. At the end of experiment, cells were removed by sonication and bone resorption evaluated by the pit assay. ( j ) Three-month-old Clcn7 WT/WT mice were injected once i.p. with 4 mg/kg of Clcn7 G213R -sticky siRNA jetPEI conjugate and sacrificed at the indicated time point. Sera were collected and evaluated for total RNA concentration by Nanodrop. ( k ) Ten-day-old Clcn7 G21R/WT mice were injected once i.p. with the indicated doses of SCR- or of Clcn7 G213R -sticky siRNA jetPEI conjugate. After 48 hours, mice were sacrificed, RNA was extracted from tibias, and evaluated by real-time RT-PCR using the primer pairs specific for the Clcn7 G213R mRNA validated in ( f ) and ( g ). In b–e, h–k data are the mean ± SD of three independent experiments or three animals/group. b–e,h,I,k : Student's t -test. j : one-way analysis of variance (ANOVA). For c–e , statistics was also performed by one way ANOVA (shown in Supplementary Table S3 ).

Article Snippet: The cationic polymer transfection reagent in vivo -jetPEI (cat# 201-50G) was from Polyplus-transfection (Illkirch, France).

Techniques: In Vitro, In Vivo, Mutagenesis, Plasmid Preparation, Stable Transfection, Transfection, Expressing, Quantitative RT-PCR, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, DNA Sequencing, Sequencing, Generated, Sonication, Injection

Journal: Molecular Therapy. Nucleic Acids

Article Title: Effective Small Interfering RNA Therapy to Treat CLCN7 -dependent Autosomal Dominant Osteopetrosis Type 2

doi: 10.1038/mtna.2015.21

Figure Lengend Snippet: In vivo treatment and safety study . Ten-day-old Clcn7 G213R/WT mice were injected i.p. with 4 mg/kg of Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 4 weeks. At the end of the experiments, mice were sacrificed and ( a ) the indicated organs were subjected to histopathological evaluation by hematoxylin/eosin staining (Bar = 100 µm for spleen and kidney, 20 µm for liver). ( b ) Sera were collected and analyzed by the Reflotron method for the indicated biomarkers of kidney and liver disease, and for the ADO2 biomarker CK. Normal values are between the two dotted lines. ( c ) RNA was extracted from the indicated organs and subjected to real time RT-PCR using primer pairs specific for the Clcn7 G213R mRNA, normalized for gapdh . ( d ) Ten day-old Clcn7 WT/WT and Clcn7 G213R/WT were treated with 4 mg/kg of scrambled- (SRC) or Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 2 and 4 weeks. At the end of the experiments, mice were sacrificed, then the serum biomarker of bone resorption, CTX, the serum osteoclast biomarker, TRAcP (5b isoform), and the CTX/TRAcP ratio were evaluated after 2 and 4 weeks of treatment. ( e ) µCT analysis of proximal tibias of mice treated with 4 mg/kg of Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 2 weeks, followed by measurements of trabecular ( f ) bone volume over total tissue volume (BV/TV), ( g ) trabecular number (Th.N), ( h ) thickness (Tb.Th), and ( i ) separation (Tb.Sp). Data are ( a,e ) representative or ( b–d, f–i ) the mean ± SD of four to seven mice per group (Student's t -test). For f–i statistics was also performed by one-way analysis of variance (shown in Supplementary Table S3 ).

Article Snippet: The cationic polymer transfection reagent in vivo -jetPEI (cat# 201-50G) was from Polyplus-transfection (Illkirch, France).

Techniques: In Vivo, Injection, Staining, Biomarker Discovery, Quantitative RT-PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Effective Small Interfering RNA Therapy to Treat CLCN7 -dependent Autosomal Dominant Osteopetrosis Type 2

doi: 10.1038/mtna.2015.21

Figure Lengend Snippet: Rescue of the bone phenotype . Ten-day-old Clcn7 WT/WT and Clcn7 G213R/WT were treated with 4 mg/kg of scrambled- (SRC) or Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 4 weeks. At the end of the experiments, mice were sacrificed and their bone phenotype analyzed. ( a ) µCT analysis of proximal tibias. ( b ) Trabecular bone volume over total tissue volume (BV/TV). ( c ) Trabecular number (Tb.N). ( d ) Trabecular thickness (Tb.Th). ( e ) Trabecular separation (TB.Sp). ( f ) Serum concentration of ParaThyroid Hormone (PTH). ( g ) Histochemical TRAcP staining to evaluate osteoclasts (purple cells). Bar = 100 µm. ( h ) Osteoclast surface over bone surface (Oc.S/BS). ( i ) Osteoclast number over bone perimeter (Oc.N/B Pm). ( j ) Transcriptional expression, by real-time RT-PCR on RNA extracted from the whole femurs of osteoclast ( Tracp and Cathepsin K ( CatK )) and osteoblast ( Alkaline phosphatase ( ALP ) and Runt-related transcription factor 2 ( Runx 2 )) genes normalized with gapdh . ( k ) Eroded surface over bone surface (ES/BS). ( l ) Representative images of the secondary spongiosa (upper panels) and measurement of cartilage area/trabecular area (lower panel). Arrows: cartilage remnants. Bar = 50 µm. Results are ( a,g,i (upper panels)) representative or ( b–f,h–i (lower panel)) the mean ± SD of three to seven mice/group (Student's t -test). In d , P > 0.2. For b–f,h–l statistics was also performed by one-way analysis of variance (shown in Supplementary Table S3 ).

Article Snippet: The cationic polymer transfection reagent in vivo -jetPEI (cat# 201-50G) was from Polyplus-transfection (Illkirch, France).

Techniques: Concentration Assay, Staining, Expressing, Quantitative RT-PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Effective Small Interfering RNA Therapy to Treat CLCN7 -dependent Autosomal Dominant Osteopetrosis Type 2

doi: 10.1038/mtna.2015.21

Figure Lengend Snippet: Cortical, growth plate, osteoblast, dynamic, and bone quality variables . Ten-day-old Clcn7 WT/WT and Clcn7 G213R/WT were treated with 4 mg/kg of scrambled- (SRC) or Clcn7 G213R -sticky siRNA jetPEI conjugate, three times a week for 4 weeks. At the end of the experiments, mice were sacrificed and their bone phenotype analyzed. ( a ) Cortical thickness (Cor.Th). ( b ) Growth plate width. ( c ) Osteoblast surface over bone surface (Ob.S/BS). ( d ) Histological images of osteoid (arrows). Bar = 5 µm. ( e ) Osteoid volume over bone volume (OV/BV). ( f ) Calcein labeling (green fluorescence) of mineral deposition (double arrowheads). Bar = 2 µm. ( g ) Mineral apposition rate (MAR). ( h ) Mineralized surface over bone surface (MS/BS). ( i ) Bone formation rate (BFR). ( j ) Total indentation distance (TDI). ( k ) First-cycle indentation distance (ID). ( l ) Touchdown distance (TDD). Results are ( d,f ) representative or ( a–c,e,g–l ) the mean ± SD of three to seven mice/group (Student's t -test). In a–c,e,g–i , P > 0.2. For a–c,e,g–l statistics was also performed by one-way analysis of variance (shown in Supplementary Table S3 ).

Article Snippet: The cationic polymer transfection reagent in vivo -jetPEI (cat# 201-50G) was from Polyplus-transfection (Illkirch, France).

Techniques: Labeling, Fluorescence

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: 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: 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: JCI Insight

Article Title: Loss of TRPV4 reduces pancreatic cancer growth and metastasis

doi: 10.1172/jci.insight.196280

Figure Lengend Snippet: ( A ) Schematic of the orthotopic experiment. ( B ) Representative images of tumors from WT, Piezo1 GFAP KO, or TRPV4-KO mice that were injected with 100,000 KPCY cells in the tail of the pancreas. Organs were collected 20 days after surgery. ( C ) Quantitative analysis of tumor size. ( D – F ) Serum levels of IL-6 ( D ), TIMP1 ( E ), and SAA ( F ) were measured by ELISA. ( G ) Saa2 / Saa1 from liver RNA was measured by RT-PCR. Statistical analyses were performed using 1-way ANOVA with Dunnett’s post hoc test. Results are expressed as mean ± SEM.

Article Snippet: RT-PCR was performed using the TaqMan assay (catalog 4331182) purchased from Life Technologies for IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), IL-11 (Hs01055414_m1), ACTA2 (Hs01099348_m1), FAP (Hs00990791_m1), MME (Hs00153510_m1, CSAR2 (Hs01933768_s1), Saa1 / Saa2 (Mm04208126_mH), S100a8 (Mm00496696_g1), and S100a9 (Mm00656925_m1).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: JCI Insight

Article Title: Loss of TRPV4 reduces pancreatic cancer growth and metastasis

doi: 10.1172/jci.insight.196280

Figure Lengend Snippet: Liver RNAs from WT or TRVP4-KO mice following orthotopic injection of KPCY cells (100,000 cells) were purified and analyzed by RNA-Seq. Upregulated WT genes versus TRPV4 KO; n = 2 animals for WT and n = 3 animals for TRPV4 KO. ( A and B ) Differentially expressed genes (DEGs list) volcano plot ( A ) and gene ontology (GO) scatter plot ( B ) listing the 10 highest upregulated pathways in WT versus TRPV4 KO: biological process (BP), cellular component (CC), and molecular function (MF). ( C ) Corresponding bar graph.

Article Snippet: RT-PCR was performed using the TaqMan assay (catalog 4331182) purchased from Life Technologies for IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), IL-11 (Hs01055414_m1), ACTA2 (Hs01099348_m1), FAP (Hs00990791_m1), MME (Hs00153510_m1, CSAR2 (Hs01933768_s1), Saa1 / Saa2 (Mm04208126_mH), S100a8 (Mm00496696_g1), and S100a9 (Mm00656925_m1).

Techniques: Injection, Purification, RNA Sequencing

Journal: JCI Insight

Article Title: Loss of TRPV4 reduces pancreatic cancer growth and metastasis

doi: 10.1172/jci.insight.196280

Figure Lengend Snippet: ( A ) mRNA levels for S100a8 and S100a9 genes measured by RT-PCR. ( B ) S100A8 + cells were counted in liver sections of WT animals ( n = 4) and TRPV4-KO animals ( n = 5). ( C ) Representative image of immunostaining of S100A8 protein in liver sections of WT and TRPV4-KO mice. Statistical analysis was performed using Student’s t test. Results were expressed as mean ± SEM. Scale bars: 100 μm. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: RT-PCR was performed using the TaqMan assay (catalog 4331182) purchased from Life Technologies for IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), IL-11 (Hs01055414_m1), ACTA2 (Hs01099348_m1), FAP (Hs00990791_m1), MME (Hs00153510_m1, CSAR2 (Hs01933768_s1), Saa1 / Saa2 (Mm04208126_mH), S100a8 (Mm00496696_g1), and S100a9 (Mm00656925_m1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunostaining

Journal: JCI Insight

Article Title: Loss of TRPV4 reduces pancreatic cancer growth and metastasis

doi: 10.1172/jci.insight.196280

Figure Lengend Snippet: ( A ) Graphical illustration of experimental design. KPC cells (100,000 cells) were injected into the pancreas, followed by injection of KPCY cells (500,000 cells) into the spleen 10 days later. ( B ) Representative images of immunostaining of mouse liver for Ki67 and GFP (marking KCPY cells) at low (upper panels) and high magnification (lower panels). Scale bars: 100 μm (top) and 50 μm (bottom). ( C and D ) Quantitative analysis of Ki67 positively stained cells and liver lesions of WT and TRPV4-KO liver. The analysis was performed on large sections of pancreatic tissues using tiling and stitching of images obtained with a Zeiss 880 Airyscan confocal microscope. Lesions were defined as an aggregate of at least 10 KPCY cells. ( E and F ) Representative images of immunostaining of liver sections with CD68 and quantitative analysis of CD68 + cells in liver sections from WT or TRPV4-KO mice. Scale bars: 100 μm. Statistical analyses were performed using Student’s t test. Animal number, n = 4–5. Results were expressed as mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: RT-PCR was performed using the TaqMan assay (catalog 4331182) purchased from Life Technologies for IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), IL-11 (Hs01055414_m1), ACTA2 (Hs01099348_m1), FAP (Hs00990791_m1), MME (Hs00153510_m1, CSAR2 (Hs01933768_s1), Saa1 / Saa2 (Mm04208126_mH), S100a8 (Mm00496696_g1), and S100a9 (Mm00656925_m1).

Techniques: Injection, Immunostaining, Staining, Microscopy

Journal: JCI Insight

Article Title: Loss of TRPV4 reduces pancreatic cancer growth and metastasis

doi: 10.1172/jci.insight.196280

Figure Lengend Snippet: ( A ) Survival curve of WT and TRPV4-KO mice following orthotopic injection of KPCY cells (10,000 cells) into the pancreas. Mice were monitored 3 times per week for signs of distress. ( B ) Pie charts of survivors versus nonsurvivors at the end of the experiment (120 days) for WT (red) and TRPV4 KO (blue) mice. ( C ) Nonsurvivor mice pancreas tumor weight WT and TRPV4 KO at the time of their death. ( D ) Representative images of livers from WT mice with metastatic tumor (upper panels) and TRPV4-KO mice (lower panels). Scale bars: 1 mm. Serial sections were stained with H&E or Masson’s trichrome dye (left panels) and immunostained with GFP (marking KCPY cells) and MPO antibodies (right panels). ( E ) Computation of macroscopic tumor, small tumors, and tumor cell clusters from liver tissues of nonsurviving animals: n = 13 for WT and n = 5 for TRPV4 KO. ( F ) Quantitative analysis of MPO + cell clusters (≥10 cells) from images of liver sections. MOP + cells present in the tumors were excluded from the cell count. Comparison of survival curves was performed using the Log-Rank (Mantel-Cox) test. Statistical analysis was performed using Student’s t test. Results were expressed as mean ± SEM. * P ≤ 0.05.

Article Snippet: RT-PCR was performed using the TaqMan assay (catalog 4331182) purchased from Life Technologies for IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), IL-11 (Hs01055414_m1), ACTA2 (Hs01099348_m1), FAP (Hs00990791_m1), MME (Hs00153510_m1, CSAR2 (Hs01933768_s1), Saa1 / Saa2 (Mm04208126_mH), S100a8 (Mm00496696_g1), and S100a9 (Mm00656925_m1).

Techniques: Injection, Staining, Cell Characterization, Comparison

Journal: JCI Insight

Article Title: Loss of TRPV4 reduces pancreatic cancer growth and metastasis

doi: 10.1172/jci.insight.196280

Figure Lengend Snippet: ( A ) Survival curve of KPC versus KPC;TRPV4-KO mice. Mice either died with weight loss (left graph) or without weight loss (right graph). Log-rank values are indicated on the graph. ( B ) Distribution of animals in each category (loss of weight or absence of weight loss). ( C ) Representative images of liver tissues from KPC mice; upper panel: immunostaining with CK19 antibody; lower panel: IHC with Ki67. Sequential slides were used for staining. Scale bar: 50 μm. ( D ) Representative images of liver tissues from KPC;TRPV4-KO mice; upper panel: immunostaining with CK19 antibody; lower panel: IHC with Ki67. Sequential slides were used for the staining. Age (days) of each mouse at time of death is listed. Arrows indicate the presence of microlesions. Scale bar: 100 μm. ( E ) Quantitative analysis of tumor microlesions in liver tissues from KPC or KPC;TRPV4-KO mice. ( F ) Human pancreatic adenocarcinoma. Left panel: overall survival curves for patients with high TRPV4 expression (upper fifteenth percentile) versus patients with low TRPV4 expression (lowest fifteenth percentile). Right panel: overall survival curves for patients with high PIEZO1 expression (upper fifteenth percentile) versus patients with low PIEZO1 expression (lowest fifteenth percentile). Comparison of survival curves was performed using the Log-Rank (Mantel-Cox) test. Statistical analysis was performed using Student’s t test. Results were expressed as mean ± SEM; ** P ≤ 0.01.

Article Snippet: RT-PCR was performed using the TaqMan assay (catalog 4331182) purchased from Life Technologies for IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), IL-11 (Hs01055414_m1), ACTA2 (Hs01099348_m1), FAP (Hs00990791_m1), MME (Hs00153510_m1, CSAR2 (Hs01933768_s1), Saa1 / Saa2 (Mm04208126_mH), S100a8 (Mm00496696_g1), and S100a9 (Mm00656925_m1).

Techniques: Immunostaining, Staining, Expressing, Comparison

Journal: Journal of neurochemistry

Article Title: Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2

doi: 10.1111/jnc.12779

Figure Lengend Snippet: Comparison of WT hTPH2 and tyrosine for serine substitution at position 41 (S41Y) expression in PC12 cells. Stable transfectants of tetracycline-regulated WT hTPH2 and S41Y PC12 cells were evaluated. (a) doxycycline (DOX)-stimulated expression of hTPH2. The same number of cells was lysed to examine expression efficiency. A total of 15-μg protein were loaded into each well. Immunoblots were probed with a TPH antibody that detects the exogenous hTPH2 and endogenous rat TH (this antibody cross-reacts with both enzymes and TH bands in the figure therefore serve as an internal control). Empty PC12 cell protein and recombinant hTPH2 were loaded as controls (the size of the recombinant protein is 63 kDa as a result of a 26-amino acid fusion segment). Immunoblots were probed with a TPH antibody that detects the exogenous hTPH2 and endogenous rat TH (the antibody cross-reacts with both enzymes). Note that DOX did not affect rat TH expression and that, while the TPH antibody recognizes both rat TH and hTPH2, it does not do so equally, so that relative expression comparisons cannot be made. (+) Indicates treatment with DOX; (−) indicates cells without DOX treatment. (b) Unlike bacterially expressed hTPH2, enzyme expressed in PC12 cells was soluble as determined from high speed supernatants (S) of cellular homogenates (H). (c) Higher S41Y expression levels are due to increased mRNA levels as determined by RT-PCR. Rat TPH1 = rTPH1; rat TPH2 = rTPH2. (d) Higher amounts of S41Y mRNA and immunoprotein are reflected in increases enzyme activity. (e) Following correction for specific amounts of hTPH2 proteins, the S41Y mutant exhibited higher inherent activity (similar to Table 1 for bacterially expressed proteins). All data are expressed as mean ± SEM (n = 3). ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: Total RNA from stably transfected PC12- hTPH2 and PC12-S41Y cells was isolated using the Qiagen RNeasy Plus Mini Kit. cDNAs were synthesized using the Omniscript reverse transcriptase kit (Qiagen, Germantown, MD, USA). qPCR was performed on a 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using primers for rat β- Actin (Rn00667869), rat TPH1 (Rn01476869), rat TPH2 (Rn00598017), and human TPH2 (Hs00998776) genes.

Techniques: Comparison, Expressing, Western Blot, Control, Recombinant, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Mutagenesis

Journal: Oncotarget

Article Title: YAP regulates PD-L1 expression in human NSCLC cells

doi: 10.18632/oncotarget.23051

Figure Lengend Snippet: ( A ) Schematic of the PD-L1 promoter region. Sequence analysis revealed a putative YAP1-TEAD co-binding site between −7911 to −7941 nucleotides upstream of the transcription start site. ( B ) ChIP assays were performed with SKLU-1 and H1299 cells in two regions (203 bp and 210 bp), which were shown by gel bands of RT-PCR products with 36 cycles. ( C ) ChIP assays were performed with A549, H1975, H1299 and SKLU-1 cells in two regions (203 bp and 210 bp), which were shown by gel bands of RT-PCR products with 30 cycles.

Article Snippet: The TaqMan Assay IDs of primer sequences for qRT-PCR were GUSB (Hs00939627 m1, Lot 1516740, 1516749 G3), PD-L1 (CD274, Hs01125301 m1, Lot 1548518, 1548524 D12) and YAP1 (Hs00902712 g1; Lot 1450809; 1450830 H6).

Techniques: Sequencing, Binding Assay, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: TGF-β-driven downregulation of the Wnt/β-Catenin transcription factor TCF7L2/TCF4 in PDGFRα + fibroblasts

doi: 10.1101/2020.01.05.895334

Figure Lengend Snippet: (A) Tcf7, Tcf7l1, Tcf7l2 , and Lef1 mRNA expression levels were analyzed by quantitative PCR in C3H/10T1/2 cells and MEFs in growing conditions. *** P <0.001; * P <0.05; n.s., not significant by one-way ANOVA with Dunnett’s post-test; n =4. (B) Skeletal muscle FAPs were FACS-isolated from the PDGFRα H2BEGFP mice. WGA staining labels lectins of the ECM. Scale bar: 50 μm. (C) Tcf7, Tcf7l1, Tcf7l2 , and Lef1 mRNA expression levels were analyzed by quantitative PCR in EGFP + FAPs in growing conditions. *** P <0.001; * P <0.05; n.s., not significant by one-way ANOVA with Dunnett’s post-test; n =3. (D) Tcf7l2 transcript abundance in the different muscle cell fractions. (n = 3–4). (E) Heat map showing gene expression of Tcf/Lef in muscle Lin - Sca1 + population (FAPs). (F) Representative western blot analysis showing TCF7L2 protein levels in gastrocnemius muscle. GAPDH was used as the loading control. (G) Confocal image of TCF7L2 immunofluorescence. Laminin-α2 (LN-α2) ( red ) and nuclei (Hoechst in blue ) were also stained. Scale bar: 10 μm. (H) Strategy used to isolate transit amplifying EGFP+ FAPs at day 3 post NTX TA injury. (I) Z-stack confocal image of cytospined EGFP+ FAPs showing TCF7L2 nuclear expression. (J) Tcf7, Tcf7l1, Tcf7l2 , and Lef1 mRNA expression levels were analyzed by quantitative PCR in cardiac fibroblasts in growing conditions. *** P <0.001; n.s., not significant by one-way ANOVA with Dunnett’s post-test; n =3. (K) Heat map showing gene expression levels of Tcf/Lef in cardiac fibroblasts. Known cardiac fibroblast marker genes ( Pdgfra and Tcf21 ) are also shown. Each column represents an individual uninjured cardiac fibroblast ( n =3). (M) Representative western blot analysis showing TCF7L2 protein levels in whole cardiac tissue. GAPDH was used as the loading control. (N) Immunofluorescence of TCF7L2 ( magenta ) in FACS-isolated heart PDGFRα-EGFP+ cells in growing conditions.

Article Snippet: Sequence information for the primers contained in the Taqman assays is provided here: Taqman probes (Thermo Fisher Scientific): Tcf7l2 mouse (Mm00501505_m1), Tcf7l1 mouse (Mm01188711_m1), Lef1 mouse (Mm00550265_m1), Tcf7 mouse (Mm00493445_m1), Runx2 mouse (Hs01047973_m1), Adipoq (Hs00605917_m1), and the housekeeping gene Hprt mouse (Mm03024075_m1).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Staining, Gene Expression, Western Blot, Control, Immunofluorescence, Marker