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The sensitivity analysis and standard curves of the mOTNRT-PCR assay. a , b , and c are the amplification curves of <t>RSV,</t> <t>HRV</t> and HMPV, respectively using serial 10-fold dilutions of the mixed recombinant plasmids from 10 8 to 10 0 copies/μL, and d is the standard curves of the mOTNRT-PCR assay. The R 2 of RSV, HRV and HMPV were 0.997, 0.994, 0.995 and the E of RSV, HRV and HMPV were 98.5, 95.7 and 92.5%, respectively
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1) Product Images from "A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus"

Article Title: A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus

Journal: Virology Journal

doi: 10.1186/s12985-018-1061-0

The sensitivity analysis and standard curves of the mOTNRT-PCR assay. a , b , and c are the amplification curves of RSV, HRV and HMPV, respectively using serial 10-fold dilutions of the mixed recombinant plasmids from 10 8 to 10 0 copies/μL, and d is the standard curves of the mOTNRT-PCR assay. The R 2 of RSV, HRV and HMPV were 0.997, 0.994, 0.995 and the E of RSV, HRV and HMPV were 98.5, 95.7 and 92.5%, respectively
Figure Legend Snippet: The sensitivity analysis and standard curves of the mOTNRT-PCR assay. a , b , and c are the amplification curves of RSV, HRV and HMPV, respectively using serial 10-fold dilutions of the mixed recombinant plasmids from 10 8 to 10 0 copies/μL, and d is the standard curves of the mOTNRT-PCR assay. The R 2 of RSV, HRV and HMPV were 0.997, 0.994, 0.995 and the E of RSV, HRV and HMPV were 98.5, 95.7 and 92.5%, respectively

Techniques Used: Polymerase Chain Reaction, Amplification, Recombinant

2) Product Images from "Complexes of Oligoribonucleotides with d-Mannitol Modulate the Innate Immune Response to Influenza A Virus H1N1 (A/FM/1/47) In Vivo"

Article Title: Complexes of Oligoribonucleotides with d-Mannitol Modulate the Innate Immune Response to Influenza A Virus H1N1 (A/FM/1/47) In Vivo

Journal: Pharmaceuticals

doi: 10.3390/ph11030073

Impaired up-regulation of the cytokines ifnε , ifnk , ifna2 , ifnb1 , and ifnγ induced by the influenza virus A/Fort Monmouth/1/1947-mouse adapted (H1N1) owing to the ORNs- d -М in vivo. Before and after infection with the influenza virus FM147 (4.0 lg LD 50 ), the BALB/c mice were treated with the ORNs- d -М. Total RNAs from the mice lungs were isolated and RT-qPCR was performed. The investigated mRNA levels were normalized to gapdh as a control. RUE: relative units of expression. ORNs- d -М control—ORNs- d -М injection into healthy mice, influenza control—infection of mice with influenza virus, ORNs- d -М + influenza—ORNs- d -М injection 24 h before influenza virus infection as prevention with ORNs- d -М, and influenza + ORNs- d -М—ORNs- d -М injection 24 h after influenza virus infection as treatment with ORNs- d -М. Data are shown as the mean ± SD for three independent experiments.
Figure Legend Snippet: Impaired up-regulation of the cytokines ifnε , ifnk , ifna2 , ifnb1 , and ifnγ induced by the influenza virus A/Fort Monmouth/1/1947-mouse adapted (H1N1) owing to the ORNs- d -М in vivo. Before and after infection with the influenza virus FM147 (4.0 lg LD 50 ), the BALB/c mice were treated with the ORNs- d -М. Total RNAs from the mice lungs were isolated and RT-qPCR was performed. The investigated mRNA levels were normalized to gapdh as a control. RUE: relative units of expression. ORNs- d -М control—ORNs- d -М injection into healthy mice, influenza control—infection of mice with influenza virus, ORNs- d -М + influenza—ORNs- d -М injection 24 h before influenza virus infection as prevention with ORNs- d -М, and influenza + ORNs- d -М—ORNs- d -М injection 24 h after influenza virus infection as treatment with ORNs- d -М. Data are shown as the mean ± SD for three independent experiments.

Techniques Used: In Vivo, Infection, Mouse Assay, Isolation, Quantitative RT-PCR, Expressing, Injection

3) Product Images from "The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis"

Article Title: The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis

Journal: Inflammatory Bowel Diseases

doi: 10.1093/ibd/izy060

Metagenomic analysis reveals Helicobacter variability with minimal effect on SAMP ileitis. A, Individual fecal metagenomic analysis of breeder mice was performed 8 months after the previously pooled fecal sampling experiment, demonstrating bacterial profiles with reproducible cluster within pairs of breeders for each individual cage. Squares highlight absence of Helicobacteraceae in Helicobacteraceae -negative SAMP colony. When present, Helicobacteraceae was highly abundant in the feces of positive mice. Analysis highlights within-cage mouse microbiota individualities, eg, Enterococcaceae. B, Boxplots, showing averages for each cage, reveal high abundance of Bacteroidaceae in SAMP mice (compared with experiment 1 in Fig. 1D ). C, The abundance of other Bacteroidetes is shown by boxplots. D, Compared with AKR and B6 mice, SAMP had a significantly higher level of Enterobacteracea (a family that contains Escherichia coli ). Notice marked abundance of dichotomous variability of Helicobacteraceae in mice. E, Gel electrophoresis of PCR amplified target regions of Helicobacter spp. within the 16S rRNA gene from fecal DNA of random mice form our SAMP and AKR colony, 4 months after the individual fecal metagenomic experiment. Notice high band intensity in SAMP (asterisks). Universal and PCR-specific primers were used to generate full or partial gene amplicons (1500 or 400 bp). F, Histological inflammation scores of distal ileum from rederived Helicobacter -negative SAMP compared with that of the SAMP colony show no differences. Notice wider variability in Helicobacter-positive mice. G, Representative snapshot of videostereomicroscopy shows unchanged morphological appearance of ileal mucosal surface in Helicobacter -negative SAMP mice. Scale = 500 um.
Figure Legend Snippet: Metagenomic analysis reveals Helicobacter variability with minimal effect on SAMP ileitis. A, Individual fecal metagenomic analysis of breeder mice was performed 8 months after the previously pooled fecal sampling experiment, demonstrating bacterial profiles with reproducible cluster within pairs of breeders for each individual cage. Squares highlight absence of Helicobacteraceae in Helicobacteraceae -negative SAMP colony. When present, Helicobacteraceae was highly abundant in the feces of positive mice. Analysis highlights within-cage mouse microbiota individualities, eg, Enterococcaceae. B, Boxplots, showing averages for each cage, reveal high abundance of Bacteroidaceae in SAMP mice (compared with experiment 1 in Fig. 1D ). C, The abundance of other Bacteroidetes is shown by boxplots. D, Compared with AKR and B6 mice, SAMP had a significantly higher level of Enterobacteracea (a family that contains Escherichia coli ). Notice marked abundance of dichotomous variability of Helicobacteraceae in mice. E, Gel electrophoresis of PCR amplified target regions of Helicobacter spp. within the 16S rRNA gene from fecal DNA of random mice form our SAMP and AKR colony, 4 months after the individual fecal metagenomic experiment. Notice high band intensity in SAMP (asterisks). Universal and PCR-specific primers were used to generate full or partial gene amplicons (1500 or 400 bp). F, Histological inflammation scores of distal ileum from rederived Helicobacter -negative SAMP compared with that of the SAMP colony show no differences. Notice wider variability in Helicobacter-positive mice. G, Representative snapshot of videostereomicroscopy shows unchanged morphological appearance of ileal mucosal surface in Helicobacter -negative SAMP mice. Scale = 500 um.

Techniques Used: Mouse Assay, Sampling, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification

4) Product Images from "Variability in the Composition of Pacific Oyster Microbiomes Across Oyster Families Exhibiting Different Levels of Susceptibility to OsHV-1 μvar Disease"

Article Title: Variability in the Composition of Pacific Oyster Microbiomes Across Oyster Families Exhibiting Different Levels of Susceptibility to OsHV-1 μvar Disease

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.00473

Heatmap of scaled OTU relative abundance for the 30 most abundant OTUs, as well as the remaining summed lowly abundant OTUs. Families ordered by OsHV-1 μvar disease-resistance. Heatmap was made using the R statistical environment using scaled data with the gplots and RColorBrewer packages ( Neuwirth, 2014 ; Warnes et al., 2016 ; R Core Team, 2017 ).
Figure Legend Snippet: Heatmap of scaled OTU relative abundance for the 30 most abundant OTUs, as well as the remaining summed lowly abundant OTUs. Families ordered by OsHV-1 μvar disease-resistance. Heatmap was made using the R statistical environment using scaled data with the gplots and RColorBrewer packages ( Neuwirth, 2014 ; Warnes et al., 2016 ; R Core Team, 2017 ).

Techniques Used:

Canonical correspondence analysis (CCA) plot using 3% relative abundance filtered data. Cupriavidus , Psychrilyobacter , Tenacibaculum , and Frankiales were found to be strongly associated with OsHV-1 μvar disease-resistance, while OTUs assigned to the Photobacterium , Vibrio , and Aliivibrio were negatively associated with OsHV-1 μvar disease-resistance. Axis 1 and 2 were able to significantly represent 53.2% of the data ( p = 0.001 for both axes with 999 permutations).
Figure Legend Snippet: Canonical correspondence analysis (CCA) plot using 3% relative abundance filtered data. Cupriavidus , Psychrilyobacter , Tenacibaculum , and Frankiales were found to be strongly associated with OsHV-1 μvar disease-resistance, while OTUs assigned to the Photobacterium , Vibrio , and Aliivibrio were negatively associated with OsHV-1 μvar disease-resistance. Axis 1 and 2 were able to significantly represent 53.2% of the data ( p = 0.001 for both axes with 999 permutations).

Techniques Used:

5) Product Images from "Over-Expression of a Maize N-Acetylglutamate Kinase Gene (ZmNAGK) Improves Drought Tolerance in Tobacco"

Article Title: Over-Expression of a Maize N-Acetylglutamate Kinase Gene (ZmNAGK) Improves Drought Tolerance in Tobacco

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.01902

Expression of ZmNAGK in leaves of maize plants exposed to different treatments. The detached maize plants were treated with (A) 10% PEG 6000 (B) 100 mM NaCl (C) 100 μM ABA (D) 50 nM BR (E) 10 mM H 2 O 2 for various times as indicated. Expression level of ZmNAGK relative to ZmActin2 was analyzed by qRT-PCR. Data are means ± SD ( n = 3). The asterisks indicate a significant difference compared to Control using the unpaired Student’s t -test ( ∗ P
Figure Legend Snippet: Expression of ZmNAGK in leaves of maize plants exposed to different treatments. The detached maize plants were treated with (A) 10% PEG 6000 (B) 100 mM NaCl (C) 100 μM ABA (D) 50 nM BR (E) 10 mM H 2 O 2 for various times as indicated. Expression level of ZmNAGK relative to ZmActin2 was analyzed by qRT-PCR. Data are means ± SD ( n = 3). The asterisks indicate a significant difference compared to Control using the unpaired Student’s t -test ( ∗ P

Techniques Used: Expressing, Quantitative RT-PCR

Expression of ZmNAGK in maize tissues. RNA was isolated from root, stem, old leaf, young leaf, female flower and male flower, and expression of ZmNAGK relative to ZmActin2 determined by qRT-PCR analysis. Data are means ± SD ( n = 3).
Figure Legend Snippet: Expression of ZmNAGK in maize tissues. RNA was isolated from root, stem, old leaf, young leaf, female flower and male flower, and expression of ZmNAGK relative to ZmActin2 determined by qRT-PCR analysis. Data are means ± SD ( n = 3).

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

Expressions of stress-related genes in leaves of ZmNAGK transgenic and vector-transformed plants in response to drought stress. (A–D) The transcript levels of NtDREB, NtERD10C, NtRD29A , and NtNCED1 under normal conditions or after 7 days drought treatment in transgenic and vector-transformed seedlings, respectively. Expression levels of these genes were relative to NtActin was analyzed by qRT-PCR. Data are means ± SD ( n = 3). The asterisks indicate a significant difference compared to vector-transformed plants using the unpaired Student’s t -test ( ∗ P
Figure Legend Snippet: Expressions of stress-related genes in leaves of ZmNAGK transgenic and vector-transformed plants in response to drought stress. (A–D) The transcript levels of NtDREB, NtERD10C, NtRD29A , and NtNCED1 under normal conditions or after 7 days drought treatment in transgenic and vector-transformed seedlings, respectively. Expression levels of these genes were relative to NtActin was analyzed by qRT-PCR. Data are means ± SD ( n = 3). The asterisks indicate a significant difference compared to vector-transformed plants using the unpaired Student’s t -test ( ∗ P

Techniques Used: Transgenic Assay, Plasmid Preparation, Transformation Assay, Expressing, Quantitative RT-PCR

6) Product Images from "Inhibitory role of adiponectin peptide I on rat choroidal neovascularization"

Article Title: Inhibitory role of adiponectin peptide I on rat choroidal neovascularization

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbamcr.2012.05.017

Effect of APNpI on VEGF expression. (A) RT-PCR products for VEGF in RPE-choroid-sclera of naïve, APNpI and PBS treated rats (n=6 rats in each group) at 10 post laser injury. APNpI decreased VEGF mRNA (327 bp) expression. A strong band for β-actin (335 bp) indicates equal amounts of RNA in each lane. (C) Densitometric analysis of PCR products. Western blot analysis of RPE-choroid tissue shows that laser treatment increases expression of VEGFA (23 kDa) in RPE-choroid compared to naïve control (B, D). Real-time PCR analysis of VEGFA mRNA expression in choroidal endothelial cells showed significant down regulation of VEGFA after APNpI (80 µg/mL) treatment (E). (F–H) Representative images of IHC staining for VEGF (green color). (I–K) Merged DIC (black and white), VEGF (green) and Isolectin IB4 (red) images helps recognize RPE cells (pigmented cells in upper part of each image) and vessels (non pigmented oval shaped structures, positively marked with Isolectin IB4 in red color) and laser injured area. Quantification of mean intensity of fluorescence in RPE (L), choroidal vessels (M) and the rest of the choroid or subretinal tissue (N) was done. VEGF expression significantly increased in all investigated structures at day 10 post laser injury (L, M, N). APNpI administration decreased expression of VEGF in RPE cells, vessels and laser injured area compared to PBS treated group. Bar for all images is 25 µm.
Figure Legend Snippet: Effect of APNpI on VEGF expression. (A) RT-PCR products for VEGF in RPE-choroid-sclera of naïve, APNpI and PBS treated rats (n=6 rats in each group) at 10 post laser injury. APNpI decreased VEGF mRNA (327 bp) expression. A strong band for β-actin (335 bp) indicates equal amounts of RNA in each lane. (C) Densitometric analysis of PCR products. Western blot analysis of RPE-choroid tissue shows that laser treatment increases expression of VEGFA (23 kDa) in RPE-choroid compared to naïve control (B, D). Real-time PCR analysis of VEGFA mRNA expression in choroidal endothelial cells showed significant down regulation of VEGFA after APNpI (80 µg/mL) treatment (E). (F–H) Representative images of IHC staining for VEGF (green color). (I–K) Merged DIC (black and white), VEGF (green) and Isolectin IB4 (red) images helps recognize RPE cells (pigmented cells in upper part of each image) and vessels (non pigmented oval shaped structures, positively marked with Isolectin IB4 in red color) and laser injured area. Quantification of mean intensity of fluorescence in RPE (L), choroidal vessels (M) and the rest of the choroid or subretinal tissue (N) was done. VEGF expression significantly increased in all investigated structures at day 10 post laser injury (L, M, N). APNpI administration decreased expression of VEGF in RPE cells, vessels and laser injured area compared to PBS treated group. Bar for all images is 25 µm.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining, Fluorescence

7) Product Images from "Neurodegeneration-associated TDP-43 Interacts with Fragile X Mental Retardation Protein (FMRP)/Staufen (STAU1) and Regulates SIRT1 Expression in Neuronal Cells *"

Article Title: Neurodegeneration-associated TDP-43 Interacts with Fragile X Mental Retardation Protein (FMRP)/Staufen (STAU1) and Regulates SIRT1 Expression in Neuronal Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.357582

TDP-43 knockdown-mediated SIRT1 down-regulation induces apoptosis and sensitizes cells to DNA toxicity. A and B , SH-SY5Ycells were transfected with indicated siRNAs and with appropriate plasmid constructs 12 h later. After 48 h of the plasmid transfection, TUNEL assay were performed with a fluorescence method. For each group, 6 different fields under fluorescence microscopy with 100-fold magnifications were randomly chosen and counted. Representative photos were showed in A and statistical analyzed in B . C–E , SH-SY5Y cells were treated as indicated with presence or absence of camptothecin (CPT, 0.2 μg/ml). 24 h later, cell viability assays were performed with a luminescent method. Error bars represent S.D. of triplicates. *, p
Figure Legend Snippet: TDP-43 knockdown-mediated SIRT1 down-regulation induces apoptosis and sensitizes cells to DNA toxicity. A and B , SH-SY5Ycells were transfected with indicated siRNAs and with appropriate plasmid constructs 12 h later. After 48 h of the plasmid transfection, TUNEL assay were performed with a fluorescence method. For each group, 6 different fields under fluorescence microscopy with 100-fold magnifications were randomly chosen and counted. Representative photos were showed in A and statistical analyzed in B . C–E , SH-SY5Y cells were treated as indicated with presence or absence of camptothecin (CPT, 0.2 μg/ml). 24 h later, cell viability assays were performed with a luminescent method. Error bars represent S.D. of triplicates. *, p

Techniques Used: Transfection, Plasmid Preparation, Construct, TUNEL Assay, Fluorescence, Microscopy, Cycling Probe Technology

FMRP and STAU1 bind to SIRT1 3′-UTR mainly depend on TDP-43. A , SH-SY5Y cells extracts were immunoprecipitated with anti-TDP-43, anti-FMRP, anti-STAU1 antibodies or control rabbit IgG, and the RIP was analyzed with quantitative RT-PCR using primers corresponding to 5′-UTR, coding region (CR) or 3′-UTR of the SIRT1 transcript. B , SH-SY5Y cells were transcfected with scrambled control siRNA, TDP-43 siRNA, FMRP siRNA, or STAU1 siRNA. RIP assays were performed with quantitative RT-PCR amplifying the 3′-UTR of SIRT1 transcript immunoprecipitated by the indicated antibodies. C , knockdown efficiency was validated by real time RT-PCR ( left panel ) and Western blotting ( right panel ). Error bars represent S.D. of triplicates. *, p
Figure Legend Snippet: FMRP and STAU1 bind to SIRT1 3′-UTR mainly depend on TDP-43. A , SH-SY5Y cells extracts were immunoprecipitated with anti-TDP-43, anti-FMRP, anti-STAU1 antibodies or control rabbit IgG, and the RIP was analyzed with quantitative RT-PCR using primers corresponding to 5′-UTR, coding region (CR) or 3′-UTR of the SIRT1 transcript. B , SH-SY5Y cells were transcfected with scrambled control siRNA, TDP-43 siRNA, FMRP siRNA, or STAU1 siRNA. RIP assays were performed with quantitative RT-PCR amplifying the 3′-UTR of SIRT1 transcript immunoprecipitated by the indicated antibodies. C , knockdown efficiency was validated by real time RT-PCR ( left panel ) and Western blotting ( right panel ). Error bars represent S.D. of triplicates. *, p

Techniques Used: Immunoprecipitation, Quantitative RT-PCR, Western Blot

Validation of SIRT1 down-regulation in TDP-43, FMRP of STAU1 silenced SH-SY5Y cells. A–C , SH-SY5Y cells were transfected with scrambled control siRNA, TDP-43 siRNA, FMRP siRNA, or STAU siRNA. Total RNAs were prepared and analyzed for SIRT1 expression by RT-PCR ( left panels ) and Western blotting ( middle panels ). The intensities of the gel bands were quantitated using the Quantity One (BioRad) program ( right panels ). The mRNA levels were normalized to those of GADPH and α-tubulin served as a loading control for the Western blotting. Error bars represent S.D. of triplicates. *, p
Figure Legend Snippet: Validation of SIRT1 down-regulation in TDP-43, FMRP of STAU1 silenced SH-SY5Y cells. A–C , SH-SY5Y cells were transfected with scrambled control siRNA, TDP-43 siRNA, FMRP siRNA, or STAU siRNA. Total RNAs were prepared and analyzed for SIRT1 expression by RT-PCR ( left panels ) and Western blotting ( middle panels ). The intensities of the gel bands were quantitated using the Quantity One (BioRad) program ( right panels ). The mRNA levels were normalized to those of GADPH and α-tubulin served as a loading control for the Western blotting. Error bars represent S.D. of triplicates. *, p

Techniques Used: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

8) Product Images from "Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells"

Article Title: Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells

Journal: Journal of Molecular Signaling

doi: 10.1186/1750-2187-8-9

RNA in MDA- MB- 231 protrusions. (A - B) Ethidium bromide (EtBr) mediated visualization of RNA in protrusions of MDA-MB-231 cells grown in Boyden chamber. (A) RNase treated cells (+ RNase) are shown for control of RNA specific EtBr staining. The left panels show DAPI staining, central pictures EtBr staining and merged pictures are shown in the right panels. Arrowheads exemplify EtBr staining in cell protrusions. Scale bar 20 μm. (B) EtBr mediated visualization of RNA localization in protrusions migrated through the 1 μm pore size membrane in the Boyden chamber. The cell bodies on the upper side of the membrane seen in (A) were removed by wiping and DAPI staining was performed in parallel to the pictures shown in (A) (left panel). EtBr staining was used to visualize cell material at the membrane lower side (arrowheads, right panel). Exposure times were increased compared to (A) visualizing also the 1 μm membrane pores. Scale bar 20 μm. (C) Determination of the relative RNA localization ratio in MDA-MB-231 protrusions by RT-qPCR analyses of candidate genes. The RNA localization ratio was calculated as the relative expression level in the protrusions fraction compared to the cell body fraction. The localization ratio of RNA in protrusions was normalized to the RNA localization ratio of ARPC3 given the value 1.
Figure Legend Snippet: RNA in MDA- MB- 231 protrusions. (A - B) Ethidium bromide (EtBr) mediated visualization of RNA in protrusions of MDA-MB-231 cells grown in Boyden chamber. (A) RNase treated cells (+ RNase) are shown for control of RNA specific EtBr staining. The left panels show DAPI staining, central pictures EtBr staining and merged pictures are shown in the right panels. Arrowheads exemplify EtBr staining in cell protrusions. Scale bar 20 μm. (B) EtBr mediated visualization of RNA localization in protrusions migrated through the 1 μm pore size membrane in the Boyden chamber. The cell bodies on the upper side of the membrane seen in (A) were removed by wiping and DAPI staining was performed in parallel to the pictures shown in (A) (left panel). EtBr staining was used to visualize cell material at the membrane lower side (arrowheads, right panel). Exposure times were increased compared to (A) visualizing also the 1 μm membrane pores. Scale bar 20 μm. (C) Determination of the relative RNA localization ratio in MDA-MB-231 protrusions by RT-qPCR analyses of candidate genes. The RNA localization ratio was calculated as the relative expression level in the protrusions fraction compared to the cell body fraction. The localization ratio of RNA in protrusions was normalized to the RNA localization ratio of ARPC3 given the value 1.

Techniques Used: Multiple Displacement Amplification, Staining, Quantitative RT-PCR, Expressing

P0071 mRNA isoform localization in MDA - MB - 231 and MCF7 cells. (A) Boyden chamber analysis of MCF7 cells. Protrusions of MCF7 cells migrated through a 1 μm micro porous membrane after 24 h assay time. α-Tubulin (green) and DAPI staining (blue) is shown without and after wipe (wipe) to remove cellular material present on the upper side of membranes. Examples of protrusions present on the Boyden chamber membrane lower side are indicated by arrowheads. Scale bar 100 μm. (B) RT-PCR analysis of mRNA localization in MCF7 and MDA-MB-231 cells. The RNA localization ratio was calculated as the relative expression in the protrusion fraction compared to the cell body fraction. The localization ratio was normalized to the RNA localization level of ARPC3 mRNA given the value 1. Pan-p0071 indicates detection of p0071 cDNA from exon 6 to exon 7. Student’s 2-Tailed t-test: *, p
Figure Legend Snippet: P0071 mRNA isoform localization in MDA - MB - 231 and MCF7 cells. (A) Boyden chamber analysis of MCF7 cells. Protrusions of MCF7 cells migrated through a 1 μm micro porous membrane after 24 h assay time. α-Tubulin (green) and DAPI staining (blue) is shown without and after wipe (wipe) to remove cellular material present on the upper side of membranes. Examples of protrusions present on the Boyden chamber membrane lower side are indicated by arrowheads. Scale bar 100 μm. (B) RT-PCR analysis of mRNA localization in MCF7 and MDA-MB-231 cells. The RNA localization ratio was calculated as the relative expression in the protrusion fraction compared to the cell body fraction. The localization ratio was normalized to the RNA localization level of ARPC3 mRNA given the value 1. Pan-p0071 indicates detection of p0071 cDNA from exon 6 to exon 7. Student’s 2-Tailed t-test: *, p

Techniques Used: Multiple Displacement Amplification, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing

DRS transcriptome analysis of purified RNA from MDA- MB -231 protrusions. (A) Comparison of DRS and RT-qPCR analyses. RNA localization ratios were normalized to ARPC3 mRNA given the value 1. (B) Annotation analysis of the 100 most MDA-MB-231 protrusion localized mRNA compared to the total group of mRNAs expressed with ≥ 5 tpm. Annotations were made using the IPA Ingenuity platform. (C) RT-qPCR confirmation analysis of selected MDA-MB-231 protrusion localized RNA identified by DRS. RNA localization ratios are normalized to ARPC3 mRNA. (D) Western blot analysis. Western blotting was performed on pooled material from three independent Boyden chamber experiments representing cell body fraction (CF) and protrusion fraction (PF). α-Tubulin Western blot analysis was used to equalize the loaded protein amounts. Subsequent western blot analyses were performed with antibodies for Histone H3, ZEB1, ANP32B and ACTB.
Figure Legend Snippet: DRS transcriptome analysis of purified RNA from MDA- MB -231 protrusions. (A) Comparison of DRS and RT-qPCR analyses. RNA localization ratios were normalized to ARPC3 mRNA given the value 1. (B) Annotation analysis of the 100 most MDA-MB-231 protrusion localized mRNA compared to the total group of mRNAs expressed with ≥ 5 tpm. Annotations were made using the IPA Ingenuity platform. (C) RT-qPCR confirmation analysis of selected MDA-MB-231 protrusion localized RNA identified by DRS. RNA localization ratios are normalized to ARPC3 mRNA. (D) Western blot analysis. Western blotting was performed on pooled material from three independent Boyden chamber experiments representing cell body fraction (CF) and protrusion fraction (PF). α-Tubulin Western blot analysis was used to equalize the loaded protein amounts. Subsequent western blot analyses were performed with antibodies for Histone H3, ZEB1, ANP32B and ACTB.

Techniques Used: Purification, Multiple Displacement Amplification, Quantitative RT-PCR, Indirect Immunoperoxidase Assay, Western Blot

9) Product Images from "SIGIRR, a Negative Regulator of TLR/IL-1R Signalling Promotes Microbiota Dependent Resistance to Colonization by Enteric Bacterial Pathogens"

Article Title: SIGIRR, a Negative Regulator of TLR/IL-1R Signalling Promotes Microbiota Dependent Resistance to Colonization by Enteric Bacterial Pathogens

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003539

IL-1R signaling is required for the exaggerated IEC responses in Sigirr −/− mice. The cecal tissues from infected Sigirr −/− mice show increased abundance of IL-1α gene transcripts (A) as compared to WT mice. The Sigirr −/− mice also express increased levels of IL-1β protein in their cecal tissues under uninfected and C. rodentium infected conditions as measured by (B) ELISA and (C) Western blot. Il-1r/Sigirr −/− mice exhibit increased (D) mortality rates, (E) elevated pathogen burdens and (F) severe mucosal damage. The severe damage suffered by the Il-1r/Sigirr −/− mice was accompanied by impaired IEC proliferation as shown by (G) immunostaining for Ki-67 and by higher IEC permeability as quantified by (H) FD4 in serum. Pathogen counts represent mucosal associated bacteria. Results are pooled from 2–4 independent infections with n = 3–4 per group. Error bars = SEM, (Student t test (Figure A, B, E), one-way ANOVA (Figure G, H), *P
Figure Legend Snippet: IL-1R signaling is required for the exaggerated IEC responses in Sigirr −/− mice. The cecal tissues from infected Sigirr −/− mice show increased abundance of IL-1α gene transcripts (A) as compared to WT mice. The Sigirr −/− mice also express increased levels of IL-1β protein in their cecal tissues under uninfected and C. rodentium infected conditions as measured by (B) ELISA and (C) Western blot. Il-1r/Sigirr −/− mice exhibit increased (D) mortality rates, (E) elevated pathogen burdens and (F) severe mucosal damage. The severe damage suffered by the Il-1r/Sigirr −/− mice was accompanied by impaired IEC proliferation as shown by (G) immunostaining for Ki-67 and by higher IEC permeability as quantified by (H) FD4 in serum. Pathogen counts represent mucosal associated bacteria. Results are pooled from 2–4 independent infections with n = 3–4 per group. Error bars = SEM, (Student t test (Figure A, B, E), one-way ANOVA (Figure G, H), *P

Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Immunostaining, Permeability

10) Product Images from "Profiling of Genes Related to Cross Protection and Competition for NbTOM1 by HLSV and TMV"

Article Title: Profiling of Genes Related to Cross Protection and Competition for NbTOM1 by HLSV and TMV

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073725

Genome organizations of HLSV and TMV and detection of viral RNA and protein levels in cross protection. ( A ) Genome organization of HLSV and TMV. Transcriptional level of HLSV ( B ) or TMV ( C ) gRNA/total viral RNA determined by quantitative real-time RT-PCR and translational level of CPs by western blot ( B and C ). Significant differences were calculated using the Student’s t -test, * and ** indicate significance at the 0.05 and 0.01 levels of confidence, respectively.
Figure Legend Snippet: Genome organizations of HLSV and TMV and detection of viral RNA and protein levels in cross protection. ( A ) Genome organization of HLSV and TMV. Transcriptional level of HLSV ( B ) or TMV ( C ) gRNA/total viral RNA determined by quantitative real-time RT-PCR and translational level of CPs by western blot ( B and C ). Significant differences were calculated using the Student’s t -test, * and ** indicate significance at the 0.05 and 0.01 levels of confidence, respectively.

Techniques Used: Quantitative RT-PCR, Western Blot

NbTOM1 transcript levels and virus accumulation with overexpression or silencing of NbTOM1 in Nicotiana benthamiana . (A) The transcriptional levels of NbTOM1 were detected in mock inoculation buffer, HLSV, TMV, HLSV+TMV (100:1) and HLSV+TMV (1:1) co-infected plants. The viral RNA levels of HLSV and TMV were determined using quantitative real-time RT-PCR with primer sequences corresponding to the coat protein genes in HLSV, or TMV or co-infected leaves. (B and C) The transcriptional levels of NbTOM1 were detected in NbTOM1 overexpressed or silenced leaves. The viral RNA levels were detected in plants first infiltrated with pGreen orpGreen- NbTOM1 (for overexpression), and pGreen or pGreen- NbTOM1 (nt1-581) (for silencing), followed by single virus (HLSV or TMV) infection or co-infection(HLSV+TMV) at 40 h post inoculation (hpi). (D) The coat proteins of HLSV and TMV were detected by western blot in NbTOM1 overexpressed or silenced leaves which were subsequently infected with single virus (HLSV or TMV) or co-infected with HLSV+TMV at 5 dpi (details see Materials Methods). Total protein from mock buffer inoculated N . benthamiana leaves was used as the negative control, while the total protein from HLSV or TMV infected leave samples which were confirmed earlier were used as positive controls.
Figure Legend Snippet: NbTOM1 transcript levels and virus accumulation with overexpression or silencing of NbTOM1 in Nicotiana benthamiana . (A) The transcriptional levels of NbTOM1 were detected in mock inoculation buffer, HLSV, TMV, HLSV+TMV (100:1) and HLSV+TMV (1:1) co-infected plants. The viral RNA levels of HLSV and TMV were determined using quantitative real-time RT-PCR with primer sequences corresponding to the coat protein genes in HLSV, or TMV or co-infected leaves. (B and C) The transcriptional levels of NbTOM1 were detected in NbTOM1 overexpressed or silenced leaves. The viral RNA levels were detected in plants first infiltrated with pGreen orpGreen- NbTOM1 (for overexpression), and pGreen or pGreen- NbTOM1 (nt1-581) (for silencing), followed by single virus (HLSV or TMV) infection or co-infection(HLSV+TMV) at 40 h post inoculation (hpi). (D) The coat proteins of HLSV and TMV were detected by western blot in NbTOM1 overexpressed or silenced leaves which were subsequently infected with single virus (HLSV or TMV) or co-infected with HLSV+TMV at 5 dpi (details see Materials Methods). Total protein from mock buffer inoculated N . benthamiana leaves was used as the negative control, while the total protein from HLSV or TMV infected leave samples which were confirmed earlier were used as positive controls.

Techniques Used: Over Expression, Infection, Quantitative RT-PCR, Western Blot, Negative Control

11) Product Images from "Caffeine and Rolipram Affect Smad Signalling and TGF-?1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells"

Article Title: Caffeine and Rolipram Affect Smad Signalling and TGF-?1 Stimulated CTGF and Transgelin Expression in Lung Epithelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097357

Effect of transgelin silencing on Smad signalling in lung epithelial cells. Transgelin was down-regulated by transgelin-specific shRNA (right side;”transgelin shRNA”) and compared with control cells, which were expressing non-specific shRNA (left side; “control shRNA”). A+B . After down-regulation of transgelin by specific shRNA, the TGF-β1–sensitive (CAGA) 12 -luciferase construct (A) or the p3TP-luciferase construct (B) were transiently transfected into A549 cells. Cells were then treated with or without TGF-β1 (10 ng/ml). Firefly luciferase activity was normalized to the activity of Renilla luciferase under control of the thymidine kinase promoter. * p
Figure Legend Snippet: Effect of transgelin silencing on Smad signalling in lung epithelial cells. Transgelin was down-regulated by transgelin-specific shRNA (right side;”transgelin shRNA”) and compared with control cells, which were expressing non-specific shRNA (left side; “control shRNA”). A+B . After down-regulation of transgelin by specific shRNA, the TGF-β1–sensitive (CAGA) 12 -luciferase construct (A) or the p3TP-luciferase construct (B) were transiently transfected into A549 cells. Cells were then treated with or without TGF-β1 (10 ng/ml). Firefly luciferase activity was normalized to the activity of Renilla luciferase under control of the thymidine kinase promoter. * p

Techniques Used: shRNA, Expressing, Luciferase, Construct, Transfection, Activity Assay

TGF-β1 increases and caffeine decreases transgelin expression in lung epithelial cells. A549 cells were incubated with TGF-β1 (A–C) or caffeine (D–F) in indicated concentrations and for various timepoints. Transgelin promoter (A+C), real-time PCR (B+E) and Western blot (C+F) analysis was performed. Means±SD of at least n = 3 independent experiments are shown. In C+F representative immunoblots of at least three independent experiments are shown. * p
Figure Legend Snippet: TGF-β1 increases and caffeine decreases transgelin expression in lung epithelial cells. A549 cells were incubated with TGF-β1 (A–C) or caffeine (D–F) in indicated concentrations and for various timepoints. Transgelin promoter (A+C), real-time PCR (B+E) and Western blot (C+F) analysis was performed. Means±SD of at least n = 3 independent experiments are shown. In C+F representative immunoblots of at least three independent experiments are shown. * p

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

Caffeine and rolipram inhibit TGF-β1 induced transgelin expression in lung epithelial cells. A+D : The transgelin promoter luciferase construct was transiently transfected into A549 cells. Cells were then treated with TGF-β1 (10 ng/ml) and/or different concentrations of caffeine (A) or rolipram (D). Firefly luciferase activity was normalized to the activity of Renilla luciferase under control of the thymidine kinase promoter. Relative luciferase activity compared to controls is shown. * p
Figure Legend Snippet: Caffeine and rolipram inhibit TGF-β1 induced transgelin expression in lung epithelial cells. A+D : The transgelin promoter luciferase construct was transiently transfected into A549 cells. Cells were then treated with TGF-β1 (10 ng/ml) and/or different concentrations of caffeine (A) or rolipram (D). Firefly luciferase activity was normalized to the activity of Renilla luciferase under control of the thymidine kinase promoter. Relative luciferase activity compared to controls is shown. * p

Techniques Used: Expressing, Luciferase, Construct, Transfection, Activity Assay

Effect of transgelin-specific shRNA on basal and TGF-β1 mediated induction of transgelin mRNA- and protein-expression in lung epithelial cells. Transgelin was down-regulated by transgelin-specific shRNA (right side; “transgelin shRNA”) compared with control cells, which were expressing non-specific shRNA (left side; “scrambled shRNA”). A549 cells were then cultured in serum reduced medium (0.2% FCS) for 24 h before treatment with TGF-β1. Total RNA and protein was isolated after incubation with TGF-β1 in different concentrations. Transgelin mRNA and protein levels were detected by real time-PCR (after 24 h) (A) or by Western blot analysis (after 48 h) (B), respectively. Means±SD of at least n = 3 independent experiments are shown. In B, a representative immunoblot of at least three independent experiments is shown. * p
Figure Legend Snippet: Effect of transgelin-specific shRNA on basal and TGF-β1 mediated induction of transgelin mRNA- and protein-expression in lung epithelial cells. Transgelin was down-regulated by transgelin-specific shRNA (right side; “transgelin shRNA”) compared with control cells, which were expressing non-specific shRNA (left side; “scrambled shRNA”). A549 cells were then cultured in serum reduced medium (0.2% FCS) for 24 h before treatment with TGF-β1. Total RNA and protein was isolated after incubation with TGF-β1 in different concentrations. Transgelin mRNA and protein levels were detected by real time-PCR (after 24 h) (A) or by Western blot analysis (after 48 h) (B), respectively. Means±SD of at least n = 3 independent experiments are shown. In B, a representative immunoblot of at least three independent experiments is shown. * p

Techniques Used: shRNA, Expressing, Cell Culture, Isolation, Incubation, Real-time Polymerase Chain Reaction, Western Blot

12) Product Images from "A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR"

Article Title: A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR

Journal: Scientific Reports

doi: 10.1038/srep04659

Duration of cell exposure to cell-lysis reagent. Cell lysates (200 μL) were prepared from MDCK-London cells (300,000/well; 24-well plate) infected with influenza virus (10,000 TCID 50 /well) by exposing them to (a) CL Buffer or (b) Bio-Rad SPR for 2–20 min at ~22°C. One μL of each resulting lysate was analyzed directly by one-step SYBR Green RT-qPCR with primers targeting the influenza virus matrix gene; total RNA was purified immediately from the remaining lysates and subjected to microfluidics-based electrophoresis using the Bio-Rad Experion system. Virtual gel images, sample RNA yields, and RNA Quality Indicators (RQIs) from a representative experiment (all derived from the Experion analysis) are shown; associated C q values from RT-qPCR are also indicated. For each condition, three independently prepared cell lysates were evaluated.
Figure Legend Snippet: Duration of cell exposure to cell-lysis reagent. Cell lysates (200 μL) were prepared from MDCK-London cells (300,000/well; 24-well plate) infected with influenza virus (10,000 TCID 50 /well) by exposing them to (a) CL Buffer or (b) Bio-Rad SPR for 2–20 min at ~22°C. One μL of each resulting lysate was analyzed directly by one-step SYBR Green RT-qPCR with primers targeting the influenza virus matrix gene; total RNA was purified immediately from the remaining lysates and subjected to microfluidics-based electrophoresis using the Bio-Rad Experion system. Virtual gel images, sample RNA yields, and RNA Quality Indicators (RQIs) from a representative experiment (all derived from the Experion analysis) are shown; associated C q values from RT-qPCR are also indicated. For each condition, three independently prepared cell lysates were evaluated.

Techniques Used: Lysis, Infection, SPR Assay, SYBR Green Assay, Quantitative RT-PCR, Purification, Electrophoresis, Derivative Assay

RT-qPCR-based microneutralization of influenza virus. In a well of a 96-well culture plate, influenza virus (A/Brisbane/59/2007; 1000 TCID 50 ) was mixed with a dilution from a 2-fold dilution series generated using a human serum sample with specific neutralizing activity. After an incubation for 1 hour at 37°C, trypsinized MDCK-London cells (30,000 per well) were added. At 6 hours post-infection, cell lysates were prepared using (a) CL Buffer or (b) Bio-Rad SPR and subjected to RT-qPCR (a single reaction per original culture well). RNA copy numbers were normalized to the mean value obtained from infected wells in the absence of neutralizing serum (virus control wells; +V/−Ab). The neutralization titer was defined as the reciprocal of the highest dilution factor of serum necessary to inhibit the PCR signal by 90% (threshold indicated by dotted line). Each serum dilution was assessed in triplicate infections; wells consisting of a replicate serum dilution series (corresponding to a row of wells in the original culture plate) are shown independently.
Figure Legend Snippet: RT-qPCR-based microneutralization of influenza virus. In a well of a 96-well culture plate, influenza virus (A/Brisbane/59/2007; 1000 TCID 50 ) was mixed with a dilution from a 2-fold dilution series generated using a human serum sample with specific neutralizing activity. After an incubation for 1 hour at 37°C, trypsinized MDCK-London cells (30,000 per well) were added. At 6 hours post-infection, cell lysates were prepared using (a) CL Buffer or (b) Bio-Rad SPR and subjected to RT-qPCR (a single reaction per original culture well). RNA copy numbers were normalized to the mean value obtained from infected wells in the absence of neutralizing serum (virus control wells; +V/−Ab). The neutralization titer was defined as the reciprocal of the highest dilution factor of serum necessary to inhibit the PCR signal by 90% (threshold indicated by dotted line). Each serum dilution was assessed in triplicate infections; wells consisting of a replicate serum dilution series (corresponding to a row of wells in the original culture plate) are shown independently.

Techniques Used: Quantitative RT-PCR, Generated, Activity Assay, Incubation, Infection, SPR Assay, Neutralization, Polymerase Chain Reaction

13) Product Images from "Stearoyl-CoA desaturase inhibition blocks formation of hepatitis C virus-induced specialized membranes"

Article Title: Stearoyl-CoA desaturase inhibition blocks formation of hepatitis C virus-induced specialized membranes

Journal: Scientific Reports

doi: 10.1038/srep04549

SCD-1 inhibition represses full-length HCV replication and infectivity. Huh7.5 cells were infected with JFH-1 T HCV (MOI = 0.1). 24 hours post-infection, infected cells were treated with varying concentrations of inhibitor A (mock, 100 nM or 500 nM). 120 hours post-treatment, intracellular HCV RNA levels were profiled via qRT-PCR (A). Supernatants were used to infect naïve Huh7.5 cells, and titres were measured (B). Viral titres are expressed as the number of focus-forming units (FFU) per ml of supernatant. Error bars represent standard deviation of the mean (n ≥ 2).
Figure Legend Snippet: SCD-1 inhibition represses full-length HCV replication and infectivity. Huh7.5 cells were infected with JFH-1 T HCV (MOI = 0.1). 24 hours post-infection, infected cells were treated with varying concentrations of inhibitor A (mock, 100 nM or 500 nM). 120 hours post-treatment, intracellular HCV RNA levels were profiled via qRT-PCR (A). Supernatants were used to infect naïve Huh7.5 cells, and titres were measured (B). Viral titres are expressed as the number of focus-forming units (FFU) per ml of supernatant. Error bars represent standard deviation of the mean (n ≥ 2).

Techniques Used: Inhibition, Infection, Quantitative RT-PCR, Standard Deviation

SCD-1 inhibition represses HCV replication. (A) A schematic depicting the full genomic replicon (FGR), subgenomic replicons (SGR), and full-length virus (JFH-1 T ) used in this study. All replicons encode for a neomycin (neo) resistance gene used as a selectable marker, and expression of HCV polyprotein is driven by encephalomyocarditis (EMCV) internal ribosomal entry site (IRES). All replicons are flanked by the HCV 5′ untranslated region (UTR) and 3′UTR. Both SGRs encode for NS3-NS5B. HCV SGR-Luc/Ubi (genotypes 1A and 1B) also encodes a luciferase reporter tag (luc) followed by a ubiquitin (ubi) gene. (B) Chemical structure of SCD-1 thiazole analogue inhibitor A . (C) Huh7 cells stably expressing HCV-SGR (genotype 1B) were treated with varying concentrations of the SCD inhibitor A . Quantitative real-time PCR (qRT-PCR) performed on RNA isolates 96 hours post-treatment demonstrates a decrease in HCV RNA levels induced by inhibiting SCD-1 in a dose-dependent manner. The error bars represent standard error of the mean (n ≥ 3). (D) Chemical structure of NS3 protease inhibitor B . (E) A dose response curve of HCV inhibition by inhibitor B after a 96 hour treatment in Huh7.5 cells stably expressing HCV-FGR (genotype 1B). RNA levels were analyzed as in (C).
Figure Legend Snippet: SCD-1 inhibition represses HCV replication. (A) A schematic depicting the full genomic replicon (FGR), subgenomic replicons (SGR), and full-length virus (JFH-1 T ) used in this study. All replicons encode for a neomycin (neo) resistance gene used as a selectable marker, and expression of HCV polyprotein is driven by encephalomyocarditis (EMCV) internal ribosomal entry site (IRES). All replicons are flanked by the HCV 5′ untranslated region (UTR) and 3′UTR. Both SGRs encode for NS3-NS5B. HCV SGR-Luc/Ubi (genotypes 1A and 1B) also encodes a luciferase reporter tag (luc) followed by a ubiquitin (ubi) gene. (B) Chemical structure of SCD-1 thiazole analogue inhibitor A . (C) Huh7 cells stably expressing HCV-SGR (genotype 1B) were treated with varying concentrations of the SCD inhibitor A . Quantitative real-time PCR (qRT-PCR) performed on RNA isolates 96 hours post-treatment demonstrates a decrease in HCV RNA levels induced by inhibiting SCD-1 in a dose-dependent manner. The error bars represent standard error of the mean (n ≥ 3). (D) Chemical structure of NS3 protease inhibitor B . (E) A dose response curve of HCV inhibition by inhibitor B after a 96 hour treatment in Huh7.5 cells stably expressing HCV-FGR (genotype 1B). RNA levels were analyzed as in (C).

Techniques Used: Inhibition, Marker, Expressing, Luciferase, Stable Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Protease Inhibitor

SCD-1 inhibition increases HCV RNA susceptibility to exogenous nucleases. (A) Huh7-SGR cells were treated with vehicle (DMSO) (Lanes 1–6) or inhibitor A (Lanes 7–11) at a concentration yielding a 50% reduction in HCV RNA levels. 96 hours post-treatment, samples were treated with a combination of digitonin (Lanes 2, 3, 5, 8, 9, 11) micrococcal nuclease (Lanes 3, 5, 6, 9, 11) or NP-40 (Lanes 4, 5, 10, 11). A representative integrity gel is shown (n = 5) illustrating 18S and 28S ribosomal RNA integrity after digitonin, micrococcal nuclease, and NP-40 treatments. Degradation of 18S and 28S rRNA bands is observed only upon permeabilizing the plasma membrane with digitonin, and treatment with exogenous micrococcal nuclease (Lanes 3, 5, 9, 11). Comparative RT-PCR was performed on HCV RNA to illustrate the relative HCV RNA abundance (cropped image below the RNA integrity gel) in the presence and absence of inhibitor A after treatments with digitonin, micrococcal nuclease, and NP-40. (B) Average HCV RNA abundance is shown from comparative PCR detecting HCV RNA. Densitometry was performed to calculate the HCV RNA abundance. All values were normalized by Lane 1 (no treatment). (C) Schematic model for SCD-1 inhibition-mediated disruption of HCV replication. Inhibition of SCD-1 alters HCV RNA's susceptibility to exogenously added nucleases by disrupting membrane HCV-associated membranes at the replication complexes. Multiple models are depicted for HCV RNA's susceptibility to endogenous nucleases (En nuc) and exogenous nucleases (Ex nuc). The size of the red arrow represents the magnitude of HCV RNA degradation by endogenous and exogenous nucleases under the different treatments including digitonin, micrococcal nucleases, and/or NP-40.
Figure Legend Snippet: SCD-1 inhibition increases HCV RNA susceptibility to exogenous nucleases. (A) Huh7-SGR cells were treated with vehicle (DMSO) (Lanes 1–6) or inhibitor A (Lanes 7–11) at a concentration yielding a 50% reduction in HCV RNA levels. 96 hours post-treatment, samples were treated with a combination of digitonin (Lanes 2, 3, 5, 8, 9, 11) micrococcal nuclease (Lanes 3, 5, 6, 9, 11) or NP-40 (Lanes 4, 5, 10, 11). A representative integrity gel is shown (n = 5) illustrating 18S and 28S ribosomal RNA integrity after digitonin, micrococcal nuclease, and NP-40 treatments. Degradation of 18S and 28S rRNA bands is observed only upon permeabilizing the plasma membrane with digitonin, and treatment with exogenous micrococcal nuclease (Lanes 3, 5, 9, 11). Comparative RT-PCR was performed on HCV RNA to illustrate the relative HCV RNA abundance (cropped image below the RNA integrity gel) in the presence and absence of inhibitor A after treatments with digitonin, micrococcal nuclease, and NP-40. (B) Average HCV RNA abundance is shown from comparative PCR detecting HCV RNA. Densitometry was performed to calculate the HCV RNA abundance. All values were normalized by Lane 1 (no treatment). (C) Schematic model for SCD-1 inhibition-mediated disruption of HCV replication. Inhibition of SCD-1 alters HCV RNA's susceptibility to exogenously added nucleases by disrupting membrane HCV-associated membranes at the replication complexes. Multiple models are depicted for HCV RNA's susceptibility to endogenous nucleases (En nuc) and exogenous nucleases (Ex nuc). The size of the red arrow represents the magnitude of HCV RNA degradation by endogenous and exogenous nucleases under the different treatments including digitonin, micrococcal nucleases, and/or NP-40.

Techniques Used: Inhibition, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

14) Product Images from "Generation of cardiac valve endocardial like cells from human pluripotent stem cells"

Article Title: Generation of cardiac valve endocardial like cells from human pluripotent stem cells

Journal: bioRxiv

doi: 10.1101/2020.04.20.050161

Cardiogenic mesoderm to endocardial endothelial cells a Schematic of hPSC-derived CPCs differentiation to endocardium progenitor cells. b The qPCR analysis of the known endocardium markers for hPSC-derived CPCs that were treated with the indicated signaling molecules for 3 days. c The qRT-PCR analysis of the known endocardium markers of hPSC-derived CPCs that were treated with VEGFA, BMP4 and bFGF for 3 days. d IF staining of day 6 hPSC-derived endocardial progenitors showing abundant expression of VE-Cad, GATA4 and NFATc1. Scale bar: 25 μm. e Quantification analysis of panel (d) by ImageJ. Bar graph represents percentage of VE-Cad, GATA4 and NFATc1 positive cells ± S.D of three independent experiments. f Western blotting using the indicated antibodies showing strong expression of VE-Cad, CD31 and NFATc1 in day 6 hPSC-derived endocardial progenitors. g Flow cytometry analysis showing percentage of VE-Cad and NFATc1 double positive cells. Day 3 hPSC-derived CPCs were treated with the indicated signaling molecules for 3 days, and stained with VE-Cad and NFATc1 antibodies. All experiments were repeated 3 times. Significant levels are: * p
Figure Legend Snippet: Cardiogenic mesoderm to endocardial endothelial cells a Schematic of hPSC-derived CPCs differentiation to endocardium progenitor cells. b The qPCR analysis of the known endocardium markers for hPSC-derived CPCs that were treated with the indicated signaling molecules for 3 days. c The qRT-PCR analysis of the known endocardium markers of hPSC-derived CPCs that were treated with VEGFA, BMP4 and bFGF for 3 days. d IF staining of day 6 hPSC-derived endocardial progenitors showing abundant expression of VE-Cad, GATA4 and NFATc1. Scale bar: 25 μm. e Quantification analysis of panel (d) by ImageJ. Bar graph represents percentage of VE-Cad, GATA4 and NFATc1 positive cells ± S.D of three independent experiments. f Western blotting using the indicated antibodies showing strong expression of VE-Cad, CD31 and NFATc1 in day 6 hPSC-derived endocardial progenitors. g Flow cytometry analysis showing percentage of VE-Cad and NFATc1 double positive cells. Day 3 hPSC-derived CPCs were treated with the indicated signaling molecules for 3 days, and stained with VE-Cad and NFATc1 antibodies. All experiments were repeated 3 times. Significant levels are: * p

Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Expressing, Western Blot, Flow Cytometry

15) Product Images from "Generation of cardiac valve endocardial like cells from human pluripotent stem cells"

Article Title: Generation of cardiac valve endocardial like cells from human pluripotent stem cells

Journal: bioRxiv

doi: 10.1101/2020.04.20.050161

Cardiogenic mesoderm to endocardial endothelial cells a Schematic of hPSC-derived CPCs differentiation to endocardium progenitor cells. b The qPCR analysis of the known endocardium markers for hPSC-derived CPCs that were treated with the indicated signaling molecules for 3 days. c The qRT-PCR analysis of the known endocardium markers of hPSC-derived CPCs that were treated with VEGFA, BMP4 and bFGF for 3 days. d IF staining of day 6 hPSC-derived endocardial progenitors showing abundant expression of VE-Cad, GATA4 and NFATc1. Scale bar: 25 μm. e Quantification analysis of panel (d) by ImageJ. Bar graph represents percentage of VE-Cad, GATA4 and NFATc1 positive cells ± S.D of three independent experiments. f Western blotting using the indicated antibodies showing strong expression of VE-Cad, CD31 and NFATc1 in day 6 hPSC-derived endocardial progenitors. g Flow cytometry analysis showing percentage of VE-Cad and NFATc1 double positive cells. Day 3 hPSC-derived CPCs were treated with the indicated signaling molecules for 3 days, and stained with VE-Cad and NFATc1 antibodies. All experiments were repeated 3 times. Significant levels are: * p
Figure Legend Snippet: Cardiogenic mesoderm to endocardial endothelial cells a Schematic of hPSC-derived CPCs differentiation to endocardium progenitor cells. b The qPCR analysis of the known endocardium markers for hPSC-derived CPCs that were treated with the indicated signaling molecules for 3 days. c The qRT-PCR analysis of the known endocardium markers of hPSC-derived CPCs that were treated with VEGFA, BMP4 and bFGF for 3 days. d IF staining of day 6 hPSC-derived endocardial progenitors showing abundant expression of VE-Cad, GATA4 and NFATc1. Scale bar: 25 μm. e Quantification analysis of panel (d) by ImageJ. Bar graph represents percentage of VE-Cad, GATA4 and NFATc1 positive cells ± S.D of three independent experiments. f Western blotting using the indicated antibodies showing strong expression of VE-Cad, CD31 and NFATc1 in day 6 hPSC-derived endocardial progenitors. g Flow cytometry analysis showing percentage of VE-Cad and NFATc1 double positive cells. Day 3 hPSC-derived CPCs were treated with the indicated signaling molecules for 3 days, and stained with VE-Cad and NFATc1 antibodies. All experiments were repeated 3 times. Significant levels are: * p

Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Expressing, Western Blot, Flow Cytometry

16) Product Images from "Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection"

Article Title: Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI128368

HOIL-1L is upregulated during IAV infection through a type I IFN receptor signaling axis. ( A and B ) AT2 cells were isolated from WT mice at 0, 3, 5, and 7 d.p.i. ( A ) HOIL-1L mRNA ( n = 5). ( B ) Representative HOIL-1L immunoblot and its quantification ( n = 4). ( C ) Representative native PAGE immunoblot of HOIL-1L and HOIP expression in AT2 cells infected in vitro with WSN ( n = 3). ( D ) HOIL-1L mRNA expression in AT2 cells from WT mice 0 and 3 d.p.i. sorted based on expression of the viral protein HA ( n = 8). ( E – I ) Representative HOIL-1L immunoblots and quantification. ( E ) A549 cells treated for 0, 8, 12, and 16 hours with conditioned medium (CM; “16*” indicates boiled CM) ( n = 4). ( F ) A549 cells treated with recombinant IFN-α ( n = 4). ( G ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with CM ( n = 3). ( H ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with WSN ( n = 3). ( I ) AT2 cells isolated from WT and CCR2 –/– mice treated in vitro with WSN ( n = 4). ( J ) Quantitative reverse transcriptase PCR quantification of HOIL-1L promoter after ChIP of IRF1 in A549 cells ( n = 4). ( K ) Representative HOIL-1L immunoblot and quantification in siControl- or siIRF1-transfected A549 cells treated with CM ( n = 4). ( L ) Proposed type I IFN pathway leading to HOIL-1L upregulation. Means ± SD overlaid with individual data points representing replicates are depicted; ** P
Figure Legend Snippet: HOIL-1L is upregulated during IAV infection through a type I IFN receptor signaling axis. ( A and B ) AT2 cells were isolated from WT mice at 0, 3, 5, and 7 d.p.i. ( A ) HOIL-1L mRNA ( n = 5). ( B ) Representative HOIL-1L immunoblot and its quantification ( n = 4). ( C ) Representative native PAGE immunoblot of HOIL-1L and HOIP expression in AT2 cells infected in vitro with WSN ( n = 3). ( D ) HOIL-1L mRNA expression in AT2 cells from WT mice 0 and 3 d.p.i. sorted based on expression of the viral protein HA ( n = 8). ( E – I ) Representative HOIL-1L immunoblots and quantification. ( E ) A549 cells treated for 0, 8, 12, and 16 hours with conditioned medium (CM; “16*” indicates boiled CM) ( n = 4). ( F ) A549 cells treated with recombinant IFN-α ( n = 4). ( G ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with CM ( n = 3). ( H ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with WSN ( n = 3). ( I ) AT2 cells isolated from WT and CCR2 –/– mice treated in vitro with WSN ( n = 4). ( J ) Quantitative reverse transcriptase PCR quantification of HOIL-1L promoter after ChIP of IRF1 in A549 cells ( n = 4). ( K ) Representative HOIL-1L immunoblot and quantification in siControl- or siIRF1-transfected A549 cells treated with CM ( n = 4). ( L ) Proposed type I IFN pathway leading to HOIL-1L upregulation. Means ± SD overlaid with individual data points representing replicates are depicted; ** P

Techniques Used: Infection, Isolation, Mouse Assay, Clear Native PAGE, Expressing, In Vitro, Western Blot, Recombinant, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Transfection

17) Product Images from "Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes, Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein"

Article Title: Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes, Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005498

CYC12 and CRK9AP are functional partners of CRK9. (A) Cumulative culture growth curves were obtained for CYC12 and CRK9AP silencing in the absence and presence of doxycycline (dox), the gene knockdown-inducing compound. For each knockdown a representative growth curve is shown. (B) Analysis of total RNA prepared from non-induced cells and cells in which CYC12 or CRK9AP were silenced for 1, 2 or 3 days. CYC12 or CRK9AP mRNA as well as α tubulin and RPB7 mRNA were analyzed by reverse transcription of oligo-dT and semi-quantitative PCR, whereas unspliced, pre-mRNA of α tubulin and RPB7 were analyzed by reverse transcription of random hexamers and by PCR using an oligonucleotide upstream of the SL addition site. rRNA was visualized by ethidium bromide staining after separation in an agarose gel. SL RNA, U2 snRNA and the Y structure intermediate were detected by primer extension assays using a SL RNA and a U2 snRNA-specific primer in the same reactions. (C) Anti-RPB1 immunoblot analysis of whole-cell lysates prepared from CRK9AP -silenced cells. Detection of the similar-sized RNA pol I subunit RPA1 served as a loading control.
Figure Legend Snippet: CYC12 and CRK9AP are functional partners of CRK9. (A) Cumulative culture growth curves were obtained for CYC12 and CRK9AP silencing in the absence and presence of doxycycline (dox), the gene knockdown-inducing compound. For each knockdown a representative growth curve is shown. (B) Analysis of total RNA prepared from non-induced cells and cells in which CYC12 or CRK9AP were silenced for 1, 2 or 3 days. CYC12 or CRK9AP mRNA as well as α tubulin and RPB7 mRNA were analyzed by reverse transcription of oligo-dT and semi-quantitative PCR, whereas unspliced, pre-mRNA of α tubulin and RPB7 were analyzed by reverse transcription of random hexamers and by PCR using an oligonucleotide upstream of the SL addition site. rRNA was visualized by ethidium bromide staining after separation in an agarose gel. SL RNA, U2 snRNA and the Y structure intermediate were detected by primer extension assays using a SL RNA and a U2 snRNA-specific primer in the same reactions. (C) Anti-RPB1 immunoblot analysis of whole-cell lysates prepared from CRK9AP -silenced cells. Detection of the similar-sized RNA pol I subunit RPA1 served as a loading control.

Techniques Used: Functional Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

CRK9AP depletion results in rapid co-loss of CRK9 and CYC12. (A) Immunoblot of whole cell lysates derived from non-induced (n.i.) and CRK9AP -silenced PF trypanosomes. The arrow indicates the gene knockdown of CRK9AP . Detection of the class I transcription factor A subunit 6 (CITFA6) served as a loading control. (B) Corresponding semi-quantitative PCR analysis of cDNA that was obtained from the same cells by reverse transcription of total RNA using oligo-dT. Relative RNA amounts were determined by ethidium bromide–stained rRNA.
Figure Legend Snippet: CRK9AP depletion results in rapid co-loss of CRK9 and CYC12. (A) Immunoblot of whole cell lysates derived from non-induced (n.i.) and CRK9AP -silenced PF trypanosomes. The arrow indicates the gene knockdown of CRK9AP . Detection of the class I transcription factor A subunit 6 (CITFA6) served as a loading control. (B) Corresponding semi-quantitative PCR analysis of cDNA that was obtained from the same cells by reverse transcription of total RNA using oligo-dT. Relative RNA amounts were determined by ethidium bromide–stained rRNA.

Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Staining

18) Product Images from "Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy"

Article Title: Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162467

Comparison different PCR methods and impact on quantification. Graphic representation of the quantification of dystrophin cDNA construct templates lacking or containing exon 51 mixed in different ratios ranging from 0% to 100% using nested PCR (40, 50 and 60 cycles), qPCR (LinRegPCR and Pfaffl methods) and ddPCR. The dashed red line indicates the theoretical template ratios. The solid black ddPCR line is almost indistinguishable from the theoretical line and both lines are superimposed. Data is based on one experiment using 2 replicates for ddPCR and 3 replicates for qPCR.
Figure Legend Snippet: Comparison different PCR methods and impact on quantification. Graphic representation of the quantification of dystrophin cDNA construct templates lacking or containing exon 51 mixed in different ratios ranging from 0% to 100% using nested PCR (40, 50 and 60 cycles), qPCR (LinRegPCR and Pfaffl methods) and ddPCR. The dashed red line indicates the theoretical template ratios. The solid black ddPCR line is almost indistinguishable from the theoretical line and both lines are superimposed. Data is based on one experiment using 2 replicates for ddPCR and 3 replicates for qPCR.

Techniques Used: Polymerase Chain Reaction, Construct, Nested PCR, Real-time Polymerase Chain Reaction

19) Product Images from "High dose ionizing radiation regulates micro RNA and gene expression changes in human peripheral blood mononuclear cells"

Article Title: High dose ionizing radiation regulates micro RNA and gene expression changes in human peripheral blood mononuclear cells

Journal: BMC Genomics

doi: 10.1186/1471-2164-15-814

Validation of the miRNA data by qPCR. qPCR revealed a strong correlation between the microarray and PCR data. Expression values were normalized relative to values in non-irradiated PBMCs. The expression of miR-99b* (A) , miR-887 (B) , and miR-4299 (C) , was significantly higher in irradiated PBMCs compared to non-irradiated PBMCs 20 hours after irradiation. Relative gene expression levels were calculated based on the mean value of four samples using the comparative Ct method. RNU44 served as internal reverence miRNA. Data are presented as the mean + SD. *p
Figure Legend Snippet: Validation of the miRNA data by qPCR. qPCR revealed a strong correlation between the microarray and PCR data. Expression values were normalized relative to values in non-irradiated PBMCs. The expression of miR-99b* (A) , miR-887 (B) , and miR-4299 (C) , was significantly higher in irradiated PBMCs compared to non-irradiated PBMCs 20 hours after irradiation. Relative gene expression levels were calculated based on the mean value of four samples using the comparative Ct method. RNU44 served as internal reverence miRNA. Data are presented as the mean + SD. *p

Techniques Used: Real-time Polymerase Chain Reaction, Microarray, Polymerase Chain Reaction, Expressing, Irradiation

20) Product Images from "Tumor Microenvironmental Changes Induced by the Sulfamate Carbonic Anhydrase IX Inhibitor S4 in a Laryngeal Tumor Model"

Article Title: Tumor Microenvironmental Changes Induced by the Sulfamate Carbonic Anhydrase IX Inhibitor S4 in a Laryngeal Tumor Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0108068

Metabolism and CAIX inhibition in SCCNij202. S4 does not influence the expression of several metabolic transporters and enzymes on the mRNA level. Abbreviations: ATP6V1C1, plasma membrane proton pump vacuolar ATPase (V-ATPase); CAIX, carbonic anhydrase IX; GLUT1, glucose transporter 1; MCT, monocarboxylate transporter; NHE1, sodium-hydrogen exchanger 1; NS, not significant; 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.
Figure Legend Snippet: Metabolism and CAIX inhibition in SCCNij202. S4 does not influence the expression of several metabolic transporters and enzymes on the mRNA level. Abbreviations: ATP6V1C1, plasma membrane proton pump vacuolar ATPase (V-ATPase); CAIX, carbonic anhydrase IX; GLUT1, glucose transporter 1; MCT, monocarboxylate transporter; NHE1, sodium-hydrogen exchanger 1; NS, not significant; 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.

Techniques Used: Inhibition, Expressing, Injection

21) Product Images from "Coordinate Regulation of Lipid Metabolism by Novel Nuclear Receptor Partnerships"

Article Title: Coordinate Regulation of Lipid Metabolism by Novel Nuclear Receptor Partnerships

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002645

NHR-49 collaborates with NHR-66 to regulate sphingolipid and lipid remodeling genes and with NHR-80 and NHR-13 to regulate fatty acid desaturase genes. (A) qRT-PCR measurement of genes involved in sphingolipid metabolism including fatty acid ceramidase, sphingosine phosphate lyase, glycosyl hydrolase and genes involved in lipid remodeling namely phospholipases, TAG lipase and O-acyltransferease expression in nhr-49(nr2041) (blue bars), nhr-66(ok940) (pink bars), nhr-80(tm1011) (green bars) and nhr-13(gk796) (purple bars) relative to wild-type animals. Values represent overall fold change of nhr-49(nr2041) , nhr-66(ok940) , nhr-80(tm1011) and nhr-13(gk796) animals with respect to wild-type worms. Expression was measured in L4 stage of development. Error bars represent SEM from 3 independent experiments. (B) qRT-PCR measurement of genes involved in fatty acid desaturases including fat-5 , fat-6 and fat-7 and genes involved in fatty acid beta-oxidation including acs-2 , cpt-5 and ech-1 in nhr-49(nr2041) (blue bars), nhr-66(ok940) (pink bars), nhr-80(tm1011) (green bars) and nhr-13(gk796) (purple bars) relative to wild-type animals. Values represent overall fold change in nhr-49(nr2041) , nhr-66(ok940) , nhr-80(tm1011) and nhr-13(gk796) animals relative to wild-type worms. Expression was measured at L4 stage of development. Error bars represent SEM from 3 independent experiments.
Figure Legend Snippet: NHR-49 collaborates with NHR-66 to regulate sphingolipid and lipid remodeling genes and with NHR-80 and NHR-13 to regulate fatty acid desaturase genes. (A) qRT-PCR measurement of genes involved in sphingolipid metabolism including fatty acid ceramidase, sphingosine phosphate lyase, glycosyl hydrolase and genes involved in lipid remodeling namely phospholipases, TAG lipase and O-acyltransferease expression in nhr-49(nr2041) (blue bars), nhr-66(ok940) (pink bars), nhr-80(tm1011) (green bars) and nhr-13(gk796) (purple bars) relative to wild-type animals. Values represent overall fold change of nhr-49(nr2041) , nhr-66(ok940) , nhr-80(tm1011) and nhr-13(gk796) animals with respect to wild-type worms. Expression was measured in L4 stage of development. Error bars represent SEM from 3 independent experiments. (B) qRT-PCR measurement of genes involved in fatty acid desaturases including fat-5 , fat-6 and fat-7 and genes involved in fatty acid beta-oxidation including acs-2 , cpt-5 and ech-1 in nhr-49(nr2041) (blue bars), nhr-66(ok940) (pink bars), nhr-80(tm1011) (green bars) and nhr-13(gk796) (purple bars) relative to wild-type animals. Values represent overall fold change in nhr-49(nr2041) , nhr-66(ok940) , nhr-80(tm1011) and nhr-13(gk796) animals relative to wild-type worms. Expression was measured at L4 stage of development. Error bars represent SEM from 3 independent experiments.

Techniques Used: Quantitative RT-PCR, Expressing, Cycling Probe Technology

22) Product Images from "Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis"

Article Title: Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis

Journal: British Journal of Cancer

doi: 10.1038/bjc.2013.535

LOX expression in mTECs. ( A ) Characterisation of mTECs and mNECs. FACS analysis of BS1-B4 lectin binding and CD31, CD105 and CD144 expression (white areas). Isotype controls are shown as black areas. All ECs were negative for the monocyte marker CD11b and the hematopoietic marker CD45. Human HB-EGF mRNA expression was found in human tumour cells but not in mouse ECs. ( B ) LOX mRNA is upregulated in mTECs, as determined by qPCR. ** P
Figure Legend Snippet: LOX expression in mTECs. ( A ) Characterisation of mTECs and mNECs. FACS analysis of BS1-B4 lectin binding and CD31, CD105 and CD144 expression (white areas). Isotype controls are shown as black areas. All ECs were negative for the monocyte marker CD11b and the hematopoietic marker CD45. Human HB-EGF mRNA expression was found in human tumour cells but not in mouse ECs. ( B ) LOX mRNA is upregulated in mTECs, as determined by qPCR. ** P

Techniques Used: Expressing, FACS, Binding Assay, Marker, Real-time Polymerase Chain Reaction

23) Product Images from "Inhibitors of Signaling Pathways That Block Reversal of HIV-1 Latency"

Article Title: Inhibitors of Signaling Pathways That Block Reversal of HIV-1 Latency

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01744-18

Quantitative PCR analysis of singly (SS) and multiply (MS) cellular HIV-1 RNA in the absence and presence of 1 μM prostratin and/or different concentrations of PF-3758309, danusertib, AZ 628, and P276-00. A total of 1 × 10 6 24ST1NLESG cells/well were exposed to prostratin (LRA) ± kinase inhibitor for 48 h, following which the total intracellular RNA from each condition was subjected to reverse transcription-quantitative PCR (RT-qPCR) analysis, as described in the Methods and Materials. Multiply spliced (MS) and singly spliced (SS) HIV-1 RNA were quantified relative to cellular IPO8 expression. Data are the average of 2 independent experiments.
Figure Legend Snippet: Quantitative PCR analysis of singly (SS) and multiply (MS) cellular HIV-1 RNA in the absence and presence of 1 μM prostratin and/or different concentrations of PF-3758309, danusertib, AZ 628, and P276-00. A total of 1 × 10 6 24ST1NLESG cells/well were exposed to prostratin (LRA) ± kinase inhibitor for 48 h, following which the total intracellular RNA from each condition was subjected to reverse transcription-quantitative PCR (RT-qPCR) analysis, as described in the Methods and Materials. Multiply spliced (MS) and singly spliced (SS) HIV-1 RNA were quantified relative to cellular IPO8 expression. Data are the average of 2 independent experiments.

Techniques Used: Real-time Polymerase Chain Reaction, Mass Spectrometry, Quantitative RT-PCR, Expressing

24) Product Images from "Helicobacter pylori modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis"

Article Title: Helicobacter pylori modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006514

H . pylori core heptose mutants are able to induce the hummingbird phenotype in human gastric AGS cells (wild type and CRISPR/Cas9 TIFA k/o) independently of IL-8 and TIFA. A ) mock-coincubated AGS wild type (wt) cells; B ) AGS wt cells coincubated with strain N6 wt bacteria; C ) AGS wt cells coincubated with strain N6 HP0858 ( hldE ) mutant bacteria; D ) AGS wt cells coincubated with N6 HP0858 complemented strain; E ) mock-coincubated AGS TIFA CRISPR/Cas9 k/o cells; F ) AGS TIFA k/o cells coincubated with N6 wt. Bacteria were coincubated with the cells for 4 h after centrifugation, fixed and microscopic images acquired. Black arrowheads designate some hummingbird phenotype cells for clarity. Size bars (white) represent 50 μm. G ), H ) Quantitation of hummingbird phenotype in bacteria-coincubated AGS wt cells ( G ) or AGS TIFA k/o cells ( H ) as shown in panels A ) through F ). 1,000 cells for each condition were quantitated for cell length using ImageJ (as detailed in Methods ). Mutants (of strain N6) coincubated with the cells shown in panels G) and H) are indicated by the respective gene numbers. Statistically significant differences between mock-coincubated and bacteria-coincubated cells are shown above the graphs (*p
Figure Legend Snippet: H . pylori core heptose mutants are able to induce the hummingbird phenotype in human gastric AGS cells (wild type and CRISPR/Cas9 TIFA k/o) independently of IL-8 and TIFA. A ) mock-coincubated AGS wild type (wt) cells; B ) AGS wt cells coincubated with strain N6 wt bacteria; C ) AGS wt cells coincubated with strain N6 HP0858 ( hldE ) mutant bacteria; D ) AGS wt cells coincubated with N6 HP0858 complemented strain; E ) mock-coincubated AGS TIFA CRISPR/Cas9 k/o cells; F ) AGS TIFA k/o cells coincubated with N6 wt. Bacteria were coincubated with the cells for 4 h after centrifugation, fixed and microscopic images acquired. Black arrowheads designate some hummingbird phenotype cells for clarity. Size bars (white) represent 50 μm. G ), H ) Quantitation of hummingbird phenotype in bacteria-coincubated AGS wt cells ( G ) or AGS TIFA k/o cells ( H ) as shown in panels A ) through F ). 1,000 cells for each condition were quantitated for cell length using ImageJ (as detailed in Methods ). Mutants (of strain N6) coincubated with the cells shown in panels G) and H) are indicated by the respective gene numbers. Statistically significant differences between mock-coincubated and bacteria-coincubated cells are shown above the graphs (*p

Techniques Used: CRISPR, Mutagenesis, Centrifugation, Quantitation Assay

Results of coincubation of parental, TIFA k/o and TIFA-complemented cell lines with H . pylori and its core heptose LPS biosynthesis mutants and detection of downstream signaling. A) AGS B) HEK293T parental and CRISPR-Cas9 TIFA k/o cells (pool) were transiently transfected with either an empty vector or a vector expressing human TIFA, by lipofectamine 2000 (HEK) or nucleofection (AGS). On the next day, parental, TIFA k/o and TIFA-complemented cells were coincubated with H . pylori of indicated genotypes (mutants indicated by respective gene numbers) at an MOI of 25 for 4 h. Cell supernatants were analyzed for IL-8 secretion by ELISA. Statistical significance of differences was determined using two-tailed, non-paired Student's t -test ( **p
Figure Legend Snippet: Results of coincubation of parental, TIFA k/o and TIFA-complemented cell lines with H . pylori and its core heptose LPS biosynthesis mutants and detection of downstream signaling. A) AGS B) HEK293T parental and CRISPR-Cas9 TIFA k/o cells (pool) were transiently transfected with either an empty vector or a vector expressing human TIFA, by lipofectamine 2000 (HEK) or nucleofection (AGS). On the next day, parental, TIFA k/o and TIFA-complemented cells were coincubated with H . pylori of indicated genotypes (mutants indicated by respective gene numbers) at an MOI of 25 for 4 h. Cell supernatants were analyzed for IL-8 secretion by ELISA. Statistical significance of differences was determined using two-tailed, non-paired Student's t -test ( **p

Techniques Used: CRISPR, Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

H . pylori IL-8 activation is abolished in AGS TIFA k/o cells, while H . pylori CagA translocation is independent of TIFA expression, IL-8 induction, and core heptose biosynthesis pathway intermediates. A) qRT-PCR detection of TIFA transcript in AGS wt and AGS TIFA k/o (KO) cells. Cells were mock-incubated or cocultured for 2 h with H . pylori strain N6 wild type (wt), isogenic cagY mutant (HP0527) and heptose pathway hldE (HP0858) or rfaD (HP0859) mutants as indicated. The TIFA transcript amounts are given in % of the mock-coincubated AGS parental cell, which was set to 100%. B) IL-8 induction in AGS CRISPR-Cas9 TIFA knock-out (KO) cells. Indicated H . pylori strains (N6 wt and isogenic core heptose mutants) were coincubated for 4 h at an MOI of 25 bacteria per cell with AGS parental cells, TIFA k/o cell single clone and TIFA k/o cell pool. HP0858 comp. is the complemented strain. Significance of differences (p) between N6 wt coincubated wild type and k/o cells were calculated by Student‘s t -test. C) CagA translocation by H . pylori N6 and isogenic cagY (HP0527; CagT4SS functional negative control) and LPS core heptose biosynthesis mutants in AGS cells and AGS TIFA KO cells. AGS cells were coincubated with H . pylori strain N6 wt or isogenic mutant bacteria for 4 h. 20 μg soluble protein (cleared cell lysates) per lane was separated on an SDS gel and blotted to nitrocellulose membrane. Western blots were incubated with antibodies as indicated. HP signifies the antiserum detection of heat-stable H . pylori surface antigens (Dako Cytomation, for antibodies see S6 Table ) and was used as a universal control for amounts of invariable H . pylori proteins in the preparations. Actin detection was used as a loading control for amounts of AGS cell proteins. D ) densitometric quantitation of CagA and p-CagA (CagA translocation) of Western blot results shown in panel C (see Methods ). The intensity values were normalized to human actin and to HP invariable protein for each condition and are depicted in % of the positive control (AGS cells coincubated with H . pylori N6 wild type bacteria), which was set to 100%.
Figure Legend Snippet: H . pylori IL-8 activation is abolished in AGS TIFA k/o cells, while H . pylori CagA translocation is independent of TIFA expression, IL-8 induction, and core heptose biosynthesis pathway intermediates. A) qRT-PCR detection of TIFA transcript in AGS wt and AGS TIFA k/o (KO) cells. Cells were mock-incubated or cocultured for 2 h with H . pylori strain N6 wild type (wt), isogenic cagY mutant (HP0527) and heptose pathway hldE (HP0858) or rfaD (HP0859) mutants as indicated. The TIFA transcript amounts are given in % of the mock-coincubated AGS parental cell, which was set to 100%. B) IL-8 induction in AGS CRISPR-Cas9 TIFA knock-out (KO) cells. Indicated H . pylori strains (N6 wt and isogenic core heptose mutants) were coincubated for 4 h at an MOI of 25 bacteria per cell with AGS parental cells, TIFA k/o cell single clone and TIFA k/o cell pool. HP0858 comp. is the complemented strain. Significance of differences (p) between N6 wt coincubated wild type and k/o cells were calculated by Student‘s t -test. C) CagA translocation by H . pylori N6 and isogenic cagY (HP0527; CagT4SS functional negative control) and LPS core heptose biosynthesis mutants in AGS cells and AGS TIFA KO cells. AGS cells were coincubated with H . pylori strain N6 wt or isogenic mutant bacteria for 4 h. 20 μg soluble protein (cleared cell lysates) per lane was separated on an SDS gel and blotted to nitrocellulose membrane. Western blots were incubated with antibodies as indicated. HP signifies the antiserum detection of heat-stable H . pylori surface antigens (Dako Cytomation, for antibodies see S6 Table ) and was used as a universal control for amounts of invariable H . pylori proteins in the preparations. Actin detection was used as a loading control for amounts of AGS cell proteins. D ) densitometric quantitation of CagA and p-CagA (CagA translocation) of Western blot results shown in panel C (see Methods ). The intensity values were normalized to human actin and to HP invariable protein for each condition and are depicted in % of the positive control (AGS cells coincubated with H . pylori N6 wild type bacteria), which was set to 100%.

Techniques Used: Activation Assay, Translocation Assay, Expressing, Quantitative RT-PCR, Incubation, Mutagenesis, CRISPR, Knock-Out, Functional Assay, Negative Control, SDS-Gel, Western Blot, Quantitation Assay, Positive Control

25) Product Images from "Time-Course RNAseq Reveals Exserohilum turcicum Effectors and Pathogenicity Determinants"

Article Title: Time-Course RNAseq Reveals Exserohilum turcicum Effectors and Pathogenicity Determinants

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.00360

Expression analysis of two candidate effectors from Exserohilum turcicum . Real-time quantitative PCR (RT-qPCR) data are shown for Ecp6 (A) and SIX13-like (B) from each Exserohilum turcicum race in planta at several time points, as well as in vitro (for race 13N). The y -axis units are the relative expression values (log-transformed mean calibrated normalized relative quantities). Analysis of molecular variance (ANOVA) and the Tukey multiple pairwise comparison was performed to identify pairwise differences in R (R Core Team, 2017 ) (R Project for Statistical Computing, RRID:SCR_001905 ). Different lowercase letters indicate significant differences between datasets. Significant pairwise differences were detected in planta for Ecp6 (A) and SIX13-like (B) . The samples collected before inoculation (0 dpi) were excluded as no fungal transcripts were detected.
Figure Legend Snippet: Expression analysis of two candidate effectors from Exserohilum turcicum . Real-time quantitative PCR (RT-qPCR) data are shown for Ecp6 (A) and SIX13-like (B) from each Exserohilum turcicum race in planta at several time points, as well as in vitro (for race 13N). The y -axis units are the relative expression values (log-transformed mean calibrated normalized relative quantities). Analysis of molecular variance (ANOVA) and the Tukey multiple pairwise comparison was performed to identify pairwise differences in R (R Core Team, 2017 ) (R Project for Statistical Computing, RRID:SCR_001905 ). Different lowercase letters indicate significant differences between datasets. Significant pairwise differences were detected in planta for Ecp6 (A) and SIX13-like (B) . The samples collected before inoculation (0 dpi) were excluded as no fungal transcripts were detected.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, In Vitro, Transformation Assay

Transcriptional profiles of Exserohilum turcicum candidate secreted effector proteins similar to known effector proteins which affect virulence. A subset of candidate effectors was selected based on similarity to known proteins on the PHI-Base or NCBI database, annotations, or literature. The scale bar indicates the read count values. Row names are given as protein identifier|Gene name/description. Column names indicate the isolate race (13N or 23N) as well as days post-inoculation (dpi). Arrows indicate candidates selected for reverse transcriptase quantitative PCR (RT-qPCR) analysis.
Figure Legend Snippet: Transcriptional profiles of Exserohilum turcicum candidate secreted effector proteins similar to known effector proteins which affect virulence. A subset of candidate effectors was selected based on similarity to known proteins on the PHI-Base or NCBI database, annotations, or literature. The scale bar indicates the read count values. Row names are given as protein identifier|Gene name/description. Column names indicate the isolate race (13N or 23N) as well as days post-inoculation (dpi). Arrows indicate candidates selected for reverse transcriptase quantitative PCR (RT-qPCR) analysis.

Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR

26) Product Images from "RNA exosome mutations in pontocerebellar hypoplasia alter ribosome biogenesis and p53 levels"

Article Title: RNA exosome mutations in pontocerebellar hypoplasia alter ribosome biogenesis and p53 levels

Journal: Life Science Alliance

doi: 10.26508/lsa.202000678

exosc8 and exosc9 homozygous mutant zebrafish have increased non-coding RNA expression and differences in gene expression. Summary of RNAseq performed on 5-dpf wild-type, exosc8 homozygous mutant and exosc9 homozygous mutant zebrafish embryos. (A) Gene set over-representation analysis of RNAs that were significantly increased or decreased in both homozygous mutant exosc8 and exosc9 zebrafish embryos compared with controls. (B) Selected GO terms and number of transcripts associated with them among the increased protein-coding RNAs in the mutant zebrafish lines. GO term analysis was performed on http://pantherdb.org/ . (C) Log 2 ratios of cumulative, normalised RNA read counts for different non-coding RNA types in exosc8 or exosc9 homozygous mutant embryos versus controls. (D) Correlation of exosc8 and exosc9 log 2 ratios of cumulative, normalised read counts for individual non-coding RNA genes. (E) Volcano plot showing the differential expression of transcripts in exosc8 and exosc9 homozygous mutants versus controls, with statistical significance ( P -value) on the y-axis versus the magnitude of change (fold change) on the x-axis. (F) Selected mRNAs where increased levels were observed in eoxsc8 and exosc9 homozygous mutant zebrafish. (F, G) Validation of changes in gene expression via qRT-PCR for the mRNAs represented in (F). Error bars represent the standard error (±SEM), and statistical analysis was performed using unpaired t tests ( exosc8 versus wt and exosc9 versus wt, respectively). Values have been normalised to a housekeeping gene and to wt fish; wt level has been set to one and is represented by the dotted line. * P -value
Figure Legend Snippet: exosc8 and exosc9 homozygous mutant zebrafish have increased non-coding RNA expression and differences in gene expression. Summary of RNAseq performed on 5-dpf wild-type, exosc8 homozygous mutant and exosc9 homozygous mutant zebrafish embryos. (A) Gene set over-representation analysis of RNAs that were significantly increased or decreased in both homozygous mutant exosc8 and exosc9 zebrafish embryos compared with controls. (B) Selected GO terms and number of transcripts associated with them among the increased protein-coding RNAs in the mutant zebrafish lines. GO term analysis was performed on http://pantherdb.org/ . (C) Log 2 ratios of cumulative, normalised RNA read counts for different non-coding RNA types in exosc8 or exosc9 homozygous mutant embryos versus controls. (D) Correlation of exosc8 and exosc9 log 2 ratios of cumulative, normalised read counts for individual non-coding RNA genes. (E) Volcano plot showing the differential expression of transcripts in exosc8 and exosc9 homozygous mutants versus controls, with statistical significance ( P -value) on the y-axis versus the magnitude of change (fold change) on the x-axis. (F) Selected mRNAs where increased levels were observed in eoxsc8 and exosc9 homozygous mutant zebrafish. (F, G) Validation of changes in gene expression via qRT-PCR for the mRNAs represented in (F). Error bars represent the standard error (±SEM), and statistical analysis was performed using unpaired t tests ( exosc8 versus wt and exosc9 versus wt, respectively). Values have been normalised to a housekeeping gene and to wt fish; wt level has been set to one and is represented by the dotted line. * P -value

Techniques Used: Mutagenesis, RNA Expression, Expressing, Quantitative RT-PCR, Fluorescence In Situ Hybridization

27) Product Images from "PRP4K is a haploinsufficient tumour suppressor negatively regulated during epithelial-to-mesenchymal transition"

Article Title: PRP4K is a haploinsufficient tumour suppressor negatively regulated during epithelial-to-mesenchymal transition

Journal: bioRxiv

doi: 10.1101/2020.04.19.043851

Overexpression of PRP4K does not increase the expression of YAP target genes in eIF3e-depleted MCF10A cells. A ) Quantitative PCR analysis of cDNA prepared from control (shCtrl) and eIF3e-depleted cells (shEIF3E-1 and shEIF3-2), with data normalized to at least two reference genes as indicated. N = 3, error bars= SEM. Significance was determined by a one-way ANOVA. B ) Quantitative PCR analysis of cDNA prepared from control (shCtrl) and eIF3e-depleted cells (shEIF3E-1 and shEIF3-2) transfected with pBluescript SK- (pBSK) or T7-tagged PRP4K, with data normalized to at least two reference genes as indicated. N = 3, error bars= SEM. Significance was determined by a one-way ANOVA. *=p
Figure Legend Snippet: Overexpression of PRP4K does not increase the expression of YAP target genes in eIF3e-depleted MCF10A cells. A ) Quantitative PCR analysis of cDNA prepared from control (shCtrl) and eIF3e-depleted cells (shEIF3E-1 and shEIF3-2), with data normalized to at least two reference genes as indicated. N = 3, error bars= SEM. Significance was determined by a one-way ANOVA. B ) Quantitative PCR analysis of cDNA prepared from control (shCtrl) and eIF3e-depleted cells (shEIF3E-1 and shEIF3-2) transfected with pBluescript SK- (pBSK) or T7-tagged PRP4K, with data normalized to at least two reference genes as indicated. N = 3, error bars= SEM. Significance was determined by a one-way ANOVA. *=p

Techniques Used: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Transfection

The induction of EMT decreases PRP4K protein expression in HMLE and MCF10A cells. A ) Phase contrast micrographs of MCF10A and HMLE cells treated with (+EMT) or without (WT) EMT-induction media for 5 days. B ) Western blot analysis of PRP4K protein expression from whole cell lysates prepared from cells treated with EMT-induction media as in A. C ) Quantitative PCR of cDNA prepared from cells treated with EMT-induction media as in A, with data normalized to at least two reference genes as indicated. N = 3, error bars= SEM. Significance was determined by a t-test. *=p
Figure Legend Snippet: The induction of EMT decreases PRP4K protein expression in HMLE and MCF10A cells. A ) Phase contrast micrographs of MCF10A and HMLE cells treated with (+EMT) or without (WT) EMT-induction media for 5 days. B ) Western blot analysis of PRP4K protein expression from whole cell lysates prepared from cells treated with EMT-induction media as in A. C ) Quantitative PCR of cDNA prepared from cells treated with EMT-induction media as in A, with data normalized to at least two reference genes as indicated. N = 3, error bars= SEM. Significance was determined by a t-test. *=p

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

The induction of EMT by depletion of eIF3e negatively regulates the translation of PRP4K in MCF10A cells. A ) Quantitative PCR of PRP4K gene ( PRPF4B ) mRNA expression in control (shCtrl) and eIF3e-depleted (shEIF3E-1 and shEIF3-2) MCF10A cells treated with 10 μg/mL Actinomycin D for the indicated time periods, with data normalized to at least two reference genes as indicated. N = 3, error bars= SEM. Significance was determined by a one-way ANOVA. B ) Western blot analysis of whole cell lysates of MCF10A shCtrl and shEIF3E cells treated with 50μg/mL cycloheximide for the indicated time periods. C ) Puromycin incorporation assay of MCF10A shCtrl and shEIF3E cells. Cells were grown for 24 h without puromycin selection and then pulsed with puromycin prior to Western blot analysis of total cell lysates (left) or following immunoprecipitation (IP) of PRP4K and detection of puromycylation of proteins and PRP4K (right).
Figure Legend Snippet: The induction of EMT by depletion of eIF3e negatively regulates the translation of PRP4K in MCF10A cells. A ) Quantitative PCR of PRP4K gene ( PRPF4B ) mRNA expression in control (shCtrl) and eIF3e-depleted (shEIF3E-1 and shEIF3-2) MCF10A cells treated with 10 μg/mL Actinomycin D for the indicated time periods, with data normalized to at least two reference genes as indicated. N = 3, error bars= SEM. Significance was determined by a one-way ANOVA. B ) Western blot analysis of whole cell lysates of MCF10A shCtrl and shEIF3E cells treated with 50μg/mL cycloheximide for the indicated time periods. C ) Puromycin incorporation assay of MCF10A shCtrl and shEIF3E cells. Cells were grown for 24 h without puromycin selection and then pulsed with puromycin prior to Western blot analysis of total cell lysates (left) or following immunoprecipitation (IP) of PRP4K and detection of puromycylation of proteins and PRP4K (right).

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Selection, Immunoprecipitation

The induction of EMT by depletion of eIF3e decreases PRP4K protein expression in MCF10A cells. A ) Phase contrast micrographs of control (shCtrl) and eIF3e-depleted cells (shEIF3E-1 and shEIF3-2). B ) Western blot analysis of whole cell lysates of MCF10A shCtrl and shEIF3E cells for markers of EMT as indicated. C ) Quantitative PCR of PRP4K gene ( PRPF4B ) mRNA expression in MCF10A shCtrl and shEIF3E cells. D ) Quantitative PCR analysis of EMT-associated gene expression (as indicated) in MCF10A shCtrl and shEIF3E cells. N = 3, error bars= SEM. Significance was determined by a t-test. *=p
Figure Legend Snippet: The induction of EMT by depletion of eIF3e decreases PRP4K protein expression in MCF10A cells. A ) Phase contrast micrographs of control (shCtrl) and eIF3e-depleted cells (shEIF3E-1 and shEIF3-2). B ) Western blot analysis of whole cell lysates of MCF10A shCtrl and shEIF3E cells for markers of EMT as indicated. C ) Quantitative PCR of PRP4K gene ( PRPF4B ) mRNA expression in MCF10A shCtrl and shEIF3E cells. D ) Quantitative PCR analysis of EMT-associated gene expression (as indicated) in MCF10A shCtrl and shEIF3E cells. N = 3, error bars= SEM. Significance was determined by a t-test. *=p

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

28) Product Images from "Low expression of organic anion-transporting polypeptide 1B3 predicts a poor prognosis in hepatocellular carcinoma"

Article Title: Low expression of organic anion-transporting polypeptide 1B3 predicts a poor prognosis in hepatocellular carcinoma

Journal: World Journal of Surgical Oncology

doi: 10.1186/s12957-020-01891-y

OATP1B3 mRNA and protein expression in HCC tissue and adjacent nontumorous tissues. a Relative expression of OATP1B3 was detected by qRT-PCR. OATP1B3 mRNA was higher in matched adjacent nontumorous tissue (ANT) than in HCC tissue (T) ( n = 30). Relative mRNA expression in the ANT group showed abnormal distribution; thus, the Wilcoxon signed-rank test was applied, and the data are represented as the median ± interquartile range. b OATP1B3 mRNA expression levels in 8 pairs of Ts and ANTs were compared. Error bars represent the SD. c Relative expression of OATP1B3 protein was detected by Western blotting. OATP1B3 expression in T was significantly lower than that in ANT ( n = 34). The data are represented as the mean ± SD, and the paired Student’s t test was applied. d Representative Western blot of OATP1B3 in T and ANT in 8 pairs of samples. Β-Actin was used as an endogenous control. a P
Figure Legend Snippet: OATP1B3 mRNA and protein expression in HCC tissue and adjacent nontumorous tissues. a Relative expression of OATP1B3 was detected by qRT-PCR. OATP1B3 mRNA was higher in matched adjacent nontumorous tissue (ANT) than in HCC tissue (T) ( n = 30). Relative mRNA expression in the ANT group showed abnormal distribution; thus, the Wilcoxon signed-rank test was applied, and the data are represented as the median ± interquartile range. b OATP1B3 mRNA expression levels in 8 pairs of Ts and ANTs were compared. Error bars represent the SD. c Relative expression of OATP1B3 protein was detected by Western blotting. OATP1B3 expression in T was significantly lower than that in ANT ( n = 34). The data are represented as the mean ± SD, and the paired Student’s t test was applied. d Representative Western blot of OATP1B3 in T and ANT in 8 pairs of samples. Β-Actin was used as an endogenous control. a P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

29) Product Images from "miR-193b exhibits mutual interaction with MYC, and suppresses growth and metastasis of osteosarcoma"

Article Title: miR-193b exhibits mutual interaction with MYC, and suppresses growth and metastasis of osteosarcoma

Journal: Oncology Reports

doi: 10.3892/or.2020.7601

miR-193b inhibits cell cycle progression and induces apoptosis. (A) Transfection of F5M2 cells with miR-193b mimic induced cell cycle arrest in G 1 phase, (B) whereas transfection of F4 cells with miR-193b inhibitor resulted in an increased percentage of cells in S and G 2 phases. (C) Upregulation of miR-193b induced apoptosis of F5M2 cells, (D) whereas downregulation of miR-193b in F4 cells inhibited apoptosis. Data are presented as the mean ± SD of at least three independent experiments. *P
Figure Legend Snippet: miR-193b inhibits cell cycle progression and induces apoptosis. (A) Transfection of F5M2 cells with miR-193b mimic induced cell cycle arrest in G 1 phase, (B) whereas transfection of F4 cells with miR-193b inhibitor resulted in an increased percentage of cells in S and G 2 phases. (C) Upregulation of miR-193b induced apoptosis of F5M2 cells, (D) whereas downregulation of miR-193b in F4 cells inhibited apoptosis. Data are presented as the mean ± SD of at least three independent experiments. *P

Techniques Used: Transfection

miR-193b inhibits migration and invasion of osteosarcoma cells and reduces the formation of lung metastasis. (A) Wound-healing assay revealed that upregulation of miR-193b inhibited cell migration in F5M2 cells, whereas downregulation of miR-193b induced the opposite result in F4 cells (magnification ×20). (B) Transwell cell invasion assay revealed that transient or stable upregulation of miR-193b inhibited invasion of F5M2 cells, whereas downregulation of miR-193b enhanced invasion of F4 cells (magnification ×40). (C) Representative images of the lungs and their corresponding H E staining pictures (magnification ×10 and ×20). Metastatic nodules in the lung were indicated by an arrow. (D) Lung weight and (E) number of microscopic pulmonary metastatic nodules were lower in mice injected with miR-193b-upregulated F5M2 cells. Data are presented as the mean ± SD of at least three independent experiments. *P
Figure Legend Snippet: miR-193b inhibits migration and invasion of osteosarcoma cells and reduces the formation of lung metastasis. (A) Wound-healing assay revealed that upregulation of miR-193b inhibited cell migration in F5M2 cells, whereas downregulation of miR-193b induced the opposite result in F4 cells (magnification ×20). (B) Transwell cell invasion assay revealed that transient or stable upregulation of miR-193b inhibited invasion of F5M2 cells, whereas downregulation of miR-193b enhanced invasion of F4 cells (magnification ×40). (C) Representative images of the lungs and their corresponding H E staining pictures (magnification ×10 and ×20). Metastatic nodules in the lung were indicated by an arrow. (D) Lung weight and (E) number of microscopic pulmonary metastatic nodules were lower in mice injected with miR-193b-upregulated F5M2 cells. Data are presented as the mean ± SD of at least three independent experiments. *P

Techniques Used: Migration, Wound Healing Assay, Invasion Assay, Staining, Mouse Assay, Injection

miR-193b inhibits proliferation via KRAS and suppresses migration and invasion via STMN1. (A) Transfection efficiency was verified after F4 and F5M2 cells were transfected with siKRAS or pMSCV-KRAS, respectively. (B) Transfection efficiency was verified after F4 and F5M2 cells were transfected with siSTMN1 or pMSCV-STMN1, respectively. (C) Co-transfection with pMSCV-KRAS reversed miR-193b-mimic induced cell cycle arrest at G 1 phase in F5M2 cells, whereas co-transfection with siKRAS reversed the miR-193b inhibitor-induced increase in percentage of cells at G 2 phase in F4 cells. (D) Transwell cell invasion assay (magnification ×40). (E) wound-healing assay (magnification ×20) revealed that co-transfection of F5M2 cells with pMSCV-STMN1 and miR-193b mimic or co-transfection of F4 cells with siSTMN1 and miR-193b inhibitor reversed the regulatory effects of miR-193b mimic or inhibitor. Data are presented as the mean ± SD of at least three independent experiments. *P
Figure Legend Snippet: miR-193b inhibits proliferation via KRAS and suppresses migration and invasion via STMN1. (A) Transfection efficiency was verified after F4 and F5M2 cells were transfected with siKRAS or pMSCV-KRAS, respectively. (B) Transfection efficiency was verified after F4 and F5M2 cells were transfected with siSTMN1 or pMSCV-STMN1, respectively. (C) Co-transfection with pMSCV-KRAS reversed miR-193b-mimic induced cell cycle arrest at G 1 phase in F5M2 cells, whereas co-transfection with siKRAS reversed the miR-193b inhibitor-induced increase in percentage of cells at G 2 phase in F4 cells. (D) Transwell cell invasion assay (magnification ×40). (E) wound-healing assay (magnification ×20) revealed that co-transfection of F5M2 cells with pMSCV-STMN1 and miR-193b mimic or co-transfection of F4 cells with siSTMN1 and miR-193b inhibitor reversed the regulatory effects of miR-193b mimic or inhibitor. Data are presented as the mean ± SD of at least three independent experiments. *P

Techniques Used: Migration, Transfection, Cotransfection, Invasion Assay, Wound Healing Assay

miR-193b suppresses KRAS and STMN1 expression through their 3′-UTRs. (A) Schematic diagram of KRAS and STMN1 3′-UTRs with the locations of predicted conserved miRNA-targeting sequences highlighted. (B) Expression levels of KRAS and STMN1 were higher in F5M2 cells compared with F4 cells. (C) Luciferase reporter assay revealed that miR-193b suppressed luciferase activities of all WT constructs. (D and E) miR-193b negatively regulated the expression of KRAS and STMN1. (F) Immunofluorescence staining revealed that tumor tissue from nude mice injected with miR-193b-upregulated F5M2 cells exhibited lower expression levels of KRAS and STMN1 compared with the control group (magnification ×20). Data are presented as the mean ± SD of at least three independent experiments. *P
Figure Legend Snippet: miR-193b suppresses KRAS and STMN1 expression through their 3′-UTRs. (A) Schematic diagram of KRAS and STMN1 3′-UTRs with the locations of predicted conserved miRNA-targeting sequences highlighted. (B) Expression levels of KRAS and STMN1 were higher in F5M2 cells compared with F4 cells. (C) Luciferase reporter assay revealed that miR-193b suppressed luciferase activities of all WT constructs. (D and E) miR-193b negatively regulated the expression of KRAS and STMN1. (F) Immunofluorescence staining revealed that tumor tissue from nude mice injected with miR-193b-upregulated F5M2 cells exhibited lower expression levels of KRAS and STMN1 compared with the control group (magnification ×20). Data are presented as the mean ± SD of at least three independent experiments. *P

Techniques Used: Expressing, Luciferase, Reporter Assay, Construct, Immunofluorescence, Staining, Mouse Assay, Injection

MYC exerts negative effects on miR-193b expression. (A) Schematic diagram showing the predicted binding site of MYC in the promoter region of miR-193b. (B) MYC expression was relatively higher in F5M2 cells compared with F4 cells. (C) Knockdown efficiency of MYC was verified in F5M2 cells. (D) Knockdown of MYC in F5M2 cells enhanced the expression of miR-193b and (E) subsequently reduced the expression of KRAS. (F) Overexpression of MYC in F4 cells (G) induced inhibition of miR-193b expression and (H) enhanced KRAS expression. Data are presented as the mean ± SD of at least three independent experiments. *P
Figure Legend Snippet: MYC exerts negative effects on miR-193b expression. (A) Schematic diagram showing the predicted binding site of MYC in the promoter region of miR-193b. (B) MYC expression was relatively higher in F5M2 cells compared with F4 cells. (C) Knockdown efficiency of MYC was verified in F5M2 cells. (D) Knockdown of MYC in F5M2 cells enhanced the expression of miR-193b and (E) subsequently reduced the expression of KRAS. (F) Overexpression of MYC in F4 cells (G) induced inhibition of miR-193b expression and (H) enhanced KRAS expression. Data are presented as the mean ± SD of at least three independent experiments. *P

Techniques Used: Expressing, Binding Assay, Over Expression, Inhibition

miR-193b forms a negative feedback loop with MYC. (A) Promoter reporter assay and (B) chromatin immunoprecipitation-quantitative PCR assay revealed that MYC could directly bind to the promoter region of miR-193b and suppress the expression of miR-193b. (C) Transfection with a miR-193b mimic decreased MYC expression in F5M2 cells, (D) whereas transfection with a miR-193b inhibitor enhanced MYC expression in F4 cells. (E) Immunofluorescence staining of xenograft tumor tissue also indicated a negative influence of miR-193b on MYC expression. Data are presented as the mean ± SD of at least three independent experiments. *P
Figure Legend Snippet: miR-193b forms a negative feedback loop with MYC. (A) Promoter reporter assay and (B) chromatin immunoprecipitation-quantitative PCR assay revealed that MYC could directly bind to the promoter region of miR-193b and suppress the expression of miR-193b. (C) Transfection with a miR-193b mimic decreased MYC expression in F5M2 cells, (D) whereas transfection with a miR-193b inhibitor enhanced MYC expression in F4 cells. (E) Immunofluorescence staining of xenograft tumor tissue also indicated a negative influence of miR-193b on MYC expression. Data are presented as the mean ± SD of at least three independent experiments. *P

Techniques Used: Reporter Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Transfection, Immunofluorescence, Staining

miR-193b suppresses proliferation and colony formation of osteosarcoma cells, and inhibits tumor growth in a xenograft model. (A) miR-193b expression was lower in F5M2 cells compared with F4 cells. (B) Overexpression of miR-193b reduced colony formation of F5M2 cells, whereas downregulation of miR-193b enhanced colony formation of F4 cells. (C) Transient and stable transfection efficiencies of miR-193b mimic or inhibitor were quantified. (D) Transient or stable transfection of miR-193b mimic in F5M2 cells inhibited proliferation, whereas transfection of miR-193b inhibitor in F4 cells enhanced cell proliferation. (E) Representative images illustrating tumor formation in a nude mouse xenograft model on day 35. (F) Tumor volume and (G) tumor weight were lower in mice injected with miR-193b-upregulated F5M2 cells. Data are presented as the mean ± SD of at least three independent experiments. *P
Figure Legend Snippet: miR-193b suppresses proliferation and colony formation of osteosarcoma cells, and inhibits tumor growth in a xenograft model. (A) miR-193b expression was lower in F5M2 cells compared with F4 cells. (B) Overexpression of miR-193b reduced colony formation of F5M2 cells, whereas downregulation of miR-193b enhanced colony formation of F4 cells. (C) Transient and stable transfection efficiencies of miR-193b mimic or inhibitor were quantified. (D) Transient or stable transfection of miR-193b mimic in F5M2 cells inhibited proliferation, whereas transfection of miR-193b inhibitor in F4 cells enhanced cell proliferation. (E) Representative images illustrating tumor formation in a nude mouse xenograft model on day 35. (F) Tumor volume and (G) tumor weight were lower in mice injected with miR-193b-upregulated F5M2 cells. Data are presented as the mean ± SD of at least three independent experiments. *P

Techniques Used: Expressing, Over Expression, Stable Transfection, Transfection, Mouse Assay, Injection

30) Product Images from "Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays"

Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0233085

ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.
Figure Legend Snippet: ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Migration, Concentration Assay

ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.
Figure Legend Snippet: ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

Techniques Used: Real-time Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Standard Deviation

Superscript IV Reverse Transcription (RT) combined with RainDance ddPCR overcomes RNA sample inhibition. (A) Two step RT-ddPCR reverse transcription step condition tests. In these tests, SIV RNA standard was spiked in 1 ug cellular RNA isolated from naïve Rhesus macaque PBMC. Various conditions such as different reverse transcriptases, RT enzyme amounts, and gene-specific (GSP) vs. random hexamer (HEX) priming methods were tested. The SIV RNA measured copies after ddPCR were compared to input copies to identify optimal RT conditions. Blue: SIV RNA input (copies); Orange: SIV RNA measured (copies). M-MLV: Moloney Murine Leukemia Virus Reverse Transcriptase; SSIII: SuperScript III Reverse Transcriptase. (B) Two step RT-ddPCR assay for SIV RNA detection. Different copy numbers of SIV RNA standard or buffer control were spiked in 1ug total RNA from naïve animal PBMC, and the samples were subject to reverse transcription (SIV gene-specific priming, RT enzyme: M-MLV, 200U/reaction), dropletization, end-point PCR and fluorescence reading/counting. (C) Linear dynamic range of the SIV RT-ddPCR RNA assay derived from quantification data of Fig 7B. (D) Severe RNA inhibition in a bone marrow aspirate sample (from Rhesus macaque 29676) in RT-qPCR analysis. Different amount of RNA sample extracted from a bone marrow aspirate sample, treated or untreated through a G-50 column as indicated, then subject to reverse transcription either using M-MLV or SuperScript IV (SSIV) as the reverse transcriptase. The cDNA was subject to nested preamplification, and qPCR step was performed as described in Methods. The discrepancy between “measured SIV RNA copies” and “Spike-in SIV RNA copies” indicates the level of inhibition under each condition. (E) LiCl treatment of RNA sample removes heparin inhibition in RT-qPCR analysis but RNA recovery is low. RNA samples from 4 bone marrow needle aspirate samples (from Rhesus macaque 28808, 29211, 28885 and 29341) were subject to additional precipitation step(s) (isopropanol, lithium chloride, or a sequential combination of both) before SIV RNA quantitation analysis by adding 1000 copies of SIV RNA standard. (F) SSIV RT combined with SIV ddPCR assay overcomes bone marrow RNA sample inhibition. RNA was isolated from the bone marrow aspirate, and subject to SSIV or SSIII reverse transcription (20 ng RNA per RT reaction) dropletization, end-point PCR and fluorescence reading/counting. No preamplification step was performed on the cDNA.
Figure Legend Snippet: Superscript IV Reverse Transcription (RT) combined with RainDance ddPCR overcomes RNA sample inhibition. (A) Two step RT-ddPCR reverse transcription step condition tests. In these tests, SIV RNA standard was spiked in 1 ug cellular RNA isolated from naïve Rhesus macaque PBMC. Various conditions such as different reverse transcriptases, RT enzyme amounts, and gene-specific (GSP) vs. random hexamer (HEX) priming methods were tested. The SIV RNA measured copies after ddPCR were compared to input copies to identify optimal RT conditions. Blue: SIV RNA input (copies); Orange: SIV RNA measured (copies). M-MLV: Moloney Murine Leukemia Virus Reverse Transcriptase; SSIII: SuperScript III Reverse Transcriptase. (B) Two step RT-ddPCR assay for SIV RNA detection. Different copy numbers of SIV RNA standard or buffer control were spiked in 1ug total RNA from naïve animal PBMC, and the samples were subject to reverse transcription (SIV gene-specific priming, RT enzyme: M-MLV, 200U/reaction), dropletization, end-point PCR and fluorescence reading/counting. (C) Linear dynamic range of the SIV RT-ddPCR RNA assay derived from quantification data of Fig 7B. (D) Severe RNA inhibition in a bone marrow aspirate sample (from Rhesus macaque 29676) in RT-qPCR analysis. Different amount of RNA sample extracted from a bone marrow aspirate sample, treated or untreated through a G-50 column as indicated, then subject to reverse transcription either using M-MLV or SuperScript IV (SSIV) as the reverse transcriptase. The cDNA was subject to nested preamplification, and qPCR step was performed as described in Methods. The discrepancy between “measured SIV RNA copies” and “Spike-in SIV RNA copies” indicates the level of inhibition under each condition. (E) LiCl treatment of RNA sample removes heparin inhibition in RT-qPCR analysis but RNA recovery is low. RNA samples from 4 bone marrow needle aspirate samples (from Rhesus macaque 28808, 29211, 28885 and 29341) were subject to additional precipitation step(s) (isopropanol, lithium chloride, or a sequential combination of both) before SIV RNA quantitation analysis by adding 1000 copies of SIV RNA standard. (F) SSIV RT combined with SIV ddPCR assay overcomes bone marrow RNA sample inhibition. RNA was isolated from the bone marrow aspirate, and subject to SSIV or SSIII reverse transcription (20 ng RNA per RT reaction) dropletization, end-point PCR and fluorescence reading/counting. No preamplification step was performed on the cDNA.

Techniques Used: Inhibition, Isolation, Random Hexamer Labeling, RNA Detection, Polymerase Chain Reaction, Fluorescence, Derivative Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Quantitation Assay

31) Product Images from "Sensitive Detection of Immunoglobulin G Stability Using in Real-Time Isothermal Differential Scanning Fluorimetry: Determinants of Protein Stability for Antibody-Based Therapeutics"

Article Title: Sensitive Detection of Immunoglobulin G Stability Using in Real-Time Isothermal Differential Scanning Fluorimetry: Determinants of Protein Stability for Antibody-Based Therapeutics

Journal: Technology in Cancer Research & Treatment

doi: 10.1177/1533034617714149

The influence of oxidation by 2,2-Azobis[2-amidinopropane]dihydrochloride (AAPH)-generated free radicals on immunoglobulin G (IgG) stability was investigated using differential scanning fluorimetry (DSF), real-time isothermal differential scanning fluorimetry (RT-iDSF), and pulse proteolysis. A, Thermal shift changes (ie, change in T m ) determined by DSF for human IgG (1 mg/mL) incubated with {0, 2.5, 5, 10, 25, or 50} mM AAPH for 1 hour at 4°C. B, The effect of oxidation on the velocity of denaturation of IgG by urea as determined by RT-iDSF. Immunoglobulin G samples were incubated with or without 50 mM AAPH for 2 hours at 4°C immediately prior to assaying. An asterisk (*) indicates significantly different from the control value ( P
Figure Legend Snippet: The influence of oxidation by 2,2-Azobis[2-amidinopropane]dihydrochloride (AAPH)-generated free radicals on immunoglobulin G (IgG) stability was investigated using differential scanning fluorimetry (DSF), real-time isothermal differential scanning fluorimetry (RT-iDSF), and pulse proteolysis. A, Thermal shift changes (ie, change in T m ) determined by DSF for human IgG (1 mg/mL) incubated with {0, 2.5, 5, 10, 25, or 50} mM AAPH for 1 hour at 4°C. B, The effect of oxidation on the velocity of denaturation of IgG by urea as determined by RT-iDSF. Immunoglobulin G samples were incubated with or without 50 mM AAPH for 2 hours at 4°C immediately prior to assaying. An asterisk (*) indicates significantly different from the control value ( P

Techniques Used: Generated, Incubation

32) Product Images from "Short Telomere Length is Associated with Aging, Central Obesity, Poor Sleep and Hypertension in Lebanese Individuals"

Article Title: Short Telomere Length is Associated with Aging, Central Obesity, Poor Sleep and Hypertension in Lebanese Individuals

Journal: Aging and Disease

doi: 10.14336/AD.2017.0310

Correlation of relative telomere length with age and waist circumference Scatter plots showing the correlation of age ( A ) and waist circumference ( B ) with the relative telomere length of peripheral leucocyte blood respectively in males and females. The grey line and (×) represent the males (n=178) and the black line and (o) represent the females (n=319). The P -values were calculated from the linear regression analyses of the relationships between age, waist circumference and RTL in males and females, and the Pearson correlation was used.
Figure Legend Snippet: Correlation of relative telomere length with age and waist circumference Scatter plots showing the correlation of age ( A ) and waist circumference ( B ) with the relative telomere length of peripheral leucocyte blood respectively in males and females. The grey line and (×) represent the males (n=178) and the black line and (o) represent the females (n=319). The P -values were calculated from the linear regression analyses of the relationships between age, waist circumference and RTL in males and females, and the Pearson correlation was used.

Techniques Used:

33) Product Images from "Conversion from long-term cultivated wheat field to Jerusalem artichoke plantation changed soil fungal communities"

Article Title: Conversion from long-term cultivated wheat field to Jerusalem artichoke plantation changed soil fungal communities

Journal: Scientific Reports

doi: 10.1038/srep41502

Soil total fungal ( a ), Fusarium ( b ) and Trichoderma ( c ) spp. community in the wheat field (W), the first (F), second (S) and third (T) cropping of Jerusalem artichoke as determined by quantitative PCR. Values (mean ± SE) with different letters are significantly different at the 0.05 probability level (Tukey’s HSD test).
Figure Legend Snippet: Soil total fungal ( a ), Fusarium ( b ) and Trichoderma ( c ) spp. community in the wheat field (W), the first (F), second (S) and third (T) cropping of Jerusalem artichoke as determined by quantitative PCR. Values (mean ± SE) with different letters are significantly different at the 0.05 probability level (Tukey’s HSD test).

Techniques Used: Real-time Polymerase Chain Reaction

34) Product Images from "Exposure of Monocytic Cells to Lipopolysaccharide Induces Coordinated Endotoxin Tolerance, Mitochondrial Biogenesis, Mitophagy, and Antioxidant Defenses"

Article Title: Exposure of Monocytic Cells to Lipopolysaccharide Induces Coordinated Endotoxin Tolerance, Mitochondrial Biogenesis, Mitophagy, and Antioxidant Defenses

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02217

Incubation of monocytes with LPS for 24 h results in reduced ability to release TNFα in response to a second LPS stimulus and increased mtDNA copy number. Blood monocytes were incubated with LPS (10 ng/ml) before measuring cytokine release and mtDNA copy number. (A) The release of TNFα in response to a second 4 h exposure to LPS (10 ng/ml) was measured by ELISA ( n = 5). (B) mtDNA copy number was determined by measuring MT-ND1 relative to B2M using qPCR ( n = 5). Data are represented as (A) mean ± standard deviation or (B) individual values with a line representing the mean. * p
Figure Legend Snippet: Incubation of monocytes with LPS for 24 h results in reduced ability to release TNFα in response to a second LPS stimulus and increased mtDNA copy number. Blood monocytes were incubated with LPS (10 ng/ml) before measuring cytokine release and mtDNA copy number. (A) The release of TNFα in response to a second 4 h exposure to LPS (10 ng/ml) was measured by ELISA ( n = 5). (B) mtDNA copy number was determined by measuring MT-ND1 relative to B2M using qPCR ( n = 5). Data are represented as (A) mean ± standard deviation or (B) individual values with a line representing the mean. * p

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Standard Deviation

Induction of mitochondrial biogenesis following exposure of THP-1 cells to LPS. THP-1 cells were incubated with LPS (100 ng/ml) for 0–72 h before assessing mitochondrial biogenesis and respiration. (A) Mitochondrial mass was assessed by measuring the uptake of NAO using flow cytometry, (positive control - glucose-free medium supplemented with 5 mM galactose) ( n = 4). (B) A colorimetric assay was used to assess the activity of the mitochondrial matrix enzyme citrate synthase ( n = 3). (C) mtDNA copy number was determined by measuring MT-ND1 relative to B2M using qPCR ( n = 5). (D) The level of TFAM protein relative to β-actin was determined by Western blot ( n = 4). (E) Scatter plot, linear regression and Pearson's correlation of the relationship between mtDNA copy number and TFAM protein levels. * p
Figure Legend Snippet: Induction of mitochondrial biogenesis following exposure of THP-1 cells to LPS. THP-1 cells were incubated with LPS (100 ng/ml) for 0–72 h before assessing mitochondrial biogenesis and respiration. (A) Mitochondrial mass was assessed by measuring the uptake of NAO using flow cytometry, (positive control - glucose-free medium supplemented with 5 mM galactose) ( n = 4). (B) A colorimetric assay was used to assess the activity of the mitochondrial matrix enzyme citrate synthase ( n = 3). (C) mtDNA copy number was determined by measuring MT-ND1 relative to B2M using qPCR ( n = 5). (D) The level of TFAM protein relative to β-actin was determined by Western blot ( n = 4). (E) Scatter plot, linear regression and Pearson's correlation of the relationship between mtDNA copy number and TFAM protein levels. * p

Techniques Used: Incubation, Flow Cytometry, Cytometry, Positive Control, Colorimetric Assay, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

35) Product Images from "Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development"

Article Title: Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0223765

In planta expression assay. a) Inoculation positions of gRNA-Cas9 construct and ChiLCV, b) qPCR assay to understand reduction of viral replication, c) qRT-PCR assay for gRNA expression analysis, d) qRT-PCR assay for Cas9 expression analysis. For qPCR and qRT-PCR, three replicates were used for each data point. The error bar denotes SEM. Stars indicate significant difference (student’s unpaired t-test) of ChiLCV expression levels between gRNA-Cas9 treated and non-treated plants (*P
Figure Legend Snippet: In planta expression assay. a) Inoculation positions of gRNA-Cas9 construct and ChiLCV, b) qPCR assay to understand reduction of viral replication, c) qRT-PCR assay for gRNA expression analysis, d) qRT-PCR assay for Cas9 expression analysis. For qPCR and qRT-PCR, three replicates were used for each data point. The error bar denotes SEM. Stars indicate significant difference (student’s unpaired t-test) of ChiLCV expression levels between gRNA-Cas9 treated and non-treated plants (*P

Techniques Used: Expressing, Construct, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

36) Product Images from "Efficacy of Olorofim (F901318) against Aspergillus fumigatus, A. nidulans, and A. tanneri in Murine Models of Profound Neutropenia and Chronic Granulomatous Disease"

Article Title: Efficacy of Olorofim (F901318) against Aspergillus fumigatus, A. nidulans, and A. tanneri in Murine Models of Profound Neutropenia and Chronic Granulomatous Disease

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00129-19

Efficacy of olorofim against A. fumigatus , A. nidulans , and A. tanneri in CD-1 mice. The CD-1 mice were treated for 9 days with either 15 mg/kg olorofim or PBS every 8 h via intraperitoneal administration, starting at 6 h postinfection (i.v.) with A. fumigatus (1 × 10 4 conidia/mouse) (A), A. nidulans (2 × 10 4 conidia/mouse) (B), and A. tanneri (1 × 10 6 conidia/mouse) (C). (A1, B1, C1) The survival of the mice ( n  = 11) was monitored for 10 days. (A2, B2, C2) On day 3 postinfection, blood samples were collected from olorofim-treated and untreated mice ( n  = 3) to measure galactomannan (GM) in centrifuged sera. GM levels were also determined from olorofim-treated surviving mice at day 9 ( n  = 3). (A3, B3, C3) In addition, kidneys were harvested and fungal burdens were estimated by measuring fungal DNA by qPCR. ****, P  ≤ 0.0001; *, P  ≤ 0.05. (A4, B4, C4) GMS-stained histology sections were prepared on day 3 postinfection. (Top and bottom left) Representative histological sections of vehicle-treated control mice; (top and bottom right) representative histological sections of olorofim-treated mice. Bars, 1 mm (A4, B4, and C4, top) and 20 μm (A4, B4, and C4, bottom); magnifications, ×25 (A4, B4, and C4, top) and ×400 (A4, B4, and C4, bottom).
Figure Legend Snippet: Efficacy of olorofim against A. fumigatus , A. nidulans , and A. tanneri in CD-1 mice. The CD-1 mice were treated for 9 days with either 15 mg/kg olorofim or PBS every 8 h via intraperitoneal administration, starting at 6 h postinfection (i.v.) with A. fumigatus (1 × 10 4 conidia/mouse) (A), A. nidulans (2 × 10 4 conidia/mouse) (B), and A. tanneri (1 × 10 6 conidia/mouse) (C). (A1, B1, C1) The survival of the mice ( n  = 11) was monitored for 10 days. (A2, B2, C2) On day 3 postinfection, blood samples were collected from olorofim-treated and untreated mice ( n  = 3) to measure galactomannan (GM) in centrifuged sera. GM levels were also determined from olorofim-treated surviving mice at day 9 ( n  = 3). (A3, B3, C3) In addition, kidneys were harvested and fungal burdens were estimated by measuring fungal DNA by qPCR. ****, P  ≤ 0.0001; *, P  ≤ 0.05. (A4, B4, C4) GMS-stained histology sections were prepared on day 3 postinfection. (Top and bottom left) Representative histological sections of vehicle-treated control mice; (top and bottom right) representative histological sections of olorofim-treated mice. Bars, 1 mm (A4, B4, and C4, top) and 20 μm (A4, B4, and C4, bottom); magnifications, ×25 (A4, B4, and C4, top) and ×400 (A4, B4, and C4, bottom).

Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Staining

Efficacy of olorofim against A. fumigatus , A. nidulans , and A. tanneri in CGD mice. CGD mice were treated for 9 days with either 15 mg/kg olorofim or PBS every 8 h via intraperitoneal administration, starting at 6 h postinfection with A. fumigatus (5 × 10 3 conidia/mouse) (A), A. nidulans (5 × 10 3 conidia/mouse) (B), and A. tanneri (2.5 × 10 6 conidia/mouse) (C), aspirated from the pharynx. (A1, B1, C1) The survival of mice ( n  = 11) was monitored for 10 days. (A2, B2, C2) On day 3 postinfection, blood samples were collected from olorofim-treated and untreated mice ( n  = 3) to measure galactomannan (GM) in centrifuged sera. GM levels were also determined from olorofim-treated mice that survived at day 9 ( n  = 3). (A3, B3, C3) Lungs from infected mice were taken at 3 days postinfection ( n  = 3), and fungal burdens were estimated. **, P  ≤ 0.01; ***, P  ≤ 0.001. (A4, B4, C4) Montaged images of H E-stained lung sections taken at 3 days postinfection show the size and frequency of infectious foci in control mice (top) and olorofim-treated mice (bottom). Bars, 2 mm; magnifications, ×25.
Figure Legend Snippet: Efficacy of olorofim against A. fumigatus , A. nidulans , and A. tanneri in CGD mice. CGD mice were treated for 9 days with either 15 mg/kg olorofim or PBS every 8 h via intraperitoneal administration, starting at 6 h postinfection with A. fumigatus (5 × 10 3 conidia/mouse) (A), A. nidulans (5 × 10 3 conidia/mouse) (B), and A. tanneri (2.5 × 10 6 conidia/mouse) (C), aspirated from the pharynx. (A1, B1, C1) The survival of mice ( n  = 11) was monitored for 10 days. (A2, B2, C2) On day 3 postinfection, blood samples were collected from olorofim-treated and untreated mice ( n  = 3) to measure galactomannan (GM) in centrifuged sera. GM levels were also determined from olorofim-treated mice that survived at day 9 ( n  = 3). (A3, B3, C3) Lungs from infected mice were taken at 3 days postinfection ( n  = 3), and fungal burdens were estimated. **, P  ≤ 0.01; ***, P  ≤ 0.001. (A4, B4, C4) Montaged images of H E-stained lung sections taken at 3 days postinfection show the size and frequency of infectious foci in control mice (top) and olorofim-treated mice (bottom). Bars, 2 mm; magnifications, ×25.

Techniques Used: Mouse Assay, Infection, Staining

37) Product Images from "Increased Drp1-Mediated Mitochondrial Fission Promotes Proliferation and Collagen Production by Right Ventricular Fibroblasts in Experimental Pulmonary Arterial Hypertension"

Article Title: Increased Drp1-Mediated Mitochondrial Fission Promotes Proliferation and Collagen Production by Right Ventricular Fibroblasts in Experimental Pulmonary Arterial Hypertension

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.00828

Compared to control, monocrotaline (MCT)-induced RVfib have upregulation of phosphorylation of dynamin-related protein 1(Drp1) at Serine 616 but no significant changes in total Drp1 or Drp1 binding partners. Representative immunofluorescence images of phosphorylated Drp1 at Serine 616 (P-Drp1-S616; red) in RVfib (A) at 20X with DAPI at blue and (B) at 63X; (C) Summary of the intensity of phosphorylated Drp1 at Serine 616 ( n = 5∼6 per group); (D) Immunoblotting on P-Drp1-S616 and its ratio to total Drp1 ( n = 4∼5 per group); (E) mRNA expression of total Drp1, fission protein 1 (Fis1), mitochondrial fission factor (MFF), and mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) in RVfib ( n = 6∼8 per group); (F) Immunoblotting on total Drp1 in RVfib ( n = 4∼6 per group). ∗ P
Figure Legend Snippet: Compared to control, monocrotaline (MCT)-induced RVfib have upregulation of phosphorylation of dynamin-related protein 1(Drp1) at Serine 616 but no significant changes in total Drp1 or Drp1 binding partners. Representative immunofluorescence images of phosphorylated Drp1 at Serine 616 (P-Drp1-S616; red) in RVfib (A) at 20X with DAPI at blue and (B) at 63X; (C) Summary of the intensity of phosphorylated Drp1 at Serine 616 ( n = 5∼6 per group); (D) Immunoblotting on P-Drp1-S616 and its ratio to total Drp1 ( n = 4∼5 per group); (E) mRNA expression of total Drp1, fission protein 1 (Fis1), mitochondrial fission factor (MFF), and mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) in RVfib ( n = 6∼8 per group); (F) Immunoblotting on total Drp1 in RVfib ( n = 4∼6 per group). ∗ P

Techniques Used: Binding Assay, Immunofluorescence, Expressing

38) Product Images from "Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia"

Article Title: Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia

Journal: Genes and immunity

doi: 10.1038/gene.2012.40

a. Combined bisulfite restriction analysis of LINE-1 in symptomatic BPH TURP samples, asymptomatic BPH, and donors. Lane 1 is a 100 base pair (bp) molecular weight marker (mwm), lane 2 is the calibrator, lanes 3–5 are symptomatic BPH TURP samples, lanes 6–8 are asymptomatic BPH, lanes 9–11 are donor and lane 12 is undigested PCR product. DNA samples were bisulfite treated, subjected to Line1 PCR and subsequent digestion with HinfI. The undigested top band at 450bp represents unmethylated DNA whereas the digestion products at 275bp and 180bp indicate methylated DNA. b. Quantification of the percent methylation compared to the calibrator of 11 symptomatic BPH from TURP, 10 asymptomatic BPH and 9 donors. Data is represented as a box and whisker plot. A statistically significant difference exists between symptomatic BPH and donors groups (p = 0.040, Mann Whitney), whereas there is no statistically significant difference between symptomatic BPH and asymptomatic BPH. c. APOBEC3G real time PCR results for symptomatic BPH, asymptomatic BPH, and donor samples. The Mann-Whitney rank-sum test was used to determine the statistically significant p value of 0.011 between symptomatic BPH and donors and the t-test was used to determine the statistically significant difference between symptomatic BPH and donors (p = 0.043).
Figure Legend Snippet: a. Combined bisulfite restriction analysis of LINE-1 in symptomatic BPH TURP samples, asymptomatic BPH, and donors. Lane 1 is a 100 base pair (bp) molecular weight marker (mwm), lane 2 is the calibrator, lanes 3–5 are symptomatic BPH TURP samples, lanes 6–8 are asymptomatic BPH, lanes 9–11 are donor and lane 12 is undigested PCR product. DNA samples were bisulfite treated, subjected to Line1 PCR and subsequent digestion with HinfI. The undigested top band at 450bp represents unmethylated DNA whereas the digestion products at 275bp and 180bp indicate methylated DNA. b. Quantification of the percent methylation compared to the calibrator of 11 symptomatic BPH from TURP, 10 asymptomatic BPH and 9 donors. Data is represented as a box and whisker plot. A statistically significant difference exists between symptomatic BPH and donors groups (p = 0.040, Mann Whitney), whereas there is no statistically significant difference between symptomatic BPH and asymptomatic BPH. c. APOBEC3G real time PCR results for symptomatic BPH, asymptomatic BPH, and donor samples. The Mann-Whitney rank-sum test was used to determine the statistically significant p value of 0.011 between symptomatic BPH and donors and the t-test was used to determine the statistically significant difference between symptomatic BPH and donors (p = 0.043).

Techniques Used: Molecular Weight, Marker, Polymerase Chain Reaction, Methylation, Whisker Assay, MANN-WHITNEY, Real-time Polymerase Chain Reaction

a. LINE1ORF2 real time PCR for symptomatic BPH, asymptomatic BPH and normal prostate tissue from organ donors. The Mann-Whitney statistical test was performed, determining that a statistically significant difference exists between symptomatic BPH and donors (p = 0.015), however there is no statistically significant difference between symptomatic and asymptomatic BPH.
Figure Legend Snippet: a. LINE1ORF2 real time PCR for symptomatic BPH, asymptomatic BPH and normal prostate tissue from organ donors. The Mann-Whitney statistical test was performed, determining that a statistically significant difference exists between symptomatic BPH and donors (p = 0.015), however there is no statistically significant difference between symptomatic and asymptomatic BPH.

Techniques Used: Real-time Polymerase Chain Reaction, MANN-WHITNEY

a. IFIT1 real time PCR with symptomatic and asymptomatic BPH samples. The Mann-Whitney statistical test was utilized for statistical analysis and determined that there was no statistically significant difference between the two groups. b. Real time PCR for IFIT1 in symptomatic BPH and asymptomatic BPH samples in which the prostate mass was less than 60g. Statistical analysis using the Mann-Whitney rank-sum test demonstrates a statistically significant difference between the two groups, p = 0.0118.
Figure Legend Snippet: a. IFIT1 real time PCR with symptomatic and asymptomatic BPH samples. The Mann-Whitney statistical test was utilized for statistical analysis and determined that there was no statistically significant difference between the two groups. b. Real time PCR for IFIT1 in symptomatic BPH and asymptomatic BPH samples in which the prostate mass was less than 60g. Statistical analysis using the Mann-Whitney rank-sum test demonstrates a statistically significant difference between the two groups, p = 0.0118.

Techniques Used: Real-time Polymerase Chain Reaction, MANN-WHITNEY

a. Correlation between IFIT1 real time PCR results (IFIT1 relative gene expression – IFIT1/GAPDH) and mass in grams of the asymptomatic BPH samples. Statistical analysis was carried out using a Pearson’s correlation, with the Pearson’s r = 0.65 and p = 0.0172. b. Correlation between APOBEC3G and IFIT1 relative gene expression (normalized to GAPDH) in the symptomatic BPH samples. Pearson’s correlation statistical analysis resulted in a Pearson’s r = 0.65 and p = 0.0066.
Figure Legend Snippet: a. Correlation between IFIT1 real time PCR results (IFIT1 relative gene expression – IFIT1/GAPDH) and mass in grams of the asymptomatic BPH samples. Statistical analysis was carried out using a Pearson’s correlation, with the Pearson’s r = 0.65 and p = 0.0172. b. Correlation between APOBEC3G and IFIT1 relative gene expression (normalized to GAPDH) in the symptomatic BPH samples. Pearson’s correlation statistical analysis resulted in a Pearson’s r = 0.65 and p = 0.0066.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

39) Product Images from "Evaluation of Primers Targeting the Diazotroph Functional Gene and Development of NifMAP – A Bioinformatics Pipeline for Analyzing nifH Amplicon Data"

Article Title: Evaluation of Primers Targeting the Diazotroph Functional Gene and Development of NifMAP – A Bioinformatics Pipeline for Analyzing nifH Amplicon Data

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00703

Average nifH copies per ng DNA ± standard error across environmental samples based on quantitative PCR using the different primer pairs. The environmental samples include: (a) beech forest soil; (b) meadow soil; (c) rhizosphere and (d) root samples of Arrhenatherum elatius ; (e) rhizosphere and (f) root samples of Oryza sativa ; (g) coastal, sub-arctic biological soil crust (BSC); (h) temperate BSC; (i) high alpine BSC; (j) semiarid BSC; (k) arid BSC; estuarine samples from the (l) Great Belt and (m) Roskilde Fjord. Numbers of copies were corrected to exclude non- nifH genes that were co-amplified using information obtained from amplicon sequencing of the samples with the specific primer pairs.
Figure Legend Snippet: Average nifH copies per ng DNA ± standard error across environmental samples based on quantitative PCR using the different primer pairs. The environmental samples include: (a) beech forest soil; (b) meadow soil; (c) rhizosphere and (d) root samples of Arrhenatherum elatius ; (e) rhizosphere and (f) root samples of Oryza sativa ; (g) coastal, sub-arctic biological soil crust (BSC); (h) temperate BSC; (i) high alpine BSC; (j) semiarid BSC; (k) arid BSC; estuarine samples from the (l) Great Belt and (m) Roskilde Fjord. Numbers of copies were corrected to exclude non- nifH genes that were co-amplified using information obtained from amplicon sequencing of the samples with the specific primer pairs.

Techniques Used: Environmental Sampling, Real-time Polymerase Chain Reaction, Amplification, Sequencing

40) Product Images from "Transcriptome-wide analysis of filarial extract-primed human monocytes reveal changes in LPS-induced PTX3 expression levels"

Article Title: Transcriptome-wide analysis of filarial extract-primed human monocytes reveal changes in LPS-induced PTX3 expression levels

Journal: Scientific Reports

doi: 10.1038/s41598-019-38985-x

B. malayi crude extract priming reduces the LPS-induced gene expression of PTX3, CCL20 and TNFRSF21 and increases the expression of metallothioneins. Fold changes of PTX3 ( a ), TNFRSF21 ( b ), CCL20 ( c ), MMP9 ( d ) as well as of the metallothioneins MT1F ( e ), MT1G ( f ), MT1H ( g ) and MT1M ( h ) of BmA primed and LPS re-stimulated human monocytes and controls (n = 11). The samples were analyzed by quantitative real-time PCR (qPCR) and fold changes in comparison to unstimulated controls are shown (asterisks). The data is presented as mean + SEM. Statistical significance was analyzed by ANOVA followed by Bonferroni Comparison Test (*p
Figure Legend Snippet: B. malayi crude extract priming reduces the LPS-induced gene expression of PTX3, CCL20 and TNFRSF21 and increases the expression of metallothioneins. Fold changes of PTX3 ( a ), TNFRSF21 ( b ), CCL20 ( c ), MMP9 ( d ) as well as of the metallothioneins MT1F ( e ), MT1G ( f ), MT1H ( g ) and MT1M ( h ) of BmA primed and LPS re-stimulated human monocytes and controls (n = 11). The samples were analyzed by quantitative real-time PCR (qPCR) and fold changes in comparison to unstimulated controls are shown (asterisks). The data is presented as mean + SEM. Statistical significance was analyzed by ANOVA followed by Bonferroni Comparison Test (*p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Related Articles

DNA Extraction:

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Article Snippet: .. Quantitative Polymerase Chain Reaction of Mucosa-Associated Bacteria DNA was isolated from ileal tissue using the Roche High Pure PCR Template Prep Kit for genomic DNA isolation. qPCR was performed using 10 ng of DNA in all reactions and primers for Eubacteria or six 16S rRNA-specific sequences for Escherichia coli , Bacteroides , Lactobacillus /Enterococcus , Eubacterium rectale/Clostridium coccoides (Erec), segmented filamentous bacteria (SFB), and mouse intestinal Bacteroides (MIB) in iTaq SYBR Green Supermix with ROX (BioRad) on an ABI prism 7900HT (ThermoFisher Scientific) using SDS2.4 software. .. Because there is no proper widely accepted “reference control bacterial population marker” to normalize qPCR microbiome data, quantitative strain-specific 16S primer amplicon data were analyzed collectively using the raw qPCR-CT values as described earlier and multivariate statistics to visualize and quantify the overall impact of the AS supplementation on the mucosa-associated microbial composition in the ileum of mice.

Real-time Polymerase Chain Reaction:

Article Title: Silencing of maternal hepatic glucocorticoid receptor is essential for normal fetal development in mice
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Article Title: Complexes of Oligoribonucleotides with d-Mannitol Modulate the Innate Immune Response to Influenza A Virus H1N1 (A/FM/1/47) In Vivo
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Article Snippet: .. Quantitative PCR (qPCR) Due to the potential role of Vibrio in OsHV-1 μvar disease dynamics ( ; ; ; ; ), quantitative PCR (qPCR) was used to examine patterns in Vibrio abundance across the RGs. qPCR was performed using an epMotion 5075l Automated Liquid Handling System on a Bio-Rad CFX384 Touch Real-Time PCR Detection System with a six-point calibration curve and negative controls on every plate. .. The calibration curve was built from a known amount of amplicon DNA measured by Qubit, followed by a 10-fold dilution to fill out the calibration curve.

Article Title: The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis
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Article Title: A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus
Article Snippet: .. Even we assume the sensitivities in the original research are comparable to the analytical sensitivities in our study, our data of clinical sensitivities are superior to the those obtained from original reports, as evidenced by the difference in the minimal Ct values for RSV (12.37 vs 17.04), HRV (12.70 vs 19.52) and HMPV (10.79 vs 13.23) between mOTNRT-PCR and corresponding RT-qPCR assays. mOTNRT-PCR assay can be carried out in one closed tube on real-time PCR machines (CFX96 Touch (Bio-Rad), Roche Light Cycler 480 or ABI 7900HT) with similar results (data not shown), indicating the wide adaptability of mOTNRT-PCR assay. ..

Transfection:

Article Title: The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma
Article Snippet: .. T cells were either transfected with RNA coding for the CSPG4-specific CAR (MCSPHL CD28-CD3ζ) [ ] or with RNA encoding the carcinoembryonic antigen (CEA)-specific CAR (CEA CD28-CD3ζ) [ ] using a GenePulser Xcell system (Bio-Rad, Hercules, CA, USA) with the square-wave protocol at 500 V for 5 ms. After transfection, T cells were immediately transferred to R10 medium. .. Surface Expression of Transfected Receptors Receptor expression on the cell surface of transfected T cells was analyzed via flow cytometry at indicated timepoints after electroporation.

Amplification:

Article Title: Silencing of maternal hepatic glucocorticoid receptor is essential for normal fetal development in mice
Article Snippet: .. Quantitative PCR One-hundred nanograms of the total RNA were reverse-transcribed and amplified according to the manufacturer’s instructions for the iScript One-Step RT-PCR kit for probes (Biorad; Hercules, CA). .. Quantitative real-time PCR (qPCR) was performed with the Biorad CFX96 sequence detection system using commercially available primer/probe sets (Applied Biosystems; Foster City, CA).

Synthesized:

Article Title: Mucor circinelloides Thrives inside the Phagosome through an Atf-Mediated Germination Pathway
Article Snippet: .. For RT-qPCR assays, cDNA was synthesized from 1 µg of total RNA using the iScript cDNA synthesis kit (Bio-Rad). .. Real-time PCR was performed in triplicate with a QuantStudio real-time PCR system (Applied Biosystems) using 2× SYBR green PCR master mix (Applied Biosystems) following the supplier’s recommendations.

Isolation:

Article Title: The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis
Article Snippet: .. Quantitative Polymerase Chain Reaction of Mucosa-Associated Bacteria DNA was isolated from ileal tissue using the Roche High Pure PCR Template Prep Kit for genomic DNA isolation. qPCR was performed using 10 ng of DNA in all reactions and primers for Eubacteria or six 16S rRNA-specific sequences for Escherichia coli , Bacteroides , Lactobacillus /Enterococcus , Eubacterium rectale/Clostridium coccoides (Erec), segmented filamentous bacteria (SFB), and mouse intestinal Bacteroides (MIB) in iTaq SYBR Green Supermix with ROX (BioRad) on an ABI prism 7900HT (ThermoFisher Scientific) using SDS2.4 software. .. Because there is no proper widely accepted “reference control bacterial population marker” to normalize qPCR microbiome data, quantitative strain-specific 16S primer amplicon data were analyzed collectively using the raw qPCR-CT values as described earlier and multivariate statistics to visualize and quantify the overall impact of the AS supplementation on the mucosa-associated microbial composition in the ileum of mice.

Quantitative RT-PCR:

Article Title: Mucor circinelloides Thrives inside the Phagosome through an Atf-Mediated Germination Pathway
Article Snippet: .. For RT-qPCR assays, cDNA was synthesized from 1 µg of total RNA using the iScript cDNA synthesis kit (Bio-Rad). .. Real-time PCR was performed in triplicate with a QuantStudio real-time PCR system (Applied Biosystems) using 2× SYBR green PCR master mix (Applied Biosystems) following the supplier’s recommendations.

Article Title: A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus
Article Snippet: .. Even we assume the sensitivities in the original research are comparable to the analytical sensitivities in our study, our data of clinical sensitivities are superior to the those obtained from original reports, as evidenced by the difference in the minimal Ct values for RSV (12.37 vs 17.04), HRV (12.70 vs 19.52) and HMPV (10.79 vs 13.23) between mOTNRT-PCR and corresponding RT-qPCR assays. mOTNRT-PCR assay can be carried out in one closed tube on real-time PCR machines (CFX96 Touch (Bio-Rad), Roche Light Cycler 480 or ABI 7900HT) with similar results (data not shown), indicating the wide adaptability of mOTNRT-PCR assay. ..

SYBR Green Assay:

Article Title: The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis
Article Snippet: .. Quantitative Polymerase Chain Reaction of Mucosa-Associated Bacteria DNA was isolated from ileal tissue using the Roche High Pure PCR Template Prep Kit for genomic DNA isolation. qPCR was performed using 10 ng of DNA in all reactions and primers for Eubacteria or six 16S rRNA-specific sequences for Escherichia coli , Bacteroides , Lactobacillus /Enterococcus , Eubacterium rectale/Clostridium coccoides (Erec), segmented filamentous bacteria (SFB), and mouse intestinal Bacteroides (MIB) in iTaq SYBR Green Supermix with ROX (BioRad) on an ABI prism 7900HT (ThermoFisher Scientific) using SDS2.4 software. .. Because there is no proper widely accepted “reference control bacterial population marker” to normalize qPCR microbiome data, quantitative strain-specific 16S primer amplicon data were analyzed collectively using the raw qPCR-CT values as described earlier and multivariate statistics to visualize and quantify the overall impact of the AS supplementation on the mucosa-associated microbial composition in the ileum of mice.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Silencing of maternal hepatic glucocorticoid receptor is essential for normal fetal development in mice
Article Snippet: .. Quantitative PCR One-hundred nanograms of the total RNA were reverse-transcribed and amplified according to the manufacturer’s instructions for the iScript One-Step RT-PCR kit for probes (Biorad; Hercules, CA). .. Quantitative real-time PCR (qPCR) was performed with the Biorad CFX96 sequence detection system using commercially available primer/probe sets (Applied Biosystems; Foster City, CA).

Mass Spectrometry:

Article Title: The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma
Article Snippet: .. T cells were either transfected with RNA coding for the CSPG4-specific CAR (MCSPHL CD28-CD3ζ) [ ] or with RNA encoding the carcinoembryonic antigen (CEA)-specific CAR (CEA CD28-CD3ζ) [ ] using a GenePulser Xcell system (Bio-Rad, Hercules, CA, USA) with the square-wave protocol at 500 V for 5 ms. After transfection, T cells were immediately transferred to R10 medium. .. Surface Expression of Transfected Receptors Receptor expression on the cell surface of transfected T cells was analyzed via flow cytometry at indicated timepoints after electroporation.

Polymerase Chain Reaction:

Article Title: The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis
Article Snippet: .. Quantitative Polymerase Chain Reaction of Mucosa-Associated Bacteria DNA was isolated from ileal tissue using the Roche High Pure PCR Template Prep Kit for genomic DNA isolation. qPCR was performed using 10 ng of DNA in all reactions and primers for Eubacteria or six 16S rRNA-specific sequences for Escherichia coli , Bacteroides , Lactobacillus /Enterococcus , Eubacterium rectale/Clostridium coccoides (Erec), segmented filamentous bacteria (SFB), and mouse intestinal Bacteroides (MIB) in iTaq SYBR Green Supermix with ROX (BioRad) on an ABI prism 7900HT (ThermoFisher Scientific) using SDS2.4 software. .. Because there is no proper widely accepted “reference control bacterial population marker” to normalize qPCR microbiome data, quantitative strain-specific 16S primer amplicon data were analyzed collectively using the raw qPCR-CT values as described earlier and multivariate statistics to visualize and quantify the overall impact of the AS supplementation on the mucosa-associated microbial composition in the ileum of mice.

Software:

Article Title: The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis
Article Snippet: .. Quantitative Polymerase Chain Reaction of Mucosa-Associated Bacteria DNA was isolated from ileal tissue using the Roche High Pure PCR Template Prep Kit for genomic DNA isolation. qPCR was performed using 10 ng of DNA in all reactions and primers for Eubacteria or six 16S rRNA-specific sequences for Escherichia coli , Bacteroides , Lactobacillus /Enterococcus , Eubacterium rectale/Clostridium coccoides (Erec), segmented filamentous bacteria (SFB), and mouse intestinal Bacteroides (MIB) in iTaq SYBR Green Supermix with ROX (BioRad) on an ABI prism 7900HT (ThermoFisher Scientific) using SDS2.4 software. .. Because there is no proper widely accepted “reference control bacterial population marker” to normalize qPCR microbiome data, quantitative strain-specific 16S primer amplicon data were analyzed collectively using the raw qPCR-CT values as described earlier and multivariate statistics to visualize and quantify the overall impact of the AS supplementation on the mucosa-associated microbial composition in the ileum of mice.

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    Bio-Rad qpcr amplifications
    ChIP analysis of <t>HSF1</t> binding sites in murine Ttr ( A ) and Hsp70 ( B ) promoters in hippocampus and liver after in vivo celastrol treatment. <t>ChIP-qPCR</t> results from treatment of murine hippocampal or hepatic cells with vehicle control (white) ( n = 4) or celastrol (gray bar) ( n = 4) are shown as percentage of input. ChIP assays were performed at least in triplicate. Multivariate analysis reveals a significant effect of celastrol on HSF1-binding activity to the Ttr promoter in the hippocampus relative to that produced by DMSO treatment. There was also an increase in binding to the Hsp70 promoter in both hippocampus and liver (Student's t test): ** p
    Qpcr Amplifications, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qpcr amplifications/product/Bio-Rad
    Average 92 stars, based on 54 article reviews
    Price from $9.99 to $1999.99
    qpcr amplifications - by Bioz Stars, 2020-09
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    92
    Bio-Rad quantitative pcr amplification
    <t>HOIL-1L</t> is upregulated during IAV infection through a type I IFN receptor signaling axis. ( A and B ) AT2 cells were isolated from WT mice at 0, 3, 5, and 7 d.p.i. ( A ) HOIL-1L mRNA ( n = 5). ( B ) Representative HOIL-1L immunoblot and its quantification ( n = 4). ( C ) Representative native PAGE immunoblot of HOIL-1L and HOIP expression in AT2 cells infected in vitro with WSN ( n = 3). ( D ) HOIL-1L mRNA expression in AT2 cells from WT mice 0 and 3 d.p.i. sorted based on expression of the viral protein HA ( n = 8). ( E – I ) Representative HOIL-1L immunoblots and quantification. ( E ) A549 cells treated for 0, 8, 12, and 16 hours with conditioned medium (CM; “16*” indicates boiled CM) ( n = 4). ( F ) A549 cells treated with recombinant IFN-α ( n = 4). ( G ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with CM ( n = 3). ( H ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with WSN ( n = 3). ( I ) AT2 cells isolated from WT and CCR2 –/– mice treated in vitro with WSN ( n = 4). ( J ) Quantitative reverse transcriptase <t>PCR</t> quantification of HOIL-1L promoter after ChIP of IRF1 in A549 cells ( n = 4). ( K ) Representative HOIL-1L immunoblot and quantification in siControl- or siIRF1-transfected A549 cells treated with CM ( n = 4). ( L ) Proposed type I IFN pathway leading to HOIL-1L upregulation. Means ± SD overlaid with individual data points representing replicates are depicted; ** P
    Quantitative Pcr Amplification, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative pcr amplification/product/Bio-Rad
    Average 92 stars, based on 51 article reviews
    Price from $9.99 to $1999.99
    quantitative pcr amplification - by Bioz Stars, 2020-09
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    ChIP analysis of HSF1 binding sites in murine Ttr ( A ) and Hsp70 ( B ) promoters in hippocampus and liver after in vivo celastrol treatment. ChIP-qPCR results from treatment of murine hippocampal or hepatic cells with vehicle control (white) ( n = 4) or celastrol (gray bar) ( n = 4) are shown as percentage of input. ChIP assays were performed at least in triplicate. Multivariate analysis reveals a significant effect of celastrol on HSF1-binding activity to the Ttr promoter in the hippocampus relative to that produced by DMSO treatment. There was also an increase in binding to the Hsp70 promoter in both hippocampus and liver (Student's t test): ** p

    Journal: The Journal of Neuroscience

    Article Title: The Systemic Amyloid Precursor Transthyretin (TTR) Behaves as a Neuronal Stress Protein Regulated by HSF1 in SH-SY5Y Human Neuroblastoma Cells and APP23 Alzheimer's Disease Model Mice

    doi: 10.1523/JNEUROSCI.4936-13.2014

    Figure Lengend Snippet: ChIP analysis of HSF1 binding sites in murine Ttr ( A ) and Hsp70 ( B ) promoters in hippocampus and liver after in vivo celastrol treatment. ChIP-qPCR results from treatment of murine hippocampal or hepatic cells with vehicle control (white) ( n = 4) or celastrol (gray bar) ( n = 4) are shown as percentage of input. ChIP assays were performed at least in triplicate. Multivariate analysis reveals a significant effect of celastrol on HSF1-binding activity to the Ttr promoter in the hippocampus relative to that produced by DMSO treatment. There was also an increase in binding to the Hsp70 promoter in both hippocampus and liver (Student's t test): ** p

    Article Snippet: The primers used to amplify the mouse genes were as follows: β- Actin , forward 5′-CAACGAGCGGTTCCGATG-3′ and reverse 5′-GCCACAGGATTCCATACCCA-3′; TTR , forward 5′- AAAAGACCTCTGAGGGATCCT-3′ and reverse 5′-GGTACAAATGGGATGCTACTGC-3′; HSF1 , forward 5′-AGTGGGAACAGCTTCCACG-3′ and reverse 5′-CCACGCAAGAAACAAGGATGC-3′. qPCR amplifications were performed using the Opticon Monitor 3 Detection System (Bio-Rad), and the data were analyzed with iCycler iQ software (Bio-Rad).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Real-time Polymerase Chain Reaction, Activity Assay, Produced

    ChIP analysis of HSF1 binding sites in human TTR promoters after 1 h 42°C heat shock treatment. ChIP-qPCR results with no treatment (white) or heat shock induction (gray) are shown as percentage of input DNA in SH-SY5Y ( A ) or HepG2 ( B ) cells (1 × 10 6 cell equivalents per IP). C , ChIP-qPCR results with Hsp70 promoter from same cells as target. Multivariate analysis reveals a significant effect of heat shock on HSF1-binding activity to the TTR promoter in the SH-SY5Y cells relative to that produced by no treatment (from ≥3 independent experiments, Student's t test): * p

    Journal: The Journal of Neuroscience

    Article Title: The Systemic Amyloid Precursor Transthyretin (TTR) Behaves as a Neuronal Stress Protein Regulated by HSF1 in SH-SY5Y Human Neuroblastoma Cells and APP23 Alzheimer's Disease Model Mice

    doi: 10.1523/JNEUROSCI.4936-13.2014

    Figure Lengend Snippet: ChIP analysis of HSF1 binding sites in human TTR promoters after 1 h 42°C heat shock treatment. ChIP-qPCR results with no treatment (white) or heat shock induction (gray) are shown as percentage of input DNA in SH-SY5Y ( A ) or HepG2 ( B ) cells (1 × 10 6 cell equivalents per IP). C , ChIP-qPCR results with Hsp70 promoter from same cells as target. Multivariate analysis reveals a significant effect of heat shock on HSF1-binding activity to the TTR promoter in the SH-SY5Y cells relative to that produced by no treatment (from ≥3 independent experiments, Student's t test): * p

    Article Snippet: The primers used to amplify the mouse genes were as follows: β- Actin , forward 5′-CAACGAGCGGTTCCGATG-3′ and reverse 5′-GCCACAGGATTCCATACCCA-3′; TTR , forward 5′- AAAAGACCTCTGAGGGATCCT-3′ and reverse 5′-GGTACAAATGGGATGCTACTGC-3′; HSF1 , forward 5′-AGTGGGAACAGCTTCCACG-3′ and reverse 5′-CCACGCAAGAAACAAGGATGC-3′. qPCR amplifications were performed using the Opticon Monitor 3 Detection System (Bio-Rad), and the data were analyzed with iCycler iQ software (Bio-Rad).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Activity Assay, Produced

    Cardiogenic mesoderm to endocardial endothelial cells a Schematic of hPSC-derived CPCs differentiation to endocardium progenitor cells. b The qPCR analysis of the known endocardium markers for hPSC-derived CPCs that were treated with the indicated signaling molecules for 3 days. c The qRT-PCR analysis of the known endocardium markers of hPSC-derived CPCs that were treated with VEGFA, BMP4 and bFGF for 3 days. d IF staining of day 6 hPSC-derived endocardial progenitors showing abundant expression of VE-Cad, GATA4 and NFATc1. Scale bar: 25 μm. e Quantification analysis of panel (d) by ImageJ. Bar graph represents percentage of VE-Cad, GATA4 and NFATc1 positive cells ± S.D of three independent experiments. f Western blotting using the indicated antibodies showing strong expression of VE-Cad, CD31 and NFATc1 in day 6 hPSC-derived endocardial progenitors. g Flow cytometry analysis showing percentage of VE-Cad and NFATc1 double positive cells. Day 3 hPSC-derived CPCs were treated with the indicated signaling molecules for 3 days, and stained with VE-Cad and NFATc1 antibodies. All experiments were repeated 3 times. Significant levels are: * p

    Journal: bioRxiv

    Article Title: Generation of cardiac valve endocardial like cells from human pluripotent stem cells

    doi: 10.1101/2020.04.20.050161

    Figure Lengend Snippet: Cardiogenic mesoderm to endocardial endothelial cells a Schematic of hPSC-derived CPCs differentiation to endocardium progenitor cells. b The qPCR analysis of the known endocardium markers for hPSC-derived CPCs that were treated with the indicated signaling molecules for 3 days. c The qRT-PCR analysis of the known endocardium markers of hPSC-derived CPCs that were treated with VEGFA, BMP4 and bFGF for 3 days. d IF staining of day 6 hPSC-derived endocardial progenitors showing abundant expression of VE-Cad, GATA4 and NFATc1. Scale bar: 25 μm. e Quantification analysis of panel (d) by ImageJ. Bar graph represents percentage of VE-Cad, GATA4 and NFATc1 positive cells ± S.D of three independent experiments. f Western blotting using the indicated antibodies showing strong expression of VE-Cad, CD31 and NFATc1 in day 6 hPSC-derived endocardial progenitors. g Flow cytometry analysis showing percentage of VE-Cad and NFATc1 double positive cells. Day 3 hPSC-derived CPCs were treated with the indicated signaling molecules for 3 days, and stained with VE-Cad and NFATc1 antibodies. All experiments were repeated 3 times. Significant levels are: * p

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed using a Bio-Rad qPCR instrument.

    Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Expressing, Western Blot, Flow Cytometry

    CYC12 and CRK9AP are functional partners of CRK9. (A) Cumulative culture growth curves were obtained for CYC12 and CRK9AP silencing in the absence and presence of doxycycline (dox), the gene knockdown-inducing compound. For each knockdown a representative growth curve is shown. (B) Analysis of total RNA prepared from non-induced cells and cells in which CYC12 or CRK9AP were silenced for 1, 2 or 3 days. CYC12 or CRK9AP mRNA as well as α tubulin and RPB7 mRNA were analyzed by reverse transcription of oligo-dT and semi-quantitative PCR, whereas unspliced, pre-mRNA of α tubulin and RPB7 were analyzed by reverse transcription of random hexamers and by PCR using an oligonucleotide upstream of the SL addition site. rRNA was visualized by ethidium bromide staining after separation in an agarose gel. SL RNA, U2 snRNA and the Y structure intermediate were detected by primer extension assays using a SL RNA and a U2 snRNA-specific primer in the same reactions. (C) Anti-RPB1 immunoblot analysis of whole-cell lysates prepared from CRK9AP -silenced cells. Detection of the similar-sized RNA pol I subunit RPA1 served as a loading control.

    Journal: PLoS Pathogens

    Article Title: Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes, Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein

    doi: 10.1371/journal.ppat.1005498

    Figure Lengend Snippet: CYC12 and CRK9AP are functional partners of CRK9. (A) Cumulative culture growth curves were obtained for CYC12 and CRK9AP silencing in the absence and presence of doxycycline (dox), the gene knockdown-inducing compound. For each knockdown a representative growth curve is shown. (B) Analysis of total RNA prepared from non-induced cells and cells in which CYC12 or CRK9AP were silenced for 1, 2 or 3 days. CYC12 or CRK9AP mRNA as well as α tubulin and RPB7 mRNA were analyzed by reverse transcription of oligo-dT and semi-quantitative PCR, whereas unspliced, pre-mRNA of α tubulin and RPB7 were analyzed by reverse transcription of random hexamers and by PCR using an oligonucleotide upstream of the SL addition site. rRNA was visualized by ethidium bromide staining after separation in an agarose gel. SL RNA, U2 snRNA and the Y structure intermediate were detected by primer extension assays using a SL RNA and a U2 snRNA-specific primer in the same reactions. (C) Anti-RPB1 immunoblot analysis of whole-cell lysates prepared from CRK9AP -silenced cells. Detection of the similar-sized RNA pol I subunit RPA1 served as a loading control.

    Article Snippet: For RNA quantifications, cDNA preparations were analyzed by qPCR assays using the SsoFast EvaGreen Supermix (BioRad) on a CFX96 cycler (BioRad) according to the manufacturer’s recommendations.

    Techniques: Functional Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

    CRK9AP depletion results in rapid co-loss of CRK9 and CYC12. (A) Immunoblot of whole cell lysates derived from non-induced (n.i.) and CRK9AP -silenced PF trypanosomes. The arrow indicates the gene knockdown of CRK9AP . Detection of the class I transcription factor A subunit 6 (CITFA6) served as a loading control. (B) Corresponding semi-quantitative PCR analysis of cDNA that was obtained from the same cells by reverse transcription of total RNA using oligo-dT. Relative RNA amounts were determined by ethidium bromide–stained rRNA.

    Journal: PLoS Pathogens

    Article Title: Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes, Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein

    doi: 10.1371/journal.ppat.1005498

    Figure Lengend Snippet: CRK9AP depletion results in rapid co-loss of CRK9 and CYC12. (A) Immunoblot of whole cell lysates derived from non-induced (n.i.) and CRK9AP -silenced PF trypanosomes. The arrow indicates the gene knockdown of CRK9AP . Detection of the class I transcription factor A subunit 6 (CITFA6) served as a loading control. (B) Corresponding semi-quantitative PCR analysis of cDNA that was obtained from the same cells by reverse transcription of total RNA using oligo-dT. Relative RNA amounts were determined by ethidium bromide–stained rRNA.

    Article Snippet: For RNA quantifications, cDNA preparations were analyzed by qPCR assays using the SsoFast EvaGreen Supermix (BioRad) on a CFX96 cycler (BioRad) according to the manufacturer’s recommendations.

    Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Staining

    HOIL-1L is upregulated during IAV infection through a type I IFN receptor signaling axis. ( A and B ) AT2 cells were isolated from WT mice at 0, 3, 5, and 7 d.p.i. ( A ) HOIL-1L mRNA ( n = 5). ( B ) Representative HOIL-1L immunoblot and its quantification ( n = 4). ( C ) Representative native PAGE immunoblot of HOIL-1L and HOIP expression in AT2 cells infected in vitro with WSN ( n = 3). ( D ) HOIL-1L mRNA expression in AT2 cells from WT mice 0 and 3 d.p.i. sorted based on expression of the viral protein HA ( n = 8). ( E – I ) Representative HOIL-1L immunoblots and quantification. ( E ) A549 cells treated for 0, 8, 12, and 16 hours with conditioned medium (CM; “16*” indicates boiled CM) ( n = 4). ( F ) A549 cells treated with recombinant IFN-α ( n = 4). ( G ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with CM ( n = 3). ( H ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with WSN ( n = 3). ( I ) AT2 cells isolated from WT and CCR2 –/– mice treated in vitro with WSN ( n = 4). ( J ) Quantitative reverse transcriptase PCR quantification of HOIL-1L promoter after ChIP of IRF1 in A549 cells ( n = 4). ( K ) Representative HOIL-1L immunoblot and quantification in siControl- or siIRF1-transfected A549 cells treated with CM ( n = 4). ( L ) Proposed type I IFN pathway leading to HOIL-1L upregulation. Means ± SD overlaid with individual data points representing replicates are depicted; ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection

    doi: 10.1172/JCI128368

    Figure Lengend Snippet: HOIL-1L is upregulated during IAV infection through a type I IFN receptor signaling axis. ( A and B ) AT2 cells were isolated from WT mice at 0, 3, 5, and 7 d.p.i. ( A ) HOIL-1L mRNA ( n = 5). ( B ) Representative HOIL-1L immunoblot and its quantification ( n = 4). ( C ) Representative native PAGE immunoblot of HOIL-1L and HOIP expression in AT2 cells infected in vitro with WSN ( n = 3). ( D ) HOIL-1L mRNA expression in AT2 cells from WT mice 0 and 3 d.p.i. sorted based on expression of the viral protein HA ( n = 8). ( E – I ) Representative HOIL-1L immunoblots and quantification. ( E ) A549 cells treated for 0, 8, 12, and 16 hours with conditioned medium (CM; “16*” indicates boiled CM) ( n = 4). ( F ) A549 cells treated with recombinant IFN-α ( n = 4). ( G ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with CM ( n = 3). ( H ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with WSN ( n = 3). ( I ) AT2 cells isolated from WT and CCR2 –/– mice treated in vitro with WSN ( n = 4). ( J ) Quantitative reverse transcriptase PCR quantification of HOIL-1L promoter after ChIP of IRF1 in A549 cells ( n = 4). ( K ) Representative HOIL-1L immunoblot and quantification in siControl- or siIRF1-transfected A549 cells treated with CM ( n = 4). ( L ) Proposed type I IFN pathway leading to HOIL-1L upregulation. Means ± SD overlaid with individual data points representing replicates are depicted; ** P

    Article Snippet: Bead eluates were subjected to proteinase digestion and quantitative PCR amplification of the HOIL-1L promoter (forward: 5′-ttagcttcagtgttccccct-3′; reverse: 5′-CAGTGGGGAGACAATGAACAA-3′) using SYBR Green (Bio-Rad, 1708880).

    Techniques: Infection, Isolation, Mouse Assay, Clear Native PAGE, Expressing, In Vitro, Western Blot, Recombinant, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Transfection