Journal: eLife

Article Title: Synapse-specific and compartmentalized expression of presynaptic homeostatic potentiation

doi: 10.7554/eLife.34338

Figure Lengend Snippet: GluRIIA knock down phenocopies GluRIIA mutants. ( A ) Representative images of NMJs on muscle six immunostained with antibodies that recognize GluRIIA, GluRIIC, and GluRIID receptor subunits in wild type ( w 1118 ), GluRIIA mutants ( w 1118 ;GluRIIA SP16 ), and G14 > GluRIIA RNAi ( w 1118 ;G14-Gal4 / +;UAS-GluRIIA RNAi / + ). ( B ) Quantification of the mean fluorescence intensity of individual GluR puncta reveals GluRIIA subunits are virtually undetectable at NMJs of both GluRIIA mutants and G14 > GluRIIA RNAi , while the essential subunits GluRIIC and GluRIID are moderately reduced, reflecting expression of the remaining GluRIIB-containing receptors. ( C ) Left: Schematic illustrating the composition of GluRIIA-containing and GluRIIB-containing postsynaptic receptor subtypes at the Drosophila NMJ. Right: Quantitative PCR analysis of GluR transcript levels for GluRIIA/B/C/D/E subunits in G14 > GluRIIA RNAi normalized to G14/+ . ( D ) Representative electrophysiological traces of EPSP and mEPSP recordings in the indicated genotypes. ( E–G ) Quantification of mEPSP amplitude ( E ), EPSP amplitude ( F ), and quantal content ( G ) in the indicated genotypes. Note that while mEPSP amplitudes are reduced to similar levels in GluRIIA mutants and G14 > GluRIIA RNAi , EPSP amplitudes remain similar to wild type because of a homeostatic increase in presynaptic glutamate release (quantal content). ( H .

Article Snippet: For analysis of pCaMKII levels, Ib and Is regions were identified using DLG and HRP on muscle 6/7 of segment A2 and A3, and only the pCamKII signal that co-localized with DLG was summated and divided by the bouton area under consideration to obtain average pCamKII. quantitative PCR: quantitative PCR (qPCR) was performed using the Luna Universal One-Step RT-qPCR Kit (NEB, E3005S) according to the manufacturer’s instructions.

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

Journal: Frontiers in Immunology

Article Title: Hypoxia Exacerbates Inflammatory Acute Lung Injury via the Toll-Like Receptor 4 Signaling Pathway

doi: 10.3389/fimmu.2018.01667

Figure Lengend Snippet: Toll-like receptor 4 (TLR4) signal pathway play an important role in the production of inflammatory cytokines. The microarray result of TLR4 expression pattern was validated by real-time quantitative PCR (RT-qPCR) (A) . The promoter activity of the TLR4 gene was detected by reporter assays in NR8383 cells (B) . The inhibitory effect of TAK-242 on the TLR4 mRNA and protein expression was detected by RT-qPCR and Western Blot in NR8383 cells (C) . The transcription levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), were detected by RT-qPCR in NR8383 cells (D) ; and their production levels in the culture medium were detected by enzyme-linked immune sorbent assay (E) . Data are presented as mean ± SD of three independent experimental repeats. * p

Article Snippet: The RT-qPCR reactions were conducted using a Bio-Rad real-time PCR system with the following modified program: initial denaturation at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 20 s. Products were verified by melting curve analysis and 1.5% agarose gel electrophoresis.

Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Hypoxia Exacerbates Inflammatory Acute Lung Injury via the Toll-Like Receptor 4 Signaling Pathway

doi: 10.3389/fimmu.2018.01667

Figure Lengend Snippet: The production of inflammatory cytokines in the bronchi alveolar lavage fluid (BALF) of rats in four groups. The inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in the BALF of rats were detected by enzyme-linked immune sorbent assay ( n = 10) (A–C) . The transcription level changes of TNF-α, IL-6, and IL-1β mRNA was determined by real-time quantitative PCR in NR8383 ( n = 3) and the total cells in BALF ( n = 5). The values of controls were normalized to 1 (D–F) . Data are presented as mean ± SD. * p

Article Snippet: The RT-qPCR reactions were conducted using a Bio-Rad real-time PCR system with the following modified program: initial denaturation at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 20 s. Products were verified by melting curve analysis and 1.5% agarose gel electrophoresis.

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Hypoxia Exacerbates Inflammatory Acute Lung Injury via the Toll-Like Receptor 4 Signaling Pathway

doi: 10.3389/fimmu.2018.01667

Figure Lengend Snippet: The effect of manipulation of hypoxia-inducible factor 1 alpha (HIF-1α) on the production of inflammatory cytokine tumor necrosis factor alpha (TNF-α). We used 50 µM PX478 treatment for 20 h (A) or transfection of a small interfering RNA (siRNA) specific for HIF-1α for 48 h (C) to downregulate the HIF-1α protein level in NR8383 cells. We used 1 mM DMOG treatment for 8 h (B) or transfection of a plasmid specific for HIF-1α for 48 h (D) to upregulate the HIF-1α protein level in NR8383 cells. Then we detected TNF-α levels in the culture media by using enzyme-linked immune sorbent assay and its mRNA expression in NR8383 cells by using real-time quantitative PCR. Data are presented as mean ± SD of three independent experimental repeats. ** p

Article Snippet: The RT-qPCR reactions were conducted using a Bio-Rad real-time PCR system with the following modified program: initial denaturation at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 20 s. Products were verified by melting curve analysis and 1.5% agarose gel electrophoresis.

Techniques: Transfection, Small Interfering RNA, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Hypoxia Exacerbates Inflammatory Acute Lung Injury via the Toll-Like Receptor 4 Signaling Pathway

doi: 10.3389/fimmu.2018.01667

Figure Lengend Snippet: Differential gene expression profiles were identified in the total cells of bronchi alveolar lavage fluid (BALF) by high-throughput microarray analysis. Before the gene chip detection, we mixed three animal samples into one pooled sample and total of three pooled samples were detected in each group. The clustering display was generated by Chip software with two-way data clustering. Each column represents an individual gene, and each row corresponds to an individual array. Gene expression values were standardized and color-coded relative to the mean (green, values less than the mean; red, values greater than the mean), the gene expression profiles of the COMB group are significant different from the other three groups (A) . We used the Scatter Plot to show the differential patterns of gene expression in the four groups (B) . We used a Venn diagram to show the distribution patterns of gene changes in lipopolysaccharides (LPS) group, HPO group, and COMB group (C) . Microarray findings of gene expression pattern were validated by using real-time quantitative PCR (RT-qPCR) (D) .

Article Snippet: The RT-qPCR reactions were conducted using a Bio-Rad real-time PCR system with the following modified program: initial denaturation at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 20 s. Products were verified by melting curve analysis and 1.5% agarose gel electrophoresis.

Techniques: Expressing, High Throughput Screening Assay, Microarray, Chromatin Immunoprecipitation, Generated, Software, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: BMC Veterinary Research

Article Title: Modulation of ovine SBD-1 expression by 17beta-estradiol in ovine oviduct epithelial cells

doi: 10.1186/1746-6148-8-143

Figure Lengend Snippet: E 2 -induced SBD-1 expression in ovine oviduct epithelial cells requires GPR30 and ERs. ( A ) The cells were treated with the GPR30 agonist G1 (10 -7 M) and E 2 (10 -8 M). ( B ) Other groups of cells were incubated with the inhibitor ICI 182,780 (Anti-ER 10 -7 M, 1 h) and then added with E 2 (10 -8 M). Furthermore, a blank control (water substituted for cDNA) was included in each qPCR reaction mixture, and a control was set up containing water instead of E 2 or/and G1/ ICI 182,178 in every group. ( C , D ) In the magnified inset, there were statistically significant changes between the two different treatments at different times from A or B. Total RNA extracted at the indicated times from the ovine oviduct epithelial cells (n = 3). Values are given as mean ± standard deviation. (*: p

Article Snippet: Quantification of SBD-1 and reference gene transcript copy number for the cDNA Samples were determined by RT-qPCR analysis carried out on the iCycler iQ5™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using gene specific primers, according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: BMC Veterinary Research

Article Title: Modulation of ovine SBD-1 expression by 17beta-estradiol in ovine oviduct epithelial cells

doi: 10.1186/1746-6148-8-143

Figure Lengend Snippet: E 2 up-regulates SBD-1 gene expression in ovine oviduct epithelial cells. Through QPCR, these primers are valid because of the single peak in every dissociation curve using SYBR® Green I. ( A , B ) Different concentrations of E 2 treatment of ovine oviduct epithelial cells increased the mRNA levels of SBD-1. The z-axis or y-axis represents the signal derived from quantitative PCR on total RNA extracted from the cells. ( C ) The x-axis shows the time during which the cells were exposed to 10 -8 M E 2 . Furthermore, a blank control (water substituted for cDNA) was included in each qPCR reaction mixture, and a control was set up containing water instead of E 2 in every group. ( E ) The greatest induction was observed in the 10 -8 M E 2 treated group at 3.5 h. Different concentrations of E 2 were added to the various groups, and total RNA extracted at the indicated time points from the ovine oviduct epithelial cells (n = 3). Values are given as mean ± standard deviation. (*: p

Article Snippet: Quantification of SBD-1 and reference gene transcript copy number for the cDNA Samples were determined by RT-qPCR analysis carried out on the iCycler iQ5™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using gene specific primers, according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Derivative Assay, Standard Deviation

Journal: BMC Veterinary Research

Article Title: Modulation of ovine SBD-1 expression by 17beta-estradiol in ovine oviduct epithelial cells

doi: 10.1186/1746-6148-8-143

Figure Lengend Snippet: E 2 -induced SBD-1 expression in ovine oviduct epithelial cells via cAMP/PKA pathway, PKC pathway and NF-κB pathway. Cells treated with the PKA (H-89, 50 μM), the PKC (H-7, 50 μM), and NF-κB inhibitors (PDTC, 50 μM) showed significantly lower SBD-1 expression compared to the control. Furthermore, a blank control (water substituted for cDNA) was included in each qPCR reaction mixture, and a control was set up containing water instead of E 2 or/and inhibitor(s) in every group. Total RNA extracted at the indicated time points from the ovine oviduct epithelial cells (n = 3). Values are given as mean ± standard deviation. (*: p

Article Snippet: Quantification of SBD-1 and reference gene transcript copy number for the cDNA Samples were determined by RT-qPCR analysis carried out on the iCycler iQ5™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using gene specific primers, according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Nature Communications

Article Title: mTORC1 accelerates retinal development via the immunoproteasome

doi: 10.1038/s41467-018-04774-9

Figure Lengend Snippet: mTORC1-induced developmental acceleration of the retina requires an immunoproteasome subunit Psmb9. a The 20S proteasomes were purified from E14.5 Tsc1-het and Tsc1-cko littermate mouse retinas by 20S Proteasome Purification Kit ® (Enzo Life Sciences), and caspase-like (β1/β1i), trypsin-like (β2/β2i), and chymotrypsin-like (β5/β5i) activities of the purified proteasomes were analyzed by measuring fluorescent intensities produced by cleaved peptide substrates Z-LLE-AMC (β1/β1i), Bz-VGF-AMC (β2/β2i), and Scu-LLVY-AMC (β5/β5i) (see details in Methods). The values are averages and error bars denote SD ( n = 3; 3 independent litters). b Relative mRNA levels of 20S proteasome subunits in E14.5 Tsc1-cko and rapamycin-treated mouse retinas were obtained by comparing real-time quantitative PCR (RT-qPCR) values with those of Tsc1-het and vehicle-treated mouse retinas at the same age. The values are averages obtained by 6 ( Tsc1-cko / Tsc1-het ) and 7 (rapamycin/vehicle) independent measurements with mRNA isolated from 4 ( Tsc1-cko / Tsc1-het ) and 6 (rapamycin/vehicle) independent batches. c Relative levels of 20S proteasome subunit proteins in the mouse retinas were also examined by WB with corresponding antibodies. d Image pixels of WB bands in Tsc1-cko samples were calculated by the ImageJ software and relative intensities were obtained by comparing the pixel numbers with those Tsc1-het samples. Values are average measurements of 4 independent WB results. e Incorporation of Psmb9 into the proteasome was examined by WB detection of Psmb9 in 20S proteasome core complex, which was isolated by the 20S Proteasome Purification Kit. P -values are obtained by Student’s t -test and shown in the graphs (*

Article Snippet: Real-time quantitative PCR (RT-qPCR) was then performed with CFX96 Real-Time System (Bio-Rad) by using iTaq Universal SYBR Green Supermix (Bio-Rad).

Techniques: Purification, Produced, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Western Blot, Software

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular mechanism(s) involved in differential expression of vitamin C transporters along the intestinal tract

doi: 10.1152/ajpgi.00369.2016

Figure Lengend Snippet: Region-specific expression of SVCT-1 and SVCT-2 mRNA in mouse and human jejunal and colonic mucosa. Real-time quantitative PCR was performed using mouse ( A and C ) and human ( B and D ) SVCT-1 and SVCT-2 gene-specific primers, and results were normalized to mouse and human β-actin, respectively. Values are means ± SE of multiple samples. * P

Article Snippet: Quantitative real-time PCR (RT-qPCR) was performed using the SYBR Green PCR kit and a real-time PCR detection system (model CFX96, Bio-Rad).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular mechanism(s) involved in differential expression of vitamin C transporters along the intestinal tract

doi: 10.1152/ajpgi.00369.2016

Figure Lengend Snippet: Mouse Slc23a1 and Slc23a2 promoter-luciferase activity in Caco-2 cells and chromatin immunoprecipitation (ChIP) quantitative PCR analysis of histone (H3) modification in mouse jejunal and colonic segments. A and B : pGL3- Slc23a1 and pGL3- Slc23a2 plasmids were transiently transfected into Caco-2 cells, firefly luciferase activity was determined, and data were normalized relative to Renilla luciferase activity. C and D : samples were scraped from mouse jejunal and colonic mucosa, and formaldehyde cross-linked chromatin was immunoprecipitated using antibodies specific to histone H3: H3K4me3, H3K9ac, and H3K27me3. DNA was purified from the immunoprecipitated complexes, and the purified DNA fragments were subjected to quantitative PCR. Results from separate PCR amplification of mouse Slc23a1 promoter spanning the region −88 to +08 bp ( C ) and mouse Slc23a2 promoter spanning the region −239 to −146 bp ( D ) were normalized relative to input DNA and expressed as percentage of enrichment relative to H3. Values are means ± SE of multiple samples. * P

Article Snippet: Quantitative real-time PCR (RT-qPCR) was performed using the SYBR Green PCR kit and a real-time PCR detection system (model CFX96, Bio-Rad).

Techniques: Luciferase, Activity Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Modification, Transfection, Immunoprecipitation, Purification, Polymerase Chain Reaction, Amplification

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular mechanism(s) involved in differential expression of vitamin C transporters along the intestinal tract

doi: 10.1152/ajpgi.00369.2016

Figure Lengend Snippet: Region-specific expression of hepatocyte nuclear factor (HNF) 1α and specificity protein 1 (Sp1) in mouse jejunal and colonic segments. A and B : Western blots of protein samples prepared from mouse jejunum and colon and densitometric quantification of blots. Blots were incubated separately with HNF1α- and Sp1-specific polyclonal antibodies. C and D : results from real-time quantitative PCR using mouse HNF1α and Sp1 gene-specific primers and cDNA synthesized from mouse jejunal and colonic RNA samples. Values are means ± SE of multiple samples. * P

Article Snippet: Quantitative real-time PCR (RT-qPCR) was performed using the SYBR Green PCR kit and a real-time PCR detection system (model CFX96, Bio-Rad).

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

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular mechanism(s) involved in differential expression of vitamin C transporters along the intestinal tract

doi: 10.1152/ajpgi.00369.2016

Figure Lengend Snippet: Region-specific expression of mouse and human SVCT-1 and SVCT-2 heterogeneous nuclear RNA (hnRNA) in jejunal and colonic mucosa. Real-time quantitative PCR was performed using mouse ( A and C ) and human ( B and D ) SVCT-1 and SVCT-2 hnRNA gene-specific primers, and results were normalized to mouse and human β-actin, respectively. Expression levels of hSVCT-1 and hSVCT-2 hnRNA were determined from a pool of 5–7 individuals. Values are means ± SE of multiple samples. * P

Article Snippet: Quantitative real-time PCR (RT-qPCR) was performed using the SYBR Green PCR kit and a real-time PCR detection system (model CFX96, Bio-Rad).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Nutrients

Article Title: Vitamin D Status Impacts Genital Mucosal Immunity and Markers of HIV-1 Susceptibility in Women

doi: 10.3390/nu12103176

Figure Lengend Snippet: Vitamin D supplementation increased mean serum 25(OH)D concentration to above adequate levels > 75 nmol/L (30 ng/mL). Women were sampled before and after 8 weeks of either weekly ( n = 10) or daily ( n = 10) high-dose vitamin D. ( A ) Grouped mean (± SD) serum 25(OH)D concentrations from both weekly and daily arms ( n = 20) before and after vitamin D supplementation. ( B ) Mean (± SD) serum 25(OH)D concentrations from weekly and daily supplementation arms. * Denotes significant difference between baseline and treatment; ** denotes significant difference between daily and weekly supplementation groups following treatment.

Article Snippet: All primers were purchased from Qiagen with catalog numbers as follows: Vitamin D Receptor (Catalog # 330001 PPH02123F), ALOX12 (Catalog # 330001 PPH05791C), ISG15 (Catalog # 330001 PPH01333F), RSAD2 (Catalog # 330001 PPH08142A), IL8 (Catalog # 330001 PPH00568A), FLG (Catalog # 330001 PPH24283A), CCL8 (Catalog # 330001 PPH01167B), CXCL11 (Catalog # 330001 PPH00506A), RPTN (Catalog # 330001 PPH18382A).

Techniques: Concentration Assay

Journal: Frontiers in Physiology

Article Title: Muscle Microbiopsy to Delineate Stem Cell Involvement in Young Patients: A Novel Approach for Children With Cerebral Palsy

doi: 10.3389/fphys.2020.00945

Figure Lengend Snippet: Altered differentiation of satellite cell-derived progenitors. (A) IF images of SC-derived progenitors from three representative TD children (top panels) and three representative patients with CP (lower panels) after 6 days of myogenic differentiation. MyHC (red); nuclei are counterstained by HOECHST (blue). Dotted circles indicate co-localization of the nuclei. Scale bar is 200 μm. (B) RT-qPCR analysis on SC-derived progenitors from TD children and CP patients for MYOMAKER, DESMIN, KIF5b , and ITGB1 expression normalized to β -ACTIN expression during myogenic differentiation at days 0, 3, and 6 ( n = 4 for each group). Data are expressed as mean and SD. Two-way ANOVA was performed ( p > 0.05).

Article Snippet: First strand cDNA synthesis was carried out based on 200 ng of total RNA ( ; ; ; ) following the protocol provided by the Superscript III First-Strand Synthesis SuperMix for RT-qPCR kit (Thermo Fisher Scientific).

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing