Structured Review

Thermo Fisher mrna
Chase inefficiency as revealed by STL1 induction. Wild-type cells (KWY165) were grown to OD 600 = 0.2, labeled with 0.2 mM 4TU for 2 hr and then washed out of the labeling media. Half of the cells were returned to the same media and the other half were resuspended in chase media containing 19.6 mM uracil. Both cultures were immediately subjected to 0.4 M NaCl salt shock and samples were collected 10 min after the salt shock. Thio-labeled mRNAs were purified and abundance of STL1 <t>thio-mRNA</t> was determined by <t>qPCR,</t> normalized to a thio-labeled spike ( srp1α (Hs) ) and then normalized to the levels of STL1 in pre-salt shocked cells (t = 0).
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1) Product Images from "Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability"

Article Title: Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability

Journal: eLife

doi: 10.7554/eLife.32536

Chase inefficiency as revealed by STL1 induction. Wild-type cells (KWY165) were grown to OD 600 = 0.2, labeled with 0.2 mM 4TU for 2 hr and then washed out of the labeling media. Half of the cells were returned to the same media and the other half were resuspended in chase media containing 19.6 mM uracil. Both cultures were immediately subjected to 0.4 M NaCl salt shock and samples were collected 10 min after the salt shock. Thio-labeled mRNAs were purified and abundance of STL1 thio-mRNA was determined by qPCR, normalized to a thio-labeled spike ( srp1α (Hs) ) and then normalized to the levels of STL1 in pre-salt shocked cells (t = 0).
Figure Legend Snippet: Chase inefficiency as revealed by STL1 induction. Wild-type cells (KWY165) were grown to OD 600 = 0.2, labeled with 0.2 mM 4TU for 2 hr and then washed out of the labeling media. Half of the cells were returned to the same media and the other half were resuspended in chase media containing 19.6 mM uracil. Both cultures were immediately subjected to 0.4 M NaCl salt shock and samples were collected 10 min after the salt shock. Thio-labeled mRNAs were purified and abundance of STL1 thio-mRNA was determined by qPCR, normalized to a thio-labeled spike ( srp1α (Hs) ) and then normalized to the levels of STL1 in pre-salt shocked cells (t = 0).

Techniques Used: Labeling, Purification, Real-time Polymerase Chain Reaction

Reproducibility of cell lysis during RNA extraction. Wild-type cells (KWY165) were grown to exponential phase and six technical replicate cell pellets of 5 OD 600 units were collected. 10 ng of in vitro transcribed rcc1 (Xl) mRNA spike was added to each sample and the cells were subjected to the RNA extraction protocol. Levels of ACT1 and the spike mRNAs were determined and the ratio of ACT1 :spike normalized to replicate one is plotted.
Figure Legend Snippet: Reproducibility of cell lysis during RNA extraction. Wild-type cells (KWY165) were grown to exponential phase and six technical replicate cell pellets of 5 OD 600 units were collected. 10 ng of in vitro transcribed rcc1 (Xl) mRNA spike was added to each sample and the cells were subjected to the RNA extraction protocol. Levels of ACT1 and the spike mRNAs were determined and the ratio of ACT1 :spike normalized to replicate one is plotted.

Techniques Used: Lysis, RNA Extraction, In Vitro

Analysis of unlabeled mRNA release during streptavidin bead purification. RNA mixtures were prepared as in Appendix1—figure 7 and total (TOT), eluate (ELU) flowthrough (FT) and wash (WASH1-WASH9) samples were analyzed by northern blot.
Figure Legend Snippet: Analysis of unlabeled mRNA release during streptavidin bead purification. RNA mixtures were prepared as in Appendix1—figure 7 and total (TOT), eluate (ELU) flowthrough (FT) and wash (WASH1-WASH9) samples were analyzed by northern blot.

Techniques Used: Purification, Northern Blot

Reproducibility of cell lysis during RNA extraction. Wild-type cells (KWY165) were grown to exponential phase and two technical replicate cell pellets of varying OD 600 units were collected. 10 ng of in vitro transcribed rcc1 (Xl) mRNA spike was added to each sample and the cells were subjected to the RNA extraction protocol. Levels of ACT1 and the spike mRNAs were determined and the ratio of ACT1 :spike is plotted. The shaded area indicates the OD 600 range in which the 4TU-chase experiments are performed.
Figure Legend Snippet: Reproducibility of cell lysis during RNA extraction. Wild-type cells (KWY165) were grown to exponential phase and two technical replicate cell pellets of varying OD 600 units were collected. 10 ng of in vitro transcribed rcc1 (Xl) mRNA spike was added to each sample and the cells were subjected to the RNA extraction protocol. Levels of ACT1 and the spike mRNAs were determined and the ratio of ACT1 :spike is plotted. The shaded area indicates the OD 600 range in which the 4TU-chase experiments are performed.

Techniques Used: Lysis, RNA Extraction, In Vitro

Analysis of streptavidin bead blocking efficiency. Wild-type cells (KWY165) were labeled with 1 mM 4TU for 2 hr. 5 ng of in vitro transcribed rcc1 (Xl) mRNA spike and 5 ng of in vitro transcribed 4TU-labeled srp1α (Hs) mRNA spike were added to 10 μg of extracted total RNA and the RNA mix was biotinylated. mRNAs were enriched for using oligo-dT beads and the mRNAs were then subjected to streptavidin bead selection. Streptavidin beads were prepared using the indicated blocking agents and the total (TOT), eluate (ELU) and flowthrough plus washes (FT + W) fractions were analyzed by northern blot.
Figure Legend Snippet: Analysis of streptavidin bead blocking efficiency. Wild-type cells (KWY165) were labeled with 1 mM 4TU for 2 hr. 5 ng of in vitro transcribed rcc1 (Xl) mRNA spike and 5 ng of in vitro transcribed 4TU-labeled srp1α (Hs) mRNA spike were added to 10 μg of extracted total RNA and the RNA mix was biotinylated. mRNAs were enriched for using oligo-dT beads and the mRNAs were then subjected to streptavidin bead selection. Streptavidin beads were prepared using the indicated blocking agents and the total (TOT), eluate (ELU) and flowthrough plus washes (FT + W) fractions were analyzed by northern blot.

Techniques Used: Blocking Assay, Labeling, In Vitro, Selection, Northern Blot

Effects of translation elongation inhibitors on mRNA stability and cell growth. ( A ) Wild-type cells (KWY165) were subjected to mRNA stability profiling immediately after addition of 0.1% DMSO or 50 μg/mL cycloheximide in 0.1% DMSO. Data were collected and plotted as in Figure 3B . ( B ) rpl28-Q38K cells (KWY7335) were subjected to mRNA stability profiling immediately after addition of 0.1% DMSO or 0.2 μg/mL cycloheximide in 0.1% DMSO. Data were collected and plotted as in Figure 3B . ( C ) Wild-type cells (KWY165) were treated with 0.1% ethanol (gray) or 1.5 μg/mL sordarin in 0.1% ethanol (blue) and growth by absorbance at 600 nm was monitored. The horizontal orange line marks t = 33 min where the growth rates diverge. ( D ) Thio-uracil containing mRNAs from the experiment described in Figure 3B were measured and levels were plotted. RNA levels were normalized to a 4TU-labeled hSRP1 spike RNA and to the time = 0 value for the mock (DMSO) treated sample. ( E ) Thio-uracil containing mRNAs from the experiment described in Figure 3C were measured and levels were plotted. RNA levels were normalized to a 4TU-labeled hSRP1 spike RNA and to the time = 0 value for the mock (EtOH) treated sample. ( F ) Wild-type cells (KWY165) were treated with 5 mM 3AT (blue) or untreated (gray) and growth by absorbance at 600 nm was monitored. ( G ) Thio-uracil containing AIM7 , CYS4 and DED1 mRNAs from the experiment described in Figure 3C–E were measured and levels were plotted. RNA levels were normalized to a 4TU-labeled hSRP1 spike RNA and to the time = 0 value for the mock (DMSO) treated sample. ( H ) Fold increase in mRNA half-life values when cells are treated with 5 mM 3AT (derived from the experiment described in Figure 3H ) were computed for groups of mRNAs binned by their histidine and glycine codon contents.
Figure Legend Snippet: Effects of translation elongation inhibitors on mRNA stability and cell growth. ( A ) Wild-type cells (KWY165) were subjected to mRNA stability profiling immediately after addition of 0.1% DMSO or 50 μg/mL cycloheximide in 0.1% DMSO. Data were collected and plotted as in Figure 3B . ( B ) rpl28-Q38K cells (KWY7335) were subjected to mRNA stability profiling immediately after addition of 0.1% DMSO or 0.2 μg/mL cycloheximide in 0.1% DMSO. Data were collected and plotted as in Figure 3B . ( C ) Wild-type cells (KWY165) were treated with 0.1% ethanol (gray) or 1.5 μg/mL sordarin in 0.1% ethanol (blue) and growth by absorbance at 600 nm was monitored. The horizontal orange line marks t = 33 min where the growth rates diverge. ( D ) Thio-uracil containing mRNAs from the experiment described in Figure 3B were measured and levels were plotted. RNA levels were normalized to a 4TU-labeled hSRP1 spike RNA and to the time = 0 value for the mock (DMSO) treated sample. ( E ) Thio-uracil containing mRNAs from the experiment described in Figure 3C were measured and levels were plotted. RNA levels were normalized to a 4TU-labeled hSRP1 spike RNA and to the time = 0 value for the mock (EtOH) treated sample. ( F ) Wild-type cells (KWY165) were treated with 5 mM 3AT (blue) or untreated (gray) and growth by absorbance at 600 nm was monitored. ( G ) Thio-uracil containing AIM7 , CYS4 and DED1 mRNAs from the experiment described in Figure 3C–E were measured and levels were plotted. RNA levels were normalized to a 4TU-labeled hSRP1 spike RNA and to the time = 0 value for the mock (DMSO) treated sample. ( H ) Fold increase in mRNA half-life values when cells are treated with 5 mM 3AT (derived from the experiment described in Figure 3H ) were computed for groups of mRNAs binned by their histidine and glycine codon contents.

Techniques Used: Labeling, Derivative Assay

2) Product Images from "The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma"

Article Title: The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19082365

NKT cells can be efficiently transfected with a chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR) using RNA electroporation. The NKT and CD8 + T cells were isolated and expanded as described in Figure 1 . After 10–11 days, the cell populations were either transfected without mRNA (mock) as controls or with mRNA encoding a CSPG4-specific CAR. ( A ) The expression kinetics of the CAR-electroporated cells at indicated timepoints are shown. The CAR expression of the NKT (green bars) and CD8 + T cells (blue bars) was detected by using an anti-IgG1 antibody. The mock-transfected cells served as controls (grey bars). The data represent the average geometric mean values of 5–7 independent experiments with SEM. The p -values were calculated by unpaired Student’s t -test and are listed in Table S3 . ( B ) Representative histograms at the peak of the CAR expression of both of the cell populations at 8 h after electroporation. The NKT cells are displayed as the green-filled histogram, and the CD8 + T cells as the blue-filled histogram. The mock-transfected cells served as controls (grey-filled histograms). The staining for the CAR expression was performed using the abovementioned antibody. The data of one representative out of 5–7 independent experiments are shown.
Figure Legend Snippet: NKT cells can be efficiently transfected with a chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR) using RNA electroporation. The NKT and CD8 + T cells were isolated and expanded as described in Figure 1 . After 10–11 days, the cell populations were either transfected without mRNA (mock) as controls or with mRNA encoding a CSPG4-specific CAR. ( A ) The expression kinetics of the CAR-electroporated cells at indicated timepoints are shown. The CAR expression of the NKT (green bars) and CD8 + T cells (blue bars) was detected by using an anti-IgG1 antibody. The mock-transfected cells served as controls (grey bars). The data represent the average geometric mean values of 5–7 independent experiments with SEM. The p -values were calculated by unpaired Student’s t -test and are listed in Table S3 . ( B ) Representative histograms at the peak of the CAR expression of both of the cell populations at 8 h after electroporation. The NKT cells are displayed as the green-filled histogram, and the CD8 + T cells as the blue-filled histogram. The mock-transfected cells served as controls (grey-filled histograms). The staining for the CAR expression was performed using the abovementioned antibody. The data of one representative out of 5–7 independent experiments are shown.

Techniques Used: Transfection, Electroporation, Isolation, Expressing, Staining

The CSPG4 CAR-transfected NKT cells secrete cytokines in an antigen-specific fashion. The NKT and CD8 + T cells were obtained as described above (see Figure 1 ). Following 10–11 days of expansion, the cells were either electroporated with mRNA coding for a carcinoembryonic antigen (CEA)-specific CAR or with mRNA encoding a CSPG4-specific CAR. The CEA CAR-transfected cells were used as controls. Then, 4 h after electroporation, the cells were co-cultured with target cells overnight. IL-2, tumor necrosis factor (TNF), and interferon-gamma (IFN-γ) production was measured in a cytometric bead array. As target cells, the TxB cell hybridoma T2.A1 (CSPG4 − , CEA − ) and the A375M melanoma cell line (CSPG4 + , CEA − ) were used. The receptor-transfected NKT cells are shown in light green bars (CEA CAR) and dark green bars (CSPG4 CAR), whereas electroporated the CD8 + T cells are displayed in light blue bars (CEA CAR) and dark blue bars (CSPG4 CAR). The average values of 4–7 independent experiments with SEM are shown. The p -values calculated by unpaired Student’s t -test are presented in Tables S6 and S7 .
Figure Legend Snippet: The CSPG4 CAR-transfected NKT cells secrete cytokines in an antigen-specific fashion. The NKT and CD8 + T cells were obtained as described above (see Figure 1 ). Following 10–11 days of expansion, the cells were either electroporated with mRNA coding for a carcinoembryonic antigen (CEA)-specific CAR or with mRNA encoding a CSPG4-specific CAR. The CEA CAR-transfected cells were used as controls. Then, 4 h after electroporation, the cells were co-cultured with target cells overnight. IL-2, tumor necrosis factor (TNF), and interferon-gamma (IFN-γ) production was measured in a cytometric bead array. As target cells, the TxB cell hybridoma T2.A1 (CSPG4 − , CEA − ) and the A375M melanoma cell line (CSPG4 + , CEA − ) were used. The receptor-transfected NKT cells are shown in light green bars (CEA CAR) and dark green bars (CSPG4 CAR), whereas electroporated the CD8 + T cells are displayed in light blue bars (CEA CAR) and dark blue bars (CSPG4 CAR). The average values of 4–7 independent experiments with SEM are shown. The p -values calculated by unpaired Student’s t -test are presented in Tables S6 and S7 .

Techniques Used: Transfection, Electroporation, Cell Culture

3) Product Images from "EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis"

Article Title: EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis

Journal: Molecular microbiology

doi: 10.1111/mmi.12718

Complementation of the espI ::Tn mutant (A) RT-PCR analysis of espI and eccD 1 cotranscription. Upper panel shows genetic arrangement of espI and eccD 1 , and primer annealing sites. Blue arrows represent the internal primers for espI , green arrows the internal primers for eccD 1 and red arrows the primers spanning the two genes. Lower panel shows RT-PCR analysis of total RNA from wild-type M. tuberculosis Erdman. 2F9 cosmid was used as a positive PCR control. - RT, no reverse transcriptase. (B) Quantitative RT-PCR analysis of mRNA levels of esxA and eccD 1. Relative expression levels were calculated using the ΔΔCt method, normalizing transcript levels to sigA signals. Data are shown as percentage relative to wild-type M. tuberculosis. (C) Immunoblots of cell lysates (CL) at 5 μg/well and culture filtrates (CF) at 10 μg/well of wild-type M. tuberculosis Erdman (WT) and espI ::Tn transformed with indicated plasmids grown in Sauton’s medium without Tween-80 for 5 days. Antibodies used are indicated. (D) Cytotoxicity assay in THP-1 cells infected with wild-type M. tuberculosis Erdman (Erd WT) (black bar) and espI ::Tn (grey bars) transformed with indicated plasmids at MOI of 5. Data represent the means and standard deviation of at least 4 independent experiments. The y-axis indicates cytotoxicity values relative to the uninfected control. Significance in difference compared to wild-type M. tuberculosis was calculated using Student’s t -test. *, p
Figure Legend Snippet: Complementation of the espI ::Tn mutant (A) RT-PCR analysis of espI and eccD 1 cotranscription. Upper panel shows genetic arrangement of espI and eccD 1 , and primer annealing sites. Blue arrows represent the internal primers for espI , green arrows the internal primers for eccD 1 and red arrows the primers spanning the two genes. Lower panel shows RT-PCR analysis of total RNA from wild-type M. tuberculosis Erdman. 2F9 cosmid was used as a positive PCR control. - RT, no reverse transcriptase. (B) Quantitative RT-PCR analysis of mRNA levels of esxA and eccD 1. Relative expression levels were calculated using the ΔΔCt method, normalizing transcript levels to sigA signals. Data are shown as percentage relative to wild-type M. tuberculosis. (C) Immunoblots of cell lysates (CL) at 5 μg/well and culture filtrates (CF) at 10 μg/well of wild-type M. tuberculosis Erdman (WT) and espI ::Tn transformed with indicated plasmids grown in Sauton’s medium without Tween-80 for 5 days. Antibodies used are indicated. (D) Cytotoxicity assay in THP-1 cells infected with wild-type M. tuberculosis Erdman (Erd WT) (black bar) and espI ::Tn (grey bars) transformed with indicated plasmids at MOI of 5. Data represent the means and standard deviation of at least 4 independent experiments. The y-axis indicates cytotoxicity values relative to the uninfected control. Significance in difference compared to wild-type M. tuberculosis was calculated using Student’s t -test. *, p

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Transformation Assay, Cytotoxicity Assay, Infection, Standard Deviation

4) Product Images from "Thyrotropin Regulates IL-6 Expression in CD34+ Fibrocytes: Clear Delineation of Its cAMP-Independent Actions"

Article Title: Thyrotropin Regulates IL-6 Expression in CD34+ Fibrocytes: Clear Delineation of Its cAMP-Independent Actions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0075100

Divergent PKCµ and PKCβII mRNA expression in pure CD34 + and CD34 − orbital fibroblast subsets. A parental strain of TAO orbital fibroblasts (containing mixed CD34 + and CD34 − cells) was sorted into pure CD34 + and CD34 − subsets by FACS as described in Experimental Procedures. These were then cultured for 48 h., RNA was isolated, andRT-PCR performed for (left panel) PKCµ and (right panel) PKCβII using the C T method. Ct values were normalized to their respective GAPDH levels. Data are expressed as the mean ± SD of three independent replicates from a single experiment, representative of three performed. PKCµ mRNA was undetectable in CD34 + subsets in all 3 studies while PKCβII mRNA levels were 44±7-fold above their respective parental fibroblast cultures.
Figure Legend Snippet: Divergent PKCµ and PKCβII mRNA expression in pure CD34 + and CD34 − orbital fibroblast subsets. A parental strain of TAO orbital fibroblasts (containing mixed CD34 + and CD34 − cells) was sorted into pure CD34 + and CD34 − subsets by FACS as described in Experimental Procedures. These were then cultured for 48 h., RNA was isolated, andRT-PCR performed for (left panel) PKCµ and (right panel) PKCβII using the C T method. Ct values were normalized to their respective GAPDH levels. Data are expressed as the mean ± SD of three independent replicates from a single experiment, representative of three performed. PKCµ mRNA was undetectable in CD34 + subsets in all 3 studies while PKCβII mRNA levels were 44±7-fold above their respective parental fibroblast cultures.

Techniques Used: Expressing, FACS, Cell Culture, Isolation, Polymerase Chain Reaction

5) Product Images from "G?12 Drives Invasion of Oral Squamous Cell Carcinoma through Up-Regulation of Proinflammatory Cytokines"

Article Title: G?12 Drives Invasion of Oral Squamous Cell Carcinoma through Up-Regulation of Proinflammatory Cytokines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066133

Gα 12 -dependent regulation of IL-6 and IL-8 in OSCC. (A) Dot plots of the linear regression analysis showing a positive correlation of gene expressions between Gα 12 and IL-6/IL-8 in OSCC tumors and normal mucosa tissues. The relative expression scales are shown by RMA value in the microarray data. (B) IL-6 and IL-8 mRNA levels are positively regulated by Gα 12 in OSCC cells. Gα 12 levels in four different OSCC cell lines (HSC-3, SCC25, OC3, and CGHNC9) were altered by the transient overexpression or RNAi knockdown of Gα 12 . The qPCR results were normalized against GAPDH. The lower panel shows the electrophoresis image of the RT-PCR products. (C) The secreted proteins of IL-6 and IL-8 are up-regulated by Gα 12 in OSCC cells. ELISA assay was used to measure IL-6 and IL-8 in the conditioned media of the Gα 12 -overexpressing or -depleted HSC-3, SCC25, OC-3, and CGHNC9 cells. (D) The LPA-induced IL-6 and IL-8 production is regulated by Gα 12 . IL-6 and IL-8 protein levels in conditioned media of OSCC cells were measured by ELISA assay. The baseline IL-6 and IL-8 levels in conditioned media from four different OSCC cell lines are shown in Figure S2 . All the quantitative results are expressed as a fold change relative to the controls. The statistical results were analyzed by t-test , * P
Figure Legend Snippet: Gα 12 -dependent regulation of IL-6 and IL-8 in OSCC. (A) Dot plots of the linear regression analysis showing a positive correlation of gene expressions between Gα 12 and IL-6/IL-8 in OSCC tumors and normal mucosa tissues. The relative expression scales are shown by RMA value in the microarray data. (B) IL-6 and IL-8 mRNA levels are positively regulated by Gα 12 in OSCC cells. Gα 12 levels in four different OSCC cell lines (HSC-3, SCC25, OC3, and CGHNC9) were altered by the transient overexpression or RNAi knockdown of Gα 12 . The qPCR results were normalized against GAPDH. The lower panel shows the electrophoresis image of the RT-PCR products. (C) The secreted proteins of IL-6 and IL-8 are up-regulated by Gα 12 in OSCC cells. ELISA assay was used to measure IL-6 and IL-8 in the conditioned media of the Gα 12 -overexpressing or -depleted HSC-3, SCC25, OC-3, and CGHNC9 cells. (D) The LPA-induced IL-6 and IL-8 production is regulated by Gα 12 . IL-6 and IL-8 protein levels in conditioned media of OSCC cells were measured by ELISA assay. The baseline IL-6 and IL-8 levels in conditioned media from four different OSCC cell lines are shown in Figure S2 . All the quantitative results are expressed as a fold change relative to the controls. The statistical results were analyzed by t-test , * P

Techniques Used: Expressing, Microarray, Over Expression, Real-time Polymerase Chain Reaction, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

6) Product Images from "Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods"

Article Title: Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084881

Developmental Potential of hiPSC-derived IM Cells Generated by the Small Molecule Method. (A) Schematic showing further in vitro and in vivo development of OSR1 + cells that were differentiated from OSR1-GFP knock-in hiPSCs (3D45), using the TTNPB method. (B) Results of RT-PCR analyses showing mRNA expression of marker genes for the developing kidney, gonad, and adrenal cortex in differentiated OSR1 + cells on culture day 14. (C) Differentiated cells stained on day 14 with antibodies or lectins against markers of IM derivatives: LTL and AQP1 for the proximal renal tubule, PDX and WT1 for glomerular podocytes, DBA and CK8 for the nephric duct and ureteric bud, SMA for smooth muscle, ECAD as an epithelial marker, and GATA6 and HSD3β as markers of gonad or adrenal cortex. (D) Immunostaining of histological sections of four-week-old hiPSC-derived IM grafts generated by the TTNPB method transplanted into immunodeficient mice (NOD. CB17- Prkdc scid /J) for human mitochondria (HuMt) (green), LTL (red), AQP1 (purple) and all nuclei (blue). (E) Immunostaining for IM derivative markers (SALL4 for the nephric duct and ureteric bud, AQP2 for the collecting duct, and GATA4 for the gonads or adrenal cortex) of histological sections of cell aggregates generated from OSR1 + cells isolated on day 6 and grown in suspension culture for an additional 8 days. (F) Renal tubule-like structures formed inside the cell aggregates on day 14 stained for ECAD (green), LTL (red), laminin (purple), and nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. (G) Section immunostaining of organ culture samples collected on day 14, for human nuclei (HuNu, green), LTL (red), laminin (purple), and all nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. The data in (B, C), (D) and (E, F) are representative of the findings of three, five and four independent experiments, respectively. Scale bars, 100 m.
Figure Legend Snippet: Developmental Potential of hiPSC-derived IM Cells Generated by the Small Molecule Method. (A) Schematic showing further in vitro and in vivo development of OSR1 + cells that were differentiated from OSR1-GFP knock-in hiPSCs (3D45), using the TTNPB method. (B) Results of RT-PCR analyses showing mRNA expression of marker genes for the developing kidney, gonad, and adrenal cortex in differentiated OSR1 + cells on culture day 14. (C) Differentiated cells stained on day 14 with antibodies or lectins against markers of IM derivatives: LTL and AQP1 for the proximal renal tubule, PDX and WT1 for glomerular podocytes, DBA and CK8 for the nephric duct and ureteric bud, SMA for smooth muscle, ECAD as an epithelial marker, and GATA6 and HSD3β as markers of gonad or adrenal cortex. (D) Immunostaining of histological sections of four-week-old hiPSC-derived IM grafts generated by the TTNPB method transplanted into immunodeficient mice (NOD. CB17- Prkdc scid /J) for human mitochondria (HuMt) (green), LTL (red), AQP1 (purple) and all nuclei (blue). (E) Immunostaining for IM derivative markers (SALL4 for the nephric duct and ureteric bud, AQP2 for the collecting duct, and GATA4 for the gonads or adrenal cortex) of histological sections of cell aggregates generated from OSR1 + cells isolated on day 6 and grown in suspension culture for an additional 8 days. (F) Renal tubule-like structures formed inside the cell aggregates on day 14 stained for ECAD (green), LTL (red), laminin (purple), and nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. (G) Section immunostaining of organ culture samples collected on day 14, for human nuclei (HuNu, green), LTL (red), laminin (purple), and all nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. The data in (B, C), (D) and (E, F) are representative of the findings of three, five and four independent experiments, respectively. Scale bars, 100 m.

Techniques Used: Derivative Assay, Generated, In Vitro, In Vivo, Knock-In, Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Staining, Immunostaining, Mouse Assay, Isolation, Organ Culture

7) Product Images from "Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods"

Article Title: Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084881

Developmental Potential of hiPSC-derived IM Cells Generated by the Small Molecule Method. (A) Schematic showing further in vitro and in vivo development of OSR1 + cells that were differentiated from OSR1-GFP knock-in hiPSCs (3D45), using the TTNPB method. (B) Results of RT-PCR analyses showing mRNA expression of marker genes for the developing kidney, gonad, and adrenal cortex in differentiated OSR1 + cells on culture day 14. (C) Differentiated cells stained on day 14 with antibodies or lectins against markers of IM derivatives: LTL and AQP1 for the proximal renal tubule, PDX and WT1 for glomerular podocytes, DBA and CK8 for the nephric duct and ureteric bud, SMA for smooth muscle, ECAD as an epithelial marker, and GATA6 and HSD3β as markers of gonad or adrenal cortex. (D) Immunostaining of histological sections of four-week-old hiPSC-derived IM grafts generated by the TTNPB method transplanted into immunodeficient mice (NOD. CB17- Prkdc scid /J) for human mitochondria (HuMt) (green), LTL (red), AQP1 (purple) and all nuclei (blue). (E) Immunostaining for IM derivative markers (SALL4 for the nephric duct and ureteric bud, AQP2 for the collecting duct, and GATA4 for the gonads or adrenal cortex) of histological sections of cell aggregates generated from OSR1 + cells isolated on day 6 and grown in suspension culture for an additional 8 days. (F) Renal tubule-like structures formed inside the cell aggregates on day 14 stained for ECAD (green), LTL (red), laminin (purple), and nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. (G) Section immunostaining of organ culture samples collected on day 14, for human nuclei (HuNu, green), LTL (red), laminin (purple), and all nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. The data in (B, C), (D) and (E, F) are representative of the findings of three, five and four independent experiments, respectively. Scale bars, 100 m.
Figure Legend Snippet: Developmental Potential of hiPSC-derived IM Cells Generated by the Small Molecule Method. (A) Schematic showing further in vitro and in vivo development of OSR1 + cells that were differentiated from OSR1-GFP knock-in hiPSCs (3D45), using the TTNPB method. (B) Results of RT-PCR analyses showing mRNA expression of marker genes for the developing kidney, gonad, and adrenal cortex in differentiated OSR1 + cells on culture day 14. (C) Differentiated cells stained on day 14 with antibodies or lectins against markers of IM derivatives: LTL and AQP1 for the proximal renal tubule, PDX and WT1 for glomerular podocytes, DBA and CK8 for the nephric duct and ureteric bud, SMA for smooth muscle, ECAD as an epithelial marker, and GATA6 and HSD3β as markers of gonad or adrenal cortex. (D) Immunostaining of histological sections of four-week-old hiPSC-derived IM grafts generated by the TTNPB method transplanted into immunodeficient mice (NOD. CB17- Prkdc scid /J) for human mitochondria (HuMt) (green), LTL (red), AQP1 (purple) and all nuclei (blue). (E) Immunostaining for IM derivative markers (SALL4 for the nephric duct and ureteric bud, AQP2 for the collecting duct, and GATA4 for the gonads or adrenal cortex) of histological sections of cell aggregates generated from OSR1 + cells isolated on day 6 and grown in suspension culture for an additional 8 days. (F) Renal tubule-like structures formed inside the cell aggregates on day 14 stained for ECAD (green), LTL (red), laminin (purple), and nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. (G) Section immunostaining of organ culture samples collected on day 14, for human nuclei (HuNu, green), LTL (red), laminin (purple), and all nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. The data in (B, C), (D) and (E, F) are representative of the findings of three, five and four independent experiments, respectively. Scale bars, 100 m.

Techniques Used: Derivative Assay, Generated, In Vitro, In Vivo, Knock-In, Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Staining, Immunostaining, Mouse Assay, Isolation, Organ Culture

8) Product Images from "Art27 Interacts with GATA4, FOG2 and NKX2.5 and Is a Novel Co-Repressor of Cardiac Genes"

Article Title: Art27 Interacts with GATA4, FOG2 and NKX2.5 and Is a Novel Co-Repressor of Cardiac Genes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095253

Art27 is a potential transcriptional regulator in cardiac developmental and adult tissues. ( A ) Northern blot on RNA derived from eight different heart tissues was probed for expression of GATA-4, FOG-2 and Art27. The membrane was stripped and re-blotted with the appropriate label. The housekeeping gene Gapdh was used as a loading control. Differential binding and/or absence of binding is indicative of probe specificity. ( B ) P19CL6 embryonal carcinoma cells with stably incorporated GFP under the control of the cardiac Mlc2v promoter [26] were induced to undergo cardiomyocyte differentiation with 1% DMSO. At 5-day increments, mRNA was isolated to assess relative expression of cardiac transcription factors Art27, GATA-4, FOG-2 and Nkx2.5 compared to housekeeping gene Gapdh. These time points correspond to various stages of cardiomyocyte differentiation as determined by systematic scoring of GFP fluorescence and beating intensity. ( C ) 293a cells were transfected with the various expression plasmids as indicated and GAL 5 LEXA 2 -Luc reporter activity was assessed. GAL(DBD)-Art27 significantly diminishes reporter activity compared to GAL(DBD)-empty expression plasmid alone showing inherent transcriptional repression activity. GAL(DBD)-SUMO-1 was used as positive control and was observed to repress the activity of the reporter as expected [49] **p
Figure Legend Snippet: Art27 is a potential transcriptional regulator in cardiac developmental and adult tissues. ( A ) Northern blot on RNA derived from eight different heart tissues was probed for expression of GATA-4, FOG-2 and Art27. The membrane was stripped and re-blotted with the appropriate label. The housekeeping gene Gapdh was used as a loading control. Differential binding and/or absence of binding is indicative of probe specificity. ( B ) P19CL6 embryonal carcinoma cells with stably incorporated GFP under the control of the cardiac Mlc2v promoter [26] were induced to undergo cardiomyocyte differentiation with 1% DMSO. At 5-day increments, mRNA was isolated to assess relative expression of cardiac transcription factors Art27, GATA-4, FOG-2 and Nkx2.5 compared to housekeeping gene Gapdh. These time points correspond to various stages of cardiomyocyte differentiation as determined by systematic scoring of GFP fluorescence and beating intensity. ( C ) 293a cells were transfected with the various expression plasmids as indicated and GAL 5 LEXA 2 -Luc reporter activity was assessed. GAL(DBD)-Art27 significantly diminishes reporter activity compared to GAL(DBD)-empty expression plasmid alone showing inherent transcriptional repression activity. GAL(DBD)-SUMO-1 was used as positive control and was observed to repress the activity of the reporter as expected [49] **p

Techniques Used: Northern Blot, Derivative Assay, Expressing, Binding Assay, Stable Transfection, Isolation, Fluorescence, Transfection, Activity Assay, Plasmid Preparation, Positive Control

Art27 knockdown de-represses endogenous cardiac genes. ( A ) Differentiation of P19CL6-Mlc2v-GFP cells was monitored by flow cytometry. The solid histogram represents GFP fluorescence of undifferentiated cells (Day 0). The solid and dotted lines show GFP fluorescence on differentiation Days 7 and 11, respectively. ( B ) Art27 knockdown relative to control. The level of Art27 mRNA was measured by qPCR in differentiated P19CL6-Mlc2v-GFP cells (Day 7) nucleofected with control siRNA (GFP siRNA) or specific Art27 siRNA. Nucleofection with Art27 siRNA resulted in a 10-fold reduction of Art27 mRNA. ( C ) Heatmap showing the relative expression of transcripts that were significantly upregulated (red) or downregulated (blue) in differentiated P19CL6-Mlc2v-GFP cells (Day 7) nucleofected with Art27 siRNA. Relevant cardiac transcripts are indicated.
Figure Legend Snippet: Art27 knockdown de-represses endogenous cardiac genes. ( A ) Differentiation of P19CL6-Mlc2v-GFP cells was monitored by flow cytometry. The solid histogram represents GFP fluorescence of undifferentiated cells (Day 0). The solid and dotted lines show GFP fluorescence on differentiation Days 7 and 11, respectively. ( B ) Art27 knockdown relative to control. The level of Art27 mRNA was measured by qPCR in differentiated P19CL6-Mlc2v-GFP cells (Day 7) nucleofected with control siRNA (GFP siRNA) or specific Art27 siRNA. Nucleofection with Art27 siRNA resulted in a 10-fold reduction of Art27 mRNA. ( C ) Heatmap showing the relative expression of transcripts that were significantly upregulated (red) or downregulated (blue) in differentiated P19CL6-Mlc2v-GFP cells (Day 7) nucleofected with Art27 siRNA. Relevant cardiac transcripts are indicated.

Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Expressing

9) Product Images from "Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development"

Article Title: Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development

Journal: BMC Genomics

doi: 10.1186/1471-2164-15-350

Life cycle of Toxoplasma gondii , and the harvest and hybridization of merozoite mRNA. (A) Life cycle of Toxoplasma gondii [ 58 ]. Green circle highlights stages for which microarray expression data exists [ 27 ]. Red circle highlights merozoite stage harvested for this study. (B) We obtained intestines from three cats (c48, c50, c52) that had been orally infected 5–6 days previously with the type II parasite TgNmBr1strain in order to isolate merozoite stage parasites. To determine that the purified forms were indeed Toxoplasma, a portion of sample c52 was labeled with mouse α-Me49 (Alexa Fluor 488, yellow). DAPI stained nuclei (blue). (C) mRNAs for two merozoite c52 (Mc52) samples and one for each of the c48 (Mc48) and c50 (Mc50) samples were labeled using the Ambion MessageAmp kit and hybridized to Affymetrix Toxoplasma GeneChips. In order to control for possible strain specific expression we also hybridized two TgNmBr1 tachyzoite mRNA samples (TNm). Data were analyzed in combination with the recently published dataset covering oocyst (Day 0 (OD0), 4 (OD4), 10 (OD10)) to tachyzoite (TD2) to bradyzoite (Day 4 (BD4), 8 (BD8), 21 (BD21)) development in the type II M4 strain [ 27 ]. Boxplots of the RMA normalized data show similar distributions and median values across the samples.
Figure Legend Snippet: Life cycle of Toxoplasma gondii , and the harvest and hybridization of merozoite mRNA. (A) Life cycle of Toxoplasma gondii [ 58 ]. Green circle highlights stages for which microarray expression data exists [ 27 ]. Red circle highlights merozoite stage harvested for this study. (B) We obtained intestines from three cats (c48, c50, c52) that had been orally infected 5–6 days previously with the type II parasite TgNmBr1strain in order to isolate merozoite stage parasites. To determine that the purified forms were indeed Toxoplasma, a portion of sample c52 was labeled with mouse α-Me49 (Alexa Fluor 488, yellow). DAPI stained nuclei (blue). (C) mRNAs for two merozoite c52 (Mc52) samples and one for each of the c48 (Mc48) and c50 (Mc50) samples were labeled using the Ambion MessageAmp kit and hybridized to Affymetrix Toxoplasma GeneChips. In order to control for possible strain specific expression we also hybridized two TgNmBr1 tachyzoite mRNA samples (TNm). Data were analyzed in combination with the recently published dataset covering oocyst (Day 0 (OD0), 4 (OD4), 10 (OD10)) to tachyzoite (TD2) to bradyzoite (Day 4 (BD4), 8 (BD8), 21 (BD21)) development in the type II M4 strain [ 27 ]. Boxplots of the RMA normalized data show similar distributions and median values across the samples.

Techniques Used: Hybridization, Microarray, Expressing, Infection, Purification, Labeling, Staining

10) Product Images from "Transfer of MicroRNAs by Embryonic Stem Cell Microvesicles"

Article Title: Transfer of MicroRNAs by Embryonic Stem Cell Microvesicles

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004722

ESMVs contain GFP mRNA and protein expressed from a GFP transgene in ESCs. (A) 300 ng of ESMV RNA from an ESC line expressing GFP were used for RT, and 35 cycles of PCR amplification were performed with the GFP primers shown in Table 1 . A 2% agarose gel was loaded with the RT-PCR products from ESMVs (lane 1), ESCs (lane 2), and a “no RT” control of ESMVs (lane 3). A 406bp band corresponding to the GFP amplicon is observed in both the ESMV and ESC lanes. (B) (Left) Box plot of relative abundance of GFP in ESMVs compared with ESCs (n = 8). (Right) Comparison of amplification curves for GFP (top) and β-actin (bottom) in ESCs (1) and ESMVs (2). Note that while quantitative RT-PCR was performed in the linear range of amplification, (panel B), the end-point PCR products shown in panel (A) are only qualitative and well outside of the linear range. (C) Immunoblot of an 8% urea-SDS polyacrylamide/Tris-glycine buffered gel loaded with 20 μg total protein/lane, using polyclonal anti-GFP antibody (1∶1000) and horse anti-rabbit secondary antibody (1∶5000). The secondary antibody was conjugated to alkaline phosphatase and visualized with BCIP/NBT. A single ∼35kD immunoreactive band corresponding to GFP in ESCs (lane 1) and ESMVs (lane 2) was detected.
Figure Legend Snippet: ESMVs contain GFP mRNA and protein expressed from a GFP transgene in ESCs. (A) 300 ng of ESMV RNA from an ESC line expressing GFP were used for RT, and 35 cycles of PCR amplification were performed with the GFP primers shown in Table 1 . A 2% agarose gel was loaded with the RT-PCR products from ESMVs (lane 1), ESCs (lane 2), and a “no RT” control of ESMVs (lane 3). A 406bp band corresponding to the GFP amplicon is observed in both the ESMV and ESC lanes. (B) (Left) Box plot of relative abundance of GFP in ESMVs compared with ESCs (n = 8). (Right) Comparison of amplification curves for GFP (top) and β-actin (bottom) in ESCs (1) and ESMVs (2). Note that while quantitative RT-PCR was performed in the linear range of amplification, (panel B), the end-point PCR products shown in panel (A) are only qualitative and well outside of the linear range. (C) Immunoblot of an 8% urea-SDS polyacrylamide/Tris-glycine buffered gel loaded with 20 μg total protein/lane, using polyclonal anti-GFP antibody (1∶1000) and horse anti-rabbit secondary antibody (1∶5000). The secondary antibody was conjugated to alkaline phosphatase and visualized with BCIP/NBT. A single ∼35kD immunoreactive band corresponding to GFP in ESCs (lane 1) and ESMVs (lane 2) was detected.

Techniques Used: Expressing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

The RNA in ESMVs is not degraded. Real time quantitative RT-PCR was used to measure the level of degradation of oct-4 and GFP mRNAs in ESMVs by comparing the 5′ and 3′ amplicon ratios of these transcripts in ESMVs with those in ESCs. Significant levels of degradation were not detected with either transcript. (A) Box plot of normalized 5′ and 3′ template values for oct-4 mRNA in ESCs and ESMVs (n = 9). (B) Box plot of normalized 5′ and 3′ template values for GFP mRNA in ESCs and ESMVs (n = 12). The boxed area represents the mean±quartile and the whiskers extend out to the minimum and maximum values. Bootstrap t-tests were performed to compare the 5′:3′ ratios for each transcript in ESCs and ESMVs. No significant difference was detected between the ESC group and ESMV group for either transcript ( p > 0.1).
Figure Legend Snippet: The RNA in ESMVs is not degraded. Real time quantitative RT-PCR was used to measure the level of degradation of oct-4 and GFP mRNAs in ESMVs by comparing the 5′ and 3′ amplicon ratios of these transcripts in ESMVs with those in ESCs. Significant levels of degradation were not detected with either transcript. (A) Box plot of normalized 5′ and 3′ template values for oct-4 mRNA in ESCs and ESMVs (n = 9). (B) Box plot of normalized 5′ and 3′ template values for GFP mRNA in ESCs and ESMVs (n = 12). The boxed area represents the mean±quartile and the whiskers extend out to the minimum and maximum values. Bootstrap t-tests were performed to compare the 5′:3′ ratios for each transcript in ESCs and ESMVs. No significant difference was detected between the ESC group and ESMV group for either transcript ( p > 0.1).

Techniques Used: Quantitative RT-PCR, Amplification

ESMVs contain RNA. ESMV total RNA lacks 28S and 18S rRNA and consists mostly of RNA below ∼2kb. (A) 1.2% denaturing agarose gel loaded with total RNA from ESMVs (lane 1) and ESCs (lane 2). (B) Digital gel images from capillary electrophoresis of total RNA from ESMVs (lane 1) and ESCs (lane 2). (C) Real time quantitative RT-PCR amplification curves for β-actin reveals much less expression in ESMVs compared with ESCs. (D) The relative abundance of several mRNAs in ESMVs compared with ESCs was determined by real time quantitative RT-PCR using the primers shown in Table 1 . The mRNAs tested include gata-4 (lane 1), jag-1 (lane 2), jag-2 (lane 3), nanog (lane 4), oct-4 (lane 5), wnt-3 (lane 6), and β-actin (lane 7). Box plot of relative abundance of all mRNA tested in ESMVs compared with ESCs (n = 8). The boxed area represents the mean±quartile and the whiskers extend out to the minimum and maximum values. Bootstrap ANOVA was performed and a significant difference was detected between all groups ( p
Figure Legend Snippet: ESMVs contain RNA. ESMV total RNA lacks 28S and 18S rRNA and consists mostly of RNA below ∼2kb. (A) 1.2% denaturing agarose gel loaded with total RNA from ESMVs (lane 1) and ESCs (lane 2). (B) Digital gel images from capillary electrophoresis of total RNA from ESMVs (lane 1) and ESCs (lane 2). (C) Real time quantitative RT-PCR amplification curves for β-actin reveals much less expression in ESMVs compared with ESCs. (D) The relative abundance of several mRNAs in ESMVs compared with ESCs was determined by real time quantitative RT-PCR using the primers shown in Table 1 . The mRNAs tested include gata-4 (lane 1), jag-1 (lane 2), jag-2 (lane 3), nanog (lane 4), oct-4 (lane 5), wnt-3 (lane 6), and β-actin (lane 7). Box plot of relative abundance of all mRNA tested in ESMVs compared with ESCs (n = 8). The boxed area represents the mean±quartile and the whiskers extend out to the minimum and maximum values. Bootstrap ANOVA was performed and a significant difference was detected between all groups ( p

Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, Quantitative RT-PCR, Amplification, Expressing

11) Product Images from "Correlational analysis for identifying genes whose regulation contributes to chronic neuropathic pain"

Article Title: Correlational analysis for identifying genes whose regulation contributes to chronic neuropathic pain

Journal: Molecular Pain

doi: 10.1186/1744-8069-5-7

L5 spinal nerve ligation (SNL) alters the expression of mRNAs in the ipsilateral L5DRG . A , Dark-field micrographs display hybridization signals for each of the six transcripts studied: Scn10a , Scn11a , P2rx3 , Trpv1 , Trpa1 and Trpm8 . DRG sections originate from naïve B6 mice (left column) and B6 mice 3 days after SNL surgery (right column). The scale bar refers to all images: 100 μm. B , Column heights (mean ± SEM, n = 4–6) indicate the proportion of DRG neuronal profiles that were positive for expression of each of the six mRNA types in each of the five strains examined. White columns refer to naïve mice; black columns refer to SNL operated mice. Statistical differences between naive and nerve injured mice are indicated with asterisks (Mann-Whitney U test) * p
Figure Legend Snippet: L5 spinal nerve ligation (SNL) alters the expression of mRNAs in the ipsilateral L5DRG . A , Dark-field micrographs display hybridization signals for each of the six transcripts studied: Scn10a , Scn11a , P2rx3 , Trpv1 , Trpa1 and Trpm8 . DRG sections originate from naïve B6 mice (left column) and B6 mice 3 days after SNL surgery (right column). The scale bar refers to all images: 100 μm. B , Column heights (mean ± SEM, n = 4–6) indicate the proportion of DRG neuronal profiles that were positive for expression of each of the six mRNA types in each of the five strains examined. White columns refer to naïve mice; black columns refer to SNL operated mice. Statistical differences between naive and nerve injured mice are indicated with asterisks (Mann-Whitney U test) * p

Techniques Used: Ligation, Expressing, Hybridization, Mouse Assay, MANN-WHITNEY

12) Product Images from "A Highly Conserved Poc1 Protein Characterized in Embryos of the Hydrozoan Clytia hemisphaerica: Localization and Functional Studies"

Article Title: A Highly Conserved Poc1 Protein Characterized in Embryos of the Hydrozoan Clytia hemisphaerica: Localization and Functional Studies

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013994

Clytia Poc1 is targeted to centrioles. Confocal images of embryos expressing fluorescent–tagged ChePoc1 protein following microinjection of mRNA constructs into the egg prior to fertilization. A. blastulae fixed at 5hpf (a–f) and planula larvae fixed at 24hpf (g,i: plane through apical ectoderm; j,k,l: plane through both endoderm (end) and ectoderm (ect)). Both fluorescent Poc1 proteins clearly localize to punctate aggregates positioned on the apical side of each ciliated ectoderm cell of the larva (bb/arrowheads: basal bodies), and randomly in blastula cells and larval endoderm (arrows). a,d,g,j: endogenous GFP showing the distribution of mitochondria in the cytoplasm of each cell (see B); b: Venus-Poc1; e,h,k: Poc1-mCherry; c,f,i,j: merged images. Scale bar: 10 µm. B. Confocal images of an unfertilized egg demonstrating that maternal endogenous Clytia GFP (a) is a natural mitochondrial marker, colocalizing with the mitochondrial dye TMRE (b) as seen in the merged image (c). C. Confocal images of larval cells expressing Poc1-mCherry following micro-injection of mRNA constructs into the egg prior to fertilization, fixed, and then processed for immunofluorescence with anti-gamma tubulin to locate centrosomes. Poc1-mCherry colocalizes with gamma-tubulin at the position of the basal body (bb/arrowheads- enlarged in insert) in ectoderm cells (a,b,c,d) and at the centrosomes of dividing cells in the endoderm (e,f,g,h) (C1/C2 = centrosomes close to prometaphase chromatin; metaphase cell in inset). a,e: DNA (Hoechst); b,f: Poc1-Cherry; c,g: gamma tubulin; d,h: merge (blue = DNA; red = Poc1-Cherry; green = gamma tubulin). Scale bars: 5 µm.
Figure Legend Snippet: Clytia Poc1 is targeted to centrioles. Confocal images of embryos expressing fluorescent–tagged ChePoc1 protein following microinjection of mRNA constructs into the egg prior to fertilization. A. blastulae fixed at 5hpf (a–f) and planula larvae fixed at 24hpf (g,i: plane through apical ectoderm; j,k,l: plane through both endoderm (end) and ectoderm (ect)). Both fluorescent Poc1 proteins clearly localize to punctate aggregates positioned on the apical side of each ciliated ectoderm cell of the larva (bb/arrowheads: basal bodies), and randomly in blastula cells and larval endoderm (arrows). a,d,g,j: endogenous GFP showing the distribution of mitochondria in the cytoplasm of each cell (see B); b: Venus-Poc1; e,h,k: Poc1-mCherry; c,f,i,j: merged images. Scale bar: 10 µm. B. Confocal images of an unfertilized egg demonstrating that maternal endogenous Clytia GFP (a) is a natural mitochondrial marker, colocalizing with the mitochondrial dye TMRE (b) as seen in the merged image (c). C. Confocal images of larval cells expressing Poc1-mCherry following micro-injection of mRNA constructs into the egg prior to fertilization, fixed, and then processed for immunofluorescence with anti-gamma tubulin to locate centrosomes. Poc1-mCherry colocalizes with gamma-tubulin at the position of the basal body (bb/arrowheads- enlarged in insert) in ectoderm cells (a,b,c,d) and at the centrosomes of dividing cells in the endoderm (e,f,g,h) (C1/C2 = centrosomes close to prometaphase chromatin; metaphase cell in inset). a,e: DNA (Hoechst); b,f: Poc1-Cherry; c,g: gamma tubulin; d,h: merge (blue = DNA; red = Poc1-Cherry; green = gamma tubulin). Scale bars: 5 µm.

Techniques Used: Expressing, Construct, Marker, Injection, Immunofluorescence

13) Product Images from "Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo"

Article Title: Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo

Journal: Oncotarget

doi:

Effect of afatinib on the expression of ABCG2 (A-B) H460/MX20 cells were treated with varying concentrations (0–2.0 μM) of afatinib for 48 h, or with 1.0 μM afatinib for 24 h, 48 h and 72 h, respectively. ABCG2 protein levels were analyzed by Western blot. GAPDH was used as a loading control. (C-D) relative quantification of the effect of afatinib on ABCG2. ABCG2 protein expression levels were normalized to GAPDH. (E-F) effect of afatinib on the ABCG2 expression at the mRNA level was determined by real time RT-PCR. The amount of ABCG2 mRNA in a given sample was normalized to the level of GAPDH in that sample. The 2 −ΔΔCt method was used to analyze the relative change. Data represent Mean ± SD. ** p
Figure Legend Snippet: Effect of afatinib on the expression of ABCG2 (A-B) H460/MX20 cells were treated with varying concentrations (0–2.0 μM) of afatinib for 48 h, or with 1.0 μM afatinib for 24 h, 48 h and 72 h, respectively. ABCG2 protein levels were analyzed by Western blot. GAPDH was used as a loading control. (C-D) relative quantification of the effect of afatinib on ABCG2. ABCG2 protein expression levels were normalized to GAPDH. (E-F) effect of afatinib on the ABCG2 expression at the mRNA level was determined by real time RT-PCR. The amount of ABCG2 mRNA in a given sample was normalized to the level of GAPDH in that sample. The 2 −ΔΔCt method was used to analyze the relative change. Data represent Mean ± SD. ** p

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

14) Product Images from "Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009"

Article Title: Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-015-0203-5

Volcano plot distribution of Clostridium butyricum CWBI 1009 mRNA transcript levels (RNA-seq) during unregulated-pH glucose fermentation. The colour code corresponds to the expression level and is presented as an RPKM value for pH 6.3. Dots corresponding to the hydA2, hydA8, hydB2, hydB3 and nifH genes are indicated.
Figure Legend Snippet: Volcano plot distribution of Clostridium butyricum CWBI 1009 mRNA transcript levels (RNA-seq) during unregulated-pH glucose fermentation. The colour code corresponds to the expression level and is presented as an RPKM value for pH 6.3. Dots corresponding to the hydA2, hydA8, hydB2, hydB3 and nifH genes are indicated.

Techniques Used: RNA Sequencing Assay, Expressing

15) Product Images from "Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules"

Article Title: Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku582

mRNA or ssDNA but not dsDNA transfections lead to stress granule assembly in puromycin-treated cells. ( A ) 1 μg of 2Luc mRNA synthesized in vitro was transfected in NRK cells using lipofectamine in the presence of 2.5 μg/ml puromycin (see ‘Materials and Methods’ section). Anti-HuR and anti-YB-1 labeling show the formation of stress granules 3 h after transfection. ( B ) NRK cells were transfected using lipofectamine (Lipo) with 1 μg of α-globin mRNA, M13 ssDNA, or linearized pUC19 dsDNA in the presence or absence of puromycin for 3 h. Benzonase treatment was performed before the formation of lipoplexes. The statistical analysis was obtained on three different samples. Results are mean ± SD ( n = 3). Both mRNA and ssDNA lead to a significant formation of stress granules in puromycin-treated cells. Anti-YB-1 labeling shows the formation of stress granules 3 h after transfection.
Figure Legend Snippet: mRNA or ssDNA but not dsDNA transfections lead to stress granule assembly in puromycin-treated cells. ( A ) 1 μg of 2Luc mRNA synthesized in vitro was transfected in NRK cells using lipofectamine in the presence of 2.5 μg/ml puromycin (see ‘Materials and Methods’ section). Anti-HuR and anti-YB-1 labeling show the formation of stress granules 3 h after transfection. ( B ) NRK cells were transfected using lipofectamine (Lipo) with 1 μg of α-globin mRNA, M13 ssDNA, or linearized pUC19 dsDNA in the presence or absence of puromycin for 3 h. Benzonase treatment was performed before the formation of lipoplexes. The statistical analysis was obtained on three different samples. Results are mean ± SD ( n = 3). Both mRNA and ssDNA lead to a significant formation of stress granules in puromycin-treated cells. Anti-YB-1 labeling shows the formation of stress granules 3 h after transfection.

Techniques Used: Transfection, Synthesized, In Vitro, Labeling

YB-1 dissociates RNA granules in vitro as revealed by atomic force microscopy. ( A ) As control, naked 2Luc mRNA (2 μg/ml) deposited on a mica surface was clearly detected by AFM. In the presence of 100 nM YB-1, single isolated mRNPs were detected. On the other hand, in the presence of 15 nM TIA-1, mRNA:TIA-1 complexes form aggregates. Interestingly, when 100 nM YB-1 was added to preformed mRNA:TIA-1 aggregates for 5 min, a clear dissociation of RNA granules into isolated mRNP was observed. Scale bar: 0.5 μm. ( B ) Statistical analyses of the particle height on the mica surface. TIA-1 (30 nM) in the absence of RNA is attracted on the negatively-charged surface and tends to form small TIA-1 aggregates (2.8 ± 0.5 nm), as expected from its self-attracting domain. In the presence of 2Luc mRNA (2 μg/ml), we noticed the appearance of large mRNA:TIA-1 aggregates (9.8 ± 3.1 nm). These large aggregates consequently contained both mRNA and TIA-1. Addition of increasing concentration of YB-1 progressively dislocates the mRNA:TIA-1 aggregates and leads to the appearance of isolated mRNPs after 5 min. Scale bar: 0.5 μm. The ‘particle analysis’ application of the nanoscope IIIa software (version 5) over at least 200 particles was used to provide mean heights and standard deviations.
Figure Legend Snippet: YB-1 dissociates RNA granules in vitro as revealed by atomic force microscopy. ( A ) As control, naked 2Luc mRNA (2 μg/ml) deposited on a mica surface was clearly detected by AFM. In the presence of 100 nM YB-1, single isolated mRNPs were detected. On the other hand, in the presence of 15 nM TIA-1, mRNA:TIA-1 complexes form aggregates. Interestingly, when 100 nM YB-1 was added to preformed mRNA:TIA-1 aggregates for 5 min, a clear dissociation of RNA granules into isolated mRNP was observed. Scale bar: 0.5 μm. ( B ) Statistical analyses of the particle height on the mica surface. TIA-1 (30 nM) in the absence of RNA is attracted on the negatively-charged surface and tends to form small TIA-1 aggregates (2.8 ± 0.5 nm), as expected from its self-attracting domain. In the presence of 2Luc mRNA (2 μg/ml), we noticed the appearance of large mRNA:TIA-1 aggregates (9.8 ± 3.1 nm). These large aggregates consequently contained both mRNA and TIA-1. Addition of increasing concentration of YB-1 progressively dislocates the mRNA:TIA-1 aggregates and leads to the appearance of isolated mRNPs after 5 min. Scale bar: 0.5 μm. The ‘particle analysis’ application of the nanoscope IIIa software (version 5) over at least 200 particles was used to provide mean heights and standard deviations.

Techniques Used: In Vitro, Microscopy, Isolation, Concentration Assay, Software

16) Product Images from "Sensing prokaryotic mRNA signifies microbial viability and promotes immunity"

Article Title: Sensing prokaryotic mRNA signifies microbial viability and promotes immunity

Journal: Nature

doi: 10.1038/nature10072

Bacterial messenger RNA constitutes an active vita -PAMP a–c and e–g . LDH, IL-1β and IL-6 at 24h. a. Total EC RNA treated with RNAse I and RNAse III, RNAse III alone, or DNAse prior to stimulation of BMDC. b. BMM treated with viable or HKEC, or HKEC with 0.1µg/ml of different bacterial RNA; ribosomal (rRNA), messenger (mRNA), small (sRNA) or eukaryotic RNA (eukRNA). c. BMDC responses. Gro-RNA; in vitro transcribed EC Gro-operon RNA. d. Predicted secondary structure of Gro-RNA. Colour code indicates base pairing probability. e. BMM treated with in vitro transcribed Gro-RNA or Gro dsRNA alone or with HKEC. f. BMDC responses. Era-RNA and DNApol-RNA; in vitro transcribed EC Era-GTPase and DNA-polymerase-III RNA, respectively. g . BMM treated with different doses of unmodified (control), or modified Gro-RNA with HKEC (5’cap, 5’ m 7 G capping; CIP, calf intestinal phosphatase; 3’poly(A), 3’-polyadenylation). a–g. #, ‘not detected’, all RNA at 10µg/ml except as noted, data represent ≥5 experiments. h. Mice vaccinated and boosted twice with viable EC, HKEC or HKEC with 30µg total purified bacterial RNA (HKEC+RNA) (vaccination regimen in suppl. Fig. 22 ). Class-specific anti- E. coli antibody serum titers at 25 days. *; p≤0.05, **; p≤0.01, ***; p≤0.001. All bars represent mean ± s.e.m.
Figure Legend Snippet: Bacterial messenger RNA constitutes an active vita -PAMP a–c and e–g . LDH, IL-1β and IL-6 at 24h. a. Total EC RNA treated with RNAse I and RNAse III, RNAse III alone, or DNAse prior to stimulation of BMDC. b. BMM treated with viable or HKEC, or HKEC with 0.1µg/ml of different bacterial RNA; ribosomal (rRNA), messenger (mRNA), small (sRNA) or eukaryotic RNA (eukRNA). c. BMDC responses. Gro-RNA; in vitro transcribed EC Gro-operon RNA. d. Predicted secondary structure of Gro-RNA. Colour code indicates base pairing probability. e. BMM treated with in vitro transcribed Gro-RNA or Gro dsRNA alone or with HKEC. f. BMDC responses. Era-RNA and DNApol-RNA; in vitro transcribed EC Era-GTPase and DNA-polymerase-III RNA, respectively. g . BMM treated with different doses of unmodified (control), or modified Gro-RNA with HKEC (5’cap, 5’ m 7 G capping; CIP, calf intestinal phosphatase; 3’poly(A), 3’-polyadenylation). a–g. #, ‘not detected’, all RNA at 10µg/ml except as noted, data represent ≥5 experiments. h. Mice vaccinated and boosted twice with viable EC, HKEC or HKEC with 30µg total purified bacterial RNA (HKEC+RNA) (vaccination regimen in suppl. Fig. 22 ). Class-specific anti- E. coli antibody serum titers at 25 days. *; p≤0.05, **; p≤0.01, ***; p≤0.001. All bars represent mean ± s.e.m.

Techniques Used: In Vitro, Modification, Mouse Assay, Purification

17) Product Images from "Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells"

Article Title: Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6606018

Knockdown of BRG1 upregulates PTEN levels in DLD-1 cells. ( A ) Results of real-time quantitative RT–PCR analysis. The cyclophilin mRNA levels were examined as a quality and quantity of control of mRNA. * P
Figure Legend Snippet: Knockdown of BRG1 upregulates PTEN levels in DLD-1 cells. ( A ) Results of real-time quantitative RT–PCR analysis. The cyclophilin mRNA levels were examined as a quality and quantity of control of mRNA. * P

Techniques Used: Quantitative RT-PCR

18) Product Images from "MicroRNA Networks in Mouse Lung Organogenesis"

Article Title: MicroRNA Networks in Mouse Lung Organogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0010854

Visualization of chromosome localization of mouse miRNAs. Chromosome localization of (A) total 521 mouse miRNAs (Sanger version 10.1) and (B) 117 significant miRNAs in different clusters (marked in different colors) involved in lung development. The scale is given to make those miRNAs having the same or very close chromosome location displayed in a vertical line. (C) Numbers of direct mRNA targets for each miRNA detected by miRNA/mRNA correlation. 30 miRNAs with multiple direct targets are annotated. MiRNAs within clusters are boxed. (D) Numbers of direct protein targets detected for each miRNA by miRNA/protein correlation. The top 30 miRNAs are annotated.
Figure Legend Snippet: Visualization of chromosome localization of mouse miRNAs. Chromosome localization of (A) total 521 mouse miRNAs (Sanger version 10.1) and (B) 117 significant miRNAs in different clusters (marked in different colors) involved in lung development. The scale is given to make those miRNAs having the same or very close chromosome location displayed in a vertical line. (C) Numbers of direct mRNA targets for each miRNA detected by miRNA/mRNA correlation. 30 miRNAs with multiple direct targets are annotated. MiRNAs within clusters are boxed. (D) Numbers of direct protein targets detected for each miRNA by miRNA/protein correlation. The top 30 miRNAs are annotated.

Techniques Used:

Scatter plots of miRNA and mRNA data by Principle Component Analysis (PCA). (A) Expression array data for mRNA. (B) Expression array data for miRNA. Samples are colored by different mouse lung development time points. The same color represents the replicate samples.
Figure Legend Snippet: Scatter plots of miRNA and mRNA data by Principle Component Analysis (PCA). (A) Expression array data for mRNA. (B) Expression array data for miRNA. Samples are colored by different mouse lung development time points. The same color represents the replicate samples.

Techniques Used: Expressing

19) Product Images from "The m6A reader Ythdf restricts axonal growth in Drosophila through target selection modulation of the Fragile X mental retardation protein"

Article Title: The m6A reader Ythdf restricts axonal growth in Drosophila through target selection modulation of the Fragile X mental retardation protein

Journal: bioRxiv

doi: 10.1101/2020.03.04.976886

Fmr1 and Ythdf physically interact and share common targets (A) Heatmap indicating the normalized forward versus inverted reverse experiment enrichments on a log 2 scale of quantitative proteomics upon pulldown of Flag-tagged Ythdf in S2R+ cells. The threshold was set to a 1-fold enrichment (dashed line). Fmr1 is co-purified with Ythdf. Known Fmr1 interacting proteins are highlighted in bold (B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with RNase T1 as indicated to remove interactions enabled by RNA. (C) GST pulldown of recombinant GST-Ythdf or GST alone mixed with recombinant 6xHis-NT-Fmr1. The purified proteins were analyzed by coomassie staining and immunoblotting using α-Ythdf and α-Fmr1 antibodies. (D) Overlap of miCLIP dataset generated in S2R+ cells defining m 6 A modified transcripts with Fmr1 and Ythdf mRNA targets determined by TRIBE in S2R+ cells. (E-F) Pulldown of biotinylated RNA probes of repetitive GGACU sequences (E) or AAACU sequences (F) incubated with E. coli lysate expressing recombinant GST-Ythdf or/and 6xHis-NT-Fmr1. Pulled down proteins were analyzed by immunoblotting using α-Ythdf and α-Fmr1 antibodies.
Figure Legend Snippet: Fmr1 and Ythdf physically interact and share common targets (A) Heatmap indicating the normalized forward versus inverted reverse experiment enrichments on a log 2 scale of quantitative proteomics upon pulldown of Flag-tagged Ythdf in S2R+ cells. The threshold was set to a 1-fold enrichment (dashed line). Fmr1 is co-purified with Ythdf. Known Fmr1 interacting proteins are highlighted in bold (B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with RNase T1 as indicated to remove interactions enabled by RNA. (C) GST pulldown of recombinant GST-Ythdf or GST alone mixed with recombinant 6xHis-NT-Fmr1. The purified proteins were analyzed by coomassie staining and immunoblotting using α-Ythdf and α-Fmr1 antibodies. (D) Overlap of miCLIP dataset generated in S2R+ cells defining m 6 A modified transcripts with Fmr1 and Ythdf mRNA targets determined by TRIBE in S2R+ cells. (E-F) Pulldown of biotinylated RNA probes of repetitive GGACU sequences (E) or AAACU sequences (F) incubated with E. coli lysate expressing recombinant GST-Ythdf or/and 6xHis-NT-Fmr1. Pulled down proteins were analyzed by immunoblotting using α-Ythdf and α-Fmr1 antibodies.

Techniques Used: Purification, Immunoprecipitation, Expressing, FLAG-tag, Recombinant, Staining, Generated, Modification, Incubation

20) Product Images from "miR-744 enhances type I interferon signaling pathway by targeting PTP1B in primary human renal mesangial cells"

Article Title: miR-744 enhances type I interferon signaling pathway by targeting PTP1B in primary human renal mesangial cells

Journal: Scientific Reports

doi: 10.1038/srep12987

miR-744 targets PTP1B , which is responsible for its regulation of type I IFN signaling pathway. ( A ) Schematic diagram of potential miR-744 binding sites in the 3′-UTR of PTP1B , predicted by RNAhybrid, and two mutant binding sites. Mutant 1 abolished the binding to miR-744 without changing the nucleotide composition of the sequence, while mutant 2 affected both the nucleotide composition of the sequence and the binding of the 3′-UTR to miR-744. ( B ) RMCs were simultaneously transfected with NC or miR-744 (200 nM) and the PTP1B 3′-UTR-containing vector (50 ng per well) or pSicheck2 vector (50 ng per well). Luciferase activity was measured 24 h after transfection. ( C ) RMCs were simultaneously transfected with NC or miR-744 (200 nM) and the PTP1B 3′-UTR-containing vector (50 ng per well) or PTP1B 3′-UTR mutant vector (50 ng per well). Luciferase activity was measured 24 h after transfection, quantified, and expressed as relative luciferase activity. ( D ) RMCs were transfected with miR-744 mimic or inhibitor and the corresponding control mimic or inhibitor for either 24 h for the mimics (left) or for 48 h for the inhibitors (right). The levels of PTP1B mRNA were detected after stimulation with type I IFN for 6 h. ( E ) RMCs were transfected with miR-744 mimic or inhibitor and the corresponding control mimic or inhibitor for either 24 h for the mimics or for 48 h for the inhibitors. PTP1B was detected in the whole-cell lysates with western blotting. The ratios of PTP1B to β-tubulin in the control-transfected cells was arbitrarily set at 1(The full-length blots/gels of PTP1B are presented in Supplementary Fig. S11 , Fig. S12 , respectively). ( F ) Induction of CCL5 after PTP1B was silenced with siRNA (200 nM) in RMCs. ( G ) Efficiency of siRNA measured with real-time PCR. ( H ) Western blotting analysis of the phosphorylation of STAT1 and STAT3 in PTP1B-silenced RMCs. Cells were treated with type I IFN (1000 U/mL) for the indicated times (The full-length blots/gels of STAT1 and STAT3 are presented in Supplementary Fig. S13 , Fig. S14 , respectively). At least three independent experiments were performed. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. NS, not significant.
Figure Legend Snippet: miR-744 targets PTP1B , which is responsible for its regulation of type I IFN signaling pathway. ( A ) Schematic diagram of potential miR-744 binding sites in the 3′-UTR of PTP1B , predicted by RNAhybrid, and two mutant binding sites. Mutant 1 abolished the binding to miR-744 without changing the nucleotide composition of the sequence, while mutant 2 affected both the nucleotide composition of the sequence and the binding of the 3′-UTR to miR-744. ( B ) RMCs were simultaneously transfected with NC or miR-744 (200 nM) and the PTP1B 3′-UTR-containing vector (50 ng per well) or pSicheck2 vector (50 ng per well). Luciferase activity was measured 24 h after transfection. ( C ) RMCs were simultaneously transfected with NC or miR-744 (200 nM) and the PTP1B 3′-UTR-containing vector (50 ng per well) or PTP1B 3′-UTR mutant vector (50 ng per well). Luciferase activity was measured 24 h after transfection, quantified, and expressed as relative luciferase activity. ( D ) RMCs were transfected with miR-744 mimic or inhibitor and the corresponding control mimic or inhibitor for either 24 h for the mimics (left) or for 48 h for the inhibitors (right). The levels of PTP1B mRNA were detected after stimulation with type I IFN for 6 h. ( E ) RMCs were transfected with miR-744 mimic or inhibitor and the corresponding control mimic or inhibitor for either 24 h for the mimics or for 48 h for the inhibitors. PTP1B was detected in the whole-cell lysates with western blotting. The ratios of PTP1B to β-tubulin in the control-transfected cells was arbitrarily set at 1(The full-length blots/gels of PTP1B are presented in Supplementary Fig. S11 , Fig. S12 , respectively). ( F ) Induction of CCL5 after PTP1B was silenced with siRNA (200 nM) in RMCs. ( G ) Efficiency of siRNA measured with real-time PCR. ( H ) Western blotting analysis of the phosphorylation of STAT1 and STAT3 in PTP1B-silenced RMCs. Cells were treated with type I IFN (1000 U/mL) for the indicated times (The full-length blots/gels of STAT1 and STAT3 are presented in Supplementary Fig. S13 , Fig. S14 , respectively). At least three independent experiments were performed. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. NS, not significant.

Techniques Used: Binding Assay, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction

21) Product Images from "Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures"

Article Title: Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2014.00012

Efficiency of cold jet to remove neuronal mRNA from co-cultures. (A) Samples from neuron-astrocyte co-cultures, with or without treatment with cold jet, were collected and mRNA levels for neuronal mRNAs were analyzed using microarray. Shown are 40 of the most down-regulated transcripts in co-culture as a result of the cold jet procedure. Each individual gene expression level was normalized (see Methods) and plotted on a Log2 scale, green representing decreased mRNA levels in CC-CJ samples compared to CC alone (B) Normalized fluorescence levels were compared and expressed as percentage of the levels present in co-culture samples. Error bars represent standard error of the mean (SEM).
Figure Legend Snippet: Efficiency of cold jet to remove neuronal mRNA from co-cultures. (A) Samples from neuron-astrocyte co-cultures, with or without treatment with cold jet, were collected and mRNA levels for neuronal mRNAs were analyzed using microarray. Shown are 40 of the most down-regulated transcripts in co-culture as a result of the cold jet procedure. Each individual gene expression level was normalized (see Methods) and plotted on a Log2 scale, green representing decreased mRNA levels in CC-CJ samples compared to CC alone (B) Normalized fluorescence levels were compared and expressed as percentage of the levels present in co-culture samples. Error bars represent standard error of the mean (SEM).

Techniques Used: Microarray, Co-Culture Assay, Expressing, Fluorescence

Microarray analysis of strongest regulated neuron-responsive astrocyte genes. Shown are 36 of the most up – and down-regulated transcripts in astrocytes when co-cultured with neurons, which were subsequent removed by cold jet treatment (CC-CJ), compared to astrocytes cultured alone (AA-CJ). Each individual gene expression level was normalized (see methods) and plotted on a Log2 scale with red representing increased and green decreased mRNA levels in CC-CJ astrocytes compared to astrocytes cultured alone (AA-CJ).
Figure Legend Snippet: Microarray analysis of strongest regulated neuron-responsive astrocyte genes. Shown are 36 of the most up – and down-regulated transcripts in astrocytes when co-cultured with neurons, which were subsequent removed by cold jet treatment (CC-CJ), compared to astrocytes cultured alone (AA-CJ). Each individual gene expression level was normalized (see methods) and plotted on a Log2 scale with red representing increased and green decreased mRNA levels in CC-CJ astrocytes compared to astrocytes cultured alone (AA-CJ).

Techniques Used: Microarray, Cell Culture, Expressing

22) Product Images from "The caveolin–cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle"

Article Title: The caveolin–cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201501046

Caveolae are essential for maintaining muscle integrity in the zebrafish. (A) Temporal expression of cavin-1a mRNA in 1.3–72-hpf zebrafish embryos. (B) Expression pattern of cavin-1a in zebrafish embryos using whole-mount mRNA in situ hybridization. Dorsal view of a 12-somite (12S) embryo shows faint labeling for cavin-1a (arrowheads). At 48 hpf, cavin-1a expression is observed exclusively within the zebrafish myotomes, shown here in dorsal (top right) and lateral (bottom left) view; anterior to left in both images. Note the lack of notochord labeling in the dorsal view. Image in bottom right represents magnification of boxed area. (C) WT and tp53 zdf1 zebrafish embryos were injected with control or cavin1a MO (CtrMO and cavin1a MO, respectively) and abnormal morphants (classified as mild or severe) imaged at 72 hpf. Arrowheads indicate cardiac edema. (D) Ruthenium red–labeled isolated muscle fibers from control MO and cavin1a MO embryos. Arrows indicate caveolae (left image) or endosomes (middle and right images). Inset represents boxed area. (E) Relative caveolae density of muscle fibers from cavin1a MO embryos was 1.4 ± 2.0% (means ± SD), compared with muscle fibers from control MO embryos. Quantitation performed on four control MO and six cavin1a MO muscle fibers. (F) EBD uptake in 96 hpf Tg(actb2:EGFP-CAAX) pc10 embryos expressing EGFP-CAAX injected with control MO or cavin1a MO and incubated in 3% MC. Inset shows higher magnification of EGFP-CAAX/EBD-positive muscle fibers. (G) 0.0 ± 0.0% of control MO and cavin1a MO embryos were EBD positive after incubation in E3 (means ± SEM; 28 control MO and 47 cavin1a MO embryos from three separate microinjections). 0.8 ± 0.8% of control MO and 17.0 ± 7.0% of cavin1a MO embryos were EBD positive after incubation in 3% MC (means ± SEM; 105 control MO and 111 cavin1a MO embryos from eight separate microinjections). (H) EBD uptake in 96 hpf embryos expressing Cav3-WT-GFP (WT) or Cav3-R26Q-GFP (R26Q) after incubation in 3% MC. Inset shows higher magnification of GFP/EBD-positive muscle fibers. (I) 0.8 ± 0.8% of WT and 2.1 ± 2.1% R26Q embryos were EBD positive after incubation in E3 (means ± SEM; 71 WT and 33 R26Q embryos from five and four clutches, respectively). 7.1 ± 2.6% of WT and 31.5 ± 5.9% R26Q embryos were EBD positive after incubation in 3% MC (130 WT and 126 R26Q embryos from seven and six clutches, respectively). Uptake was performed in two separate founder lines for both WT and R26Q to ensure that phenotypes observed were not caused by Tol2 integration sites. *, P ≤ 0.05; **, P ≤ 0.01. ns, not significant. Bars: (B and C) 200 µm; (D, main images) 1 µm; (D, inset) 200 nm; (F and H, main images and insets) 50 µm.
Figure Legend Snippet: Caveolae are essential for maintaining muscle integrity in the zebrafish. (A) Temporal expression of cavin-1a mRNA in 1.3–72-hpf zebrafish embryos. (B) Expression pattern of cavin-1a in zebrafish embryos using whole-mount mRNA in situ hybridization. Dorsal view of a 12-somite (12S) embryo shows faint labeling for cavin-1a (arrowheads). At 48 hpf, cavin-1a expression is observed exclusively within the zebrafish myotomes, shown here in dorsal (top right) and lateral (bottom left) view; anterior to left in both images. Note the lack of notochord labeling in the dorsal view. Image in bottom right represents magnification of boxed area. (C) WT and tp53 zdf1 zebrafish embryos were injected with control or cavin1a MO (CtrMO and cavin1a MO, respectively) and abnormal morphants (classified as mild or severe) imaged at 72 hpf. Arrowheads indicate cardiac edema. (D) Ruthenium red–labeled isolated muscle fibers from control MO and cavin1a MO embryos. Arrows indicate caveolae (left image) or endosomes (middle and right images). Inset represents boxed area. (E) Relative caveolae density of muscle fibers from cavin1a MO embryos was 1.4 ± 2.0% (means ± SD), compared with muscle fibers from control MO embryos. Quantitation performed on four control MO and six cavin1a MO muscle fibers. (F) EBD uptake in 96 hpf Tg(actb2:EGFP-CAAX) pc10 embryos expressing EGFP-CAAX injected with control MO or cavin1a MO and incubated in 3% MC. Inset shows higher magnification of EGFP-CAAX/EBD-positive muscle fibers. (G) 0.0 ± 0.0% of control MO and cavin1a MO embryos were EBD positive after incubation in E3 (means ± SEM; 28 control MO and 47 cavin1a MO embryos from three separate microinjections). 0.8 ± 0.8% of control MO and 17.0 ± 7.0% of cavin1a MO embryos were EBD positive after incubation in 3% MC (means ± SEM; 105 control MO and 111 cavin1a MO embryos from eight separate microinjections). (H) EBD uptake in 96 hpf embryos expressing Cav3-WT-GFP (WT) or Cav3-R26Q-GFP (R26Q) after incubation in 3% MC. Inset shows higher magnification of GFP/EBD-positive muscle fibers. (I) 0.8 ± 0.8% of WT and 2.1 ± 2.1% R26Q embryos were EBD positive after incubation in E3 (means ± SEM; 71 WT and 33 R26Q embryos from five and four clutches, respectively). 7.1 ± 2.6% of WT and 31.5 ± 5.9% R26Q embryos were EBD positive after incubation in 3% MC (130 WT and 126 R26Q embryos from seven and six clutches, respectively). Uptake was performed in two separate founder lines for both WT and R26Q to ensure that phenotypes observed were not caused by Tol2 integration sites. *, P ≤ 0.05; **, P ≤ 0.01. ns, not significant. Bars: (B and C) 200 µm; (D, main images) 1 µm; (D, inset) 200 nm; (F and H, main images and insets) 50 µm.

Techniques Used: Expressing, In Situ Hybridization, Labeling, Injection, Isolation, Quantitation Assay, Incubation

23) Product Images from "The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma"

Article Title: The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma

Journal: Nature genetics

doi: 10.1038/ng.2938

ACVR1 mutations in DIPG activate BMP signaling a. Missense ACVR1 substitutions in DIPG were clustered in the glycine/serine rich domain (G/S) or kinase domain. Each red circle indicates a DIPG carrying the specified mutation, and an * indicates mutations previously found as germline mutations in individuals with FOP. The extracellular domain (EC) and transmembrane domain (TM) did not contain mutations. b. ACVR1 mutations ventralize zebrafish embryos. Graph shows the percentage of embryos exhibiting a dorsalized or ventralized phenotype. Embryos injected with wild-type ACVR1 mRNA (WT) showed a dorsalized phenotype, while embryos injected with mutant ACVR1 mRNA showed a ventralized phenotype (increasing severity from left to right). R258G had the least severe effect, resulting only in the V3-V4 ventralized phenotype, whereas G328V had the most severe effect with 90% of embryos showing the V5 ventralized phenotype. The number of embryos examined is shown on top. c. Representative phenotype images of zebrafish embryos injected with the indicated ACVR1 mRNA. Untreated mutants R258G, G328E, G328W, and R206H have little to no dorsal structures, and G356D and G328V are more severely affected. Treatment with LDN-193189 (LDN) reversed the ventralization effects in the ACVR1 mutants, as can be seen by the partial rescue of dorsal structures (i.e. head) for R258G (100%, n=20), G328E (83%, n=23), G328W (100%, n=20), R206H (100%, n=27), and the reduced severity of ventralization without the formation of dorsal structures for G356D and G328V. Scale bar is 200 μm. d. ACVR1 mutations drive increased levels of phospho-SMAD1/5 in primary astrocyte cultures. Western blots from lysates of primary astrocytes isolated from brainstem of neonatal Tp53 conditional knockout mice, transduced with retroviruses expressing FLAG-tagged ACVR1 wild-type, or indicated mutants, and serum starved for 2 hours. Quantitation of the ratio of phospho-SMAD/Total SMAD normalized to the empty vector control is shown below.
Figure Legend Snippet: ACVR1 mutations in DIPG activate BMP signaling a. Missense ACVR1 substitutions in DIPG were clustered in the glycine/serine rich domain (G/S) or kinase domain. Each red circle indicates a DIPG carrying the specified mutation, and an * indicates mutations previously found as germline mutations in individuals with FOP. The extracellular domain (EC) and transmembrane domain (TM) did not contain mutations. b. ACVR1 mutations ventralize zebrafish embryos. Graph shows the percentage of embryos exhibiting a dorsalized or ventralized phenotype. Embryos injected with wild-type ACVR1 mRNA (WT) showed a dorsalized phenotype, while embryos injected with mutant ACVR1 mRNA showed a ventralized phenotype (increasing severity from left to right). R258G had the least severe effect, resulting only in the V3-V4 ventralized phenotype, whereas G328V had the most severe effect with 90% of embryos showing the V5 ventralized phenotype. The number of embryos examined is shown on top. c. Representative phenotype images of zebrafish embryos injected with the indicated ACVR1 mRNA. Untreated mutants R258G, G328E, G328W, and R206H have little to no dorsal structures, and G356D and G328V are more severely affected. Treatment with LDN-193189 (LDN) reversed the ventralization effects in the ACVR1 mutants, as can be seen by the partial rescue of dorsal structures (i.e. head) for R258G (100%, n=20), G328E (83%, n=23), G328W (100%, n=20), R206H (100%, n=27), and the reduced severity of ventralization without the formation of dorsal structures for G356D and G328V. Scale bar is 200 μm. d. ACVR1 mutations drive increased levels of phospho-SMAD1/5 in primary astrocyte cultures. Western blots from lysates of primary astrocytes isolated from brainstem of neonatal Tp53 conditional knockout mice, transduced with retroviruses expressing FLAG-tagged ACVR1 wild-type, or indicated mutants, and serum starved for 2 hours. Quantitation of the ratio of phospho-SMAD/Total SMAD normalized to the empty vector control is shown below.

Techniques Used: Mutagenesis, Injection, Western Blot, Isolation, Knock-Out, Mouse Assay, Transduction, Expressing, Quantitation Assay, Plasmid Preparation

24) Product Images from "MiR-221 promotes stemness of breast cancer cells by targeting DNMT3b"

Article Title: MiR-221 promotes stemness of breast cancer cells by targeting DNMT3b

Journal: Oncotarget

doi:

DNMT3b is a direct target of miR-221 A. Predicted alignment between the miR-221 sequence and the 3′UTR of DNMT3b . Luciferase assay showed that reporter activity was inhibited in T47D cells only in the presence of wild type DNMT3b and not with a mutated 3′UTR. The data represent the results of two independent experiments. B. MiR-221 transfection downregulated DNMT3b mRNA and protein levels, as assessed by qRT-PCR and Western blotting. C. Anti-miR-221 transfection upregulated the levels of DNMT3b. D. DNMT3b mRNA and protein were downregulated in T47D stem cells compared to differentiated cells. E. DNMT3b mRNA and protein were downregulated in T47D cells stably infected with a miR-221 lentivirus. In B, C, D, E data are mean values ± SD from three independent experiments. Significance was calculated using Student's t -test. *, p
Figure Legend Snippet: DNMT3b is a direct target of miR-221 A. Predicted alignment between the miR-221 sequence and the 3′UTR of DNMT3b . Luciferase assay showed that reporter activity was inhibited in T47D cells only in the presence of wild type DNMT3b and not with a mutated 3′UTR. The data represent the results of two independent experiments. B. MiR-221 transfection downregulated DNMT3b mRNA and protein levels, as assessed by qRT-PCR and Western blotting. C. Anti-miR-221 transfection upregulated the levels of DNMT3b. D. DNMT3b mRNA and protein were downregulated in T47D stem cells compared to differentiated cells. E. DNMT3b mRNA and protein were downregulated in T47D cells stably infected with a miR-221 lentivirus. In B, C, D, E data are mean values ± SD from three independent experiments. Significance was calculated using Student's t -test. *, p

Techniques Used: Sequencing, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Stable Transfection, Infection

25) Product Images from "RIPK3 Is Largely Dispensable for RIG-I-Like Receptor- and Type I Interferon-Driven Transcriptional Responses to Influenza A Virus in Murine Fibroblasts"

Article Title: RIPK3 Is Largely Dispensable for RIG-I-Like Receptor- and Type I Interferon-Driven Transcriptional Responses to Influenza A Virus in Murine Fibroblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0158774

Modest post-transcriptional role for RIPK3 in production of IFN-β upon RLR stimulation. (A) Ripk3 +/+ or ripk3 -/- MEFs were infected with WT PR8 (m.o.i. = 2), PR8-ΔNS1 (m.o.i. = 1), or transfected with virus mimetic poly(I:C) for the indicated times, and IFN-β in supernatants of cultured cells was quantified by ELISA. Ripk3 +/+ MEFs were infected with PR8-ΔNS1 in the presence or absence of the RIPK3 kinase inhibitor GSK’872 (5μM), for the indicated times, and secretion of IFN-β in supernatants of cell culture was quantified by ELISA. (B) IFN-α in supernatants of cell culture was also quantified by ELISA. (C) Ripk3 +/+ MEFs treated with or without RIPK3 inhibitor (GSK’872, 5μM), or ripk3 -/- MEFs, were infected with PR8-ΔNS1 (m.o.i. = 1), for the indicated times, and ifnb1 mRNA levels determined from DNA microarray data output. Fold-induction was calculated using basal levels (time = 0) of each condition as baseline, which was normalized to 1. Error bars represent mean +/- S.D. NS = not statistically significant; ** p
Figure Legend Snippet: Modest post-transcriptional role for RIPK3 in production of IFN-β upon RLR stimulation. (A) Ripk3 +/+ or ripk3 -/- MEFs were infected with WT PR8 (m.o.i. = 2), PR8-ΔNS1 (m.o.i. = 1), or transfected with virus mimetic poly(I:C) for the indicated times, and IFN-β in supernatants of cultured cells was quantified by ELISA. Ripk3 +/+ MEFs were infected with PR8-ΔNS1 in the presence or absence of the RIPK3 kinase inhibitor GSK’872 (5μM), for the indicated times, and secretion of IFN-β in supernatants of cell culture was quantified by ELISA. (B) IFN-α in supernatants of cell culture was also quantified by ELISA. (C) Ripk3 +/+ MEFs treated with or without RIPK3 inhibitor (GSK’872, 5μM), or ripk3 -/- MEFs, were infected with PR8-ΔNS1 (m.o.i. = 1), for the indicated times, and ifnb1 mRNA levels determined from DNA microarray data output. Fold-induction was calculated using basal levels (time = 0) of each condition as baseline, which was normalized to 1. Error bars represent mean +/- S.D. NS = not statistically significant; ** p

Techniques Used: Infection, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Microarray

26) Product Images from "Over-Expressed miR-224 Promotes the Progression of Cervical Cancer via Targeting RASSF8"

Article Title: Over-Expressed miR-224 Promotes the Progression of Cervical Cancer via Targeting RASSF8

Journal: PLoS ONE

doi: 10.1371/journal.pone.0162378

Protein and mRNA levels of RASSF8 were decreased in cervical cancer tissues. (A) Protein level of RASSF8 in normal cervical epithelium and cervical cancer specimens was measured by IHC. The positive staining of RASSF8 was in the nuclear and more strongly in normal cervical epithelium. The expression levels of RASSF8 were determined by assessing its staining using software image pro-plus 6.0. The results were showed as integrated optical density (IOD)/area. Bar represented the average values of corresponding protein levels in normal cervical epithelium and cancer tumors ±s.e. (B) Real-time PCR validated the mRNA levels of RASSF8 in cervical cancer tissues compared with tht in normal cervical tissues. Mean ± s.e. **P
Figure Legend Snippet: Protein and mRNA levels of RASSF8 were decreased in cervical cancer tissues. (A) Protein level of RASSF8 in normal cervical epithelium and cervical cancer specimens was measured by IHC. The positive staining of RASSF8 was in the nuclear and more strongly in normal cervical epithelium. The expression levels of RASSF8 were determined by assessing its staining using software image pro-plus 6.0. The results were showed as integrated optical density (IOD)/area. Bar represented the average values of corresponding protein levels in normal cervical epithelium and cancer tumors ±s.e. (B) Real-time PCR validated the mRNA levels of RASSF8 in cervical cancer tissues compared with tht in normal cervical tissues. Mean ± s.e. **P

Techniques Used: Immunohistochemistry, Staining, Expressing, Software, Real-time Polymerase Chain Reaction

miR-224 suppressed RASSF8 expression and directly targeted 3'UTR of RASSF8 mRNA. (A,B) At 72h after transfected with 100 nM of miRNA mimic and negative control, the endogenous protein levels of RASSF8 in SiHa and CaSki cells were measured by western blot. Bars indicated the relative protein levels that were normalized to GAPDH. Data were presented as mean±s.d. (n¼3) *P
Figure Legend Snippet: miR-224 suppressed RASSF8 expression and directly targeted 3'UTR of RASSF8 mRNA. (A,B) At 72h after transfected with 100 nM of miRNA mimic and negative control, the endogenous protein levels of RASSF8 in SiHa and CaSki cells were measured by western blot. Bars indicated the relative protein levels that were normalized to GAPDH. Data were presented as mean±s.d. (n¼3) *P

Techniques Used: Expressing, Transfection, Negative Control, Western Blot

27) Product Images from "CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages"

Article Title: CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20141836

CCR2 signaling regulates CCL3 expression in MAMs. (A) Levels of Ccl3 mRNA (left) and CCL3 protein (right) in BMDMs isolated from WT or Ccr2 −/− mice were assessed by RT-PCR ( n = 6 per genotype, 6 independent experiments) and ELISA ( n = 3 per genotype, two independent experiments). Data are means ± SEM. *, P
Figure Legend Snippet: CCR2 signaling regulates CCL3 expression in MAMs. (A) Levels of Ccl3 mRNA (left) and CCL3 protein (right) in BMDMs isolated from WT or Ccr2 −/− mice were assessed by RT-PCR ( n = 6 per genotype, 6 independent experiments) and ELISA ( n = 3 per genotype, two independent experiments). Data are means ± SEM. *, P

Techniques Used: Expressing, Micro-arrays for Mass Spectrometry, Isolation, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

The CCL3–CCR1 axis is required for accumulation of MAMs and after extravasation of cancer cells. (A) Levels of chemokine receptors were assessed by flow cytometry in circulating inflammatory monocytes (IMs) and lung MAMs, as indicated. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection ( n = 3). Dotted lines and shaded areas show control isotype matched IgG for each cell type. Representative histograms from three independent experiments are shown. (B) Levels of Ccr1 and Ccr5 mRNA were assessed in IMs, MAMs, and E0771-LG cancer cells. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection ( n = 4 ) . Data are means ± SEM. *, P
Figure Legend Snippet: The CCL3–CCR1 axis is required for accumulation of MAMs and after extravasation of cancer cells. (A) Levels of chemokine receptors were assessed by flow cytometry in circulating inflammatory monocytes (IMs) and lung MAMs, as indicated. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection ( n = 3). Dotted lines and shaded areas show control isotype matched IgG for each cell type. Representative histograms from three independent experiments are shown. (B) Levels of Ccr1 and Ccr5 mRNA were assessed in IMs, MAMs, and E0771-LG cancer cells. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection ( n = 4 ) . Data are means ± SEM. *, P

Techniques Used: Micro-arrays for Mass Spectrometry, Flow Cytometry, Cytometry, Isolation, Mouse Assay, Injection

28) Product Images from "SOX9 Regulates Multiple Genes in Chondrocytes, Including Genes Encoding ECM Proteins, ECM Modification Enzymes, Receptors, and Transporters"

Article Title: SOX9 Regulates Multiple Genes in Chondrocytes, Including Genes Encoding ECM Proteins, ECM Modification Enzymes, Receptors, and Transporters

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107577

Decrease of Sox9 expression in mouse Sox9 flox/flox primary rib chondrocytes infected with Ad-CMV -Cre . Primary rib chondrocytes obtained from 4-day-old Sox9 flox/flox mice were cultured for 24 hours and then infected with Ad-CMV -Cre or control vector Ad-CMV-Null at 200 moi. In these cells, exon 2 through exon 3 of the Sox9 gene was flanked with loxP sites. Forty-eight hours after infection, the Sox9 mRNA levels in cells infected with Ad-CMV-Cre, measured by RT-qPCR, were decreased by more than 8-fold compared to the levels in cells infected with Ad-CMV-Null.
Figure Legend Snippet: Decrease of Sox9 expression in mouse Sox9 flox/flox primary rib chondrocytes infected with Ad-CMV -Cre . Primary rib chondrocytes obtained from 4-day-old Sox9 flox/flox mice were cultured for 24 hours and then infected with Ad-CMV -Cre or control vector Ad-CMV-Null at 200 moi. In these cells, exon 2 through exon 3 of the Sox9 gene was flanked with loxP sites. Forty-eight hours after infection, the Sox9 mRNA levels in cells infected with Ad-CMV-Cre, measured by RT-qPCR, were decreased by more than 8-fold compared to the levels in cells infected with Ad-CMV-Null.

Techniques Used: Expressing, Infection, Mouse Assay, Cell Culture, Plasmid Preparation, Quantitative RT-PCR

29) Product Images from "miRNA regulation of Sdf1 chemokine signaling provides genetic robustness to germ cell migration"

Article Title: miRNA regulation of Sdf1 chemokine signaling provides genetic robustness to germ cell migration

Journal: Nature genetics

doi: 10.1038/ng.758

Blocking miR-430-mediated repression of sdf1a and cxcr7b causes PGC mislocalization and expanded sdf1a expression. ( a ) Schematic representation of the experimental setup. Injecting the TP (purple) blocks miRNA-mediated repression, increasing mRNA expression and leading to mislocalization of cells. Co-injecting a morpholino to reduce translation of the target gene (red, AUG MO) rescues the mislocalization phenotype. The inset shows the region of the embryo depicted in the panels (b, d-h). ( b, d-g ) Whole mount in situ of nanos mRNA, labeling PGCs in 24 hpf embryos. Bracket shows correct localization of PGCs. Arrowheads identify mislocalized PGCs. ( c ) Quantification of the percentage of embryos with mislocalized PGCs in each experimental condition as indicated. A significantly increased number of TP-injected embryos have mislocalized PGCs (*, p=1.185 ×10 −7 , sdf1a-TP; p=2.52×10 −7 , cxcr7b-TP; two-sided Fisher’s exact test). Error bars show ± s.d. ( d, e ) Representative images of PGC mislocalization are shown. ( f, g ) Co-injection of a low level of the corresponding AUG MO rescues the TP phenotype ( sdf1a AUG MO, 0.01 pmol; cxcr7b AUG MO , 0.045 pmol). ( h ) In situ hybridization to detect sdf1a mRNA. The trunk of embryos at 20 hpf, 22 hpf, and 24 hpf are wild type, injected with sdf1a-TP, or MZ dicer . Brackets illustrate the extension of the sdf1a expression domain along the pronephric region. ( i ) qPCR for sdf1a in 24 hpf wild type, sdf1a-TP-injected embryos, and MZ dicer . An increase in expression was observed in the absence of miR-430-mediated repression. ( j ) Schematic summary of Sdf1a tail expression and the resulting PGC mislocalization.
Figure Legend Snippet: Blocking miR-430-mediated repression of sdf1a and cxcr7b causes PGC mislocalization and expanded sdf1a expression. ( a ) Schematic representation of the experimental setup. Injecting the TP (purple) blocks miRNA-mediated repression, increasing mRNA expression and leading to mislocalization of cells. Co-injecting a morpholino to reduce translation of the target gene (red, AUG MO) rescues the mislocalization phenotype. The inset shows the region of the embryo depicted in the panels (b, d-h). ( b, d-g ) Whole mount in situ of nanos mRNA, labeling PGCs in 24 hpf embryos. Bracket shows correct localization of PGCs. Arrowheads identify mislocalized PGCs. ( c ) Quantification of the percentage of embryos with mislocalized PGCs in each experimental condition as indicated. A significantly increased number of TP-injected embryos have mislocalized PGCs (*, p=1.185 ×10 −7 , sdf1a-TP; p=2.52×10 −7 , cxcr7b-TP; two-sided Fisher’s exact test). Error bars show ± s.d. ( d, e ) Representative images of PGC mislocalization are shown. ( f, g ) Co-injection of a low level of the corresponding AUG MO rescues the TP phenotype ( sdf1a AUG MO, 0.01 pmol; cxcr7b AUG MO , 0.045 pmol). ( h ) In situ hybridization to detect sdf1a mRNA. The trunk of embryos at 20 hpf, 22 hpf, and 24 hpf are wild type, injected with sdf1a-TP, or MZ dicer . Brackets illustrate the extension of the sdf1a expression domain along the pronephric region. ( i ) qPCR for sdf1a in 24 hpf wild type, sdf1a-TP-injected embryos, and MZ dicer . An increase in expression was observed in the absence of miR-430-mediated repression. ( j ) Schematic summary of Sdf1a tail expression and the resulting PGC mislocalization.

Techniques Used: Blocking Assay, Pyrolysis Gas Chromatography, Expressing, In Situ, Labeling, Injection, In Situ Hybridization, Real-time Polymerase Chain Reaction

miR-430 and Cxcr7b act in a functionally redundant manner to refine Sdf1a expression. ( a, d ) Quantification of PGC mislocalization. Embryos were injected at the one-cell stage with a morpholino targeting the start site (AUG MO) of cxcr7b (a) or sdf1a (d). These AUG MOs were injected at low concentrations, which were insufficient to completely knockdown the transcript and caused a weak mislocalization phenotype. (a) Co-injecting sdf1a-TP and cxcr7b AUG MO causes significantly more mismigration that injection of sdf1a-TP or the same amount of the AUG MO alone (*, p=4.08×10 −3 , sdf1a-TP + 45 fmol to 45 fmol alone; p=4.87×10 −3 , sdf1a-TP + 90 fmol to 90 fmol alone; two-tailed Fisher’s exact test), suggesting that miR-430 regulation of sdf1a mRNA can partially compensate for a reduction of cxcr7b . Similarly, co-injecting cxcr7b-TP and sdf1a AUG MO significantly enhances the mislocalization phenotype (*, p=8.93×10 −4 , cxcr7b-TP + 10 fmol to 10 fmol alone; p=0.016, cxcr7b-TP + 10 fmol to 10 fmol alone; two-tailed Fisher’s exact test), suggesting that regulation of cxcr7b by miR-430 prevents excessive clearance of the sdf1a . Data are shown as mean ± S.D. ( b, e ) nanos in situ at 24 hpf to visualize the location of germ cells. Brackets indicate correctly localized PGCs, and arrowheads show mislocalized cells. ( c, f ) Scheme representing the predicted effect of the experimental conditions on Sdf1a and Cxcr7b shown in panels (b and e). The added effect of removing miR-430 targeting and modulation by Cxcr7b supports a functional redundancy of miR-430 and Cxcr7b.
Figure Legend Snippet: miR-430 and Cxcr7b act in a functionally redundant manner to refine Sdf1a expression. ( a, d ) Quantification of PGC mislocalization. Embryos were injected at the one-cell stage with a morpholino targeting the start site (AUG MO) of cxcr7b (a) or sdf1a (d). These AUG MOs were injected at low concentrations, which were insufficient to completely knockdown the transcript and caused a weak mislocalization phenotype. (a) Co-injecting sdf1a-TP and cxcr7b AUG MO causes significantly more mismigration that injection of sdf1a-TP or the same amount of the AUG MO alone (*, p=4.08×10 −3 , sdf1a-TP + 45 fmol to 45 fmol alone; p=4.87×10 −3 , sdf1a-TP + 90 fmol to 90 fmol alone; two-tailed Fisher’s exact test), suggesting that miR-430 regulation of sdf1a mRNA can partially compensate for a reduction of cxcr7b . Similarly, co-injecting cxcr7b-TP and sdf1a AUG MO significantly enhances the mislocalization phenotype (*, p=8.93×10 −4 , cxcr7b-TP + 10 fmol to 10 fmol alone; p=0.016, cxcr7b-TP + 10 fmol to 10 fmol alone; two-tailed Fisher’s exact test), suggesting that regulation of cxcr7b by miR-430 prevents excessive clearance of the sdf1a . Data are shown as mean ± S.D. ( b, e ) nanos in situ at 24 hpf to visualize the location of germ cells. Brackets indicate correctly localized PGCs, and arrowheads show mislocalized cells. ( c, f ) Scheme representing the predicted effect of the experimental conditions on Sdf1a and Cxcr7b shown in panels (b and e). The added effect of removing miR-430 targeting and modulation by Cxcr7b supports a functional redundancy of miR-430 and Cxcr7b.

Techniques Used: Activated Clotting Time Assay, Expressing, Pyrolysis Gas Chromatography, Injection, Two Tailed Test, In Situ, Functional Assay

30) Product Images from "A New Anti-Depressive Strategy for the Elderly: Ablation of FKBP5/FKBP51"

Article Title: A New Anti-Depressive Strategy for the Elderly: Ablation of FKBP5/FKBP51

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024840

Distribution and confirmation of gene knockout. (A) Representative images of β-gal staining in horizontal brain slices of 5.5 and 20 month old FKBP5−/− mice. (B) Western blot analysis of 20-month old wildtype and FKBP5−/− whole brain homogenates. (C) FKBP5 primer-specific PCR of cDNA synthesized from brain-isolated mRNA by reverse transcription.
Figure Legend Snippet: Distribution and confirmation of gene knockout. (A) Representative images of β-gal staining in horizontal brain slices of 5.5 and 20 month old FKBP5−/− mice. (B) Western blot analysis of 20-month old wildtype and FKBP5−/− whole brain homogenates. (C) FKBP5 primer-specific PCR of cDNA synthesized from brain-isolated mRNA by reverse transcription.

Techniques Used: Gene Knockout, Staining, Mouse Assay, Western Blot, Polymerase Chain Reaction, Synthesized, Isolation

31) Product Images from "Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters"

Article Title: Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters

Journal: Cell Death & Disease

doi: 10.1038/cddis.2011.61

Breast epithelial cells have a heterogeneous expression of ABC transporters. ( a ) Representative graphs showing relative mRNA expression of ABC transporters in immortalized (MCF10A and HBL100), non-invasive (MCF7 and T47D), and invasive (MDAMB231 and MDAMB435) breast epithelial cell lines as analyzed by RT-PCR, n =3. ( b ) Representative graphs showing relative mRNA expression of ABC transporters in primary normal (NB), DCIS, and IDC breast samples as analyzed by RT-PCR. n =3 for NB, n =6 for DCIS, and n =3 for IDC samples
Figure Legend Snippet: Breast epithelial cells have a heterogeneous expression of ABC transporters. ( a ) Representative graphs showing relative mRNA expression of ABC transporters in immortalized (MCF10A and HBL100), non-invasive (MCF7 and T47D), and invasive (MDAMB231 and MDAMB435) breast epithelial cell lines as analyzed by RT-PCR, n =3. ( b ) Representative graphs showing relative mRNA expression of ABC transporters in primary normal (NB), DCIS, and IDC breast samples as analyzed by RT-PCR. n =3 for NB, n =6 for DCIS, and n =3 for IDC samples

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

32) Product Images from "Lineage specific composition of cyclin D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4, CDK6 and individual D-type cyclins in differentiating cells of embryonic origin"

Article Title: Lineage specific composition of cyclin D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4, CDK6 and individual D-type cyclins in differentiating cells of embryonic origin

Journal: Cell Proliferation

doi: 10.1111/j.1365-2184.2008.00556.x

Expression of D-type cyclins Cells were cultured according to protocols outlined in Fig. 1a . (a) Amounts of cyclins D1, D2 and D3 in protein samples were quantified by Western blotting. Data are representative of four independent replicates. (b) Expression of mRNA coding for p27, cyclins D1, D2 and D3 was quantified using quantitative real-time RT-PCR. Data represent means ± SDs of four independent replicates.
Figure Legend Snippet: Expression of D-type cyclins Cells were cultured according to protocols outlined in Fig. 1a . (a) Amounts of cyclins D1, D2 and D3 in protein samples were quantified by Western blotting. Data are representative of four independent replicates. (b) Expression of mRNA coding for p27, cyclins D1, D2 and D3 was quantified using quantitative real-time RT-PCR. Data represent means ± SDs of four independent replicates.

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

33) Product Images from "Mechanisms of RNAi: mRNA cleavage fragments may indicate stalled RISC"

Article Title: Mechanisms of RNAi: mRNA cleavage fragments may indicate stalled RISC

Journal: Journal of RNAi and Gene Silencing : An International Journal of RNA and Gene Targeting Research

doi:

(A) Graphical representation of Fen1 mRNA and the siRNA positions. (B) Screening of 10 siRNAs against Fen1 at 12 hr post-transfection by northern blot assay. Upper panel: Fen1 target mRNA. Lower panel: GAPDH loading control.
Figure Legend Snippet: (A) Graphical representation of Fen1 mRNA and the siRNA positions. (B) Screening of 10 siRNAs against Fen1 at 12 hr post-transfection by northern blot assay. Upper panel: Fen1 target mRNA. Lower panel: GAPDH loading control.

Techniques Used: Transfection, Northern Blot

Diagrams of putative mRNA fragment ends after siRNA cleavage. Cleavage point as defined by Martinez and Tuschl (2004) . Shown are sense sequences for the five siRNAs with considerable cleavage activity at 12 hr post-transfection.
Figure Legend Snippet: Diagrams of putative mRNA fragment ends after siRNA cleavage. Cleavage point as defined by Martinez and Tuschl (2004) . Shown are sense sequences for the five siRNAs with considerable cleavage activity at 12 hr post-transfection.

Techniques Used: Activity Assay, Transfection

34) Product Images from "A useful approach to total analysis of RISC-associated RNA"

Article Title: A useful approach to total analysis of RISC-associated RNA

Journal: BMC Research Notes

doi: 10.1186/1756-0500-2-169

Size distribution of synthesized cDNA and categories of cDNAs isolated from HeLa cells . A: Size distribution pattern of synthesized cDNA. The amount and size distribution of cDNA were determined with the Bioanalyzer DNA 1000 Kit. The red line indicates cDNA synthesized from anti-Ago2 immunoprecipitates, while the blue line indicates that from control mouse IgG. B: Composition of isolated cDNA clones. The sequences of 91 clones were examined by BLAST search and categorized as mRNA, Alu RNA embedded mRNA, free Alu RNA, or hits in the genome. The percentage recovery of each categorized cDNA clone is shown. Alu RNA embedded mRNA (14.3%, not indicated in the figure) is included in the mRNA area (total 80%) and separated by a broken line.
Figure Legend Snippet: Size distribution of synthesized cDNA and categories of cDNAs isolated from HeLa cells . A: Size distribution pattern of synthesized cDNA. The amount and size distribution of cDNA were determined with the Bioanalyzer DNA 1000 Kit. The red line indicates cDNA synthesized from anti-Ago2 immunoprecipitates, while the blue line indicates that from control mouse IgG. B: Composition of isolated cDNA clones. The sequences of 91 clones were examined by BLAST search and categorized as mRNA, Alu RNA embedded mRNA, free Alu RNA, or hits in the genome. The percentage recovery of each categorized cDNA clone is shown. Alu RNA embedded mRNA (14.3%, not indicated in the figure) is included in the mRNA area (total 80%) and separated by a broken line.

Techniques Used: Synthesized, Isolation, Clone Assay

Changes in mRNA level in total and Ago2-associated RNA after miR-122 transfection . Seventeen clones with high score percentile for the miR-122 target site ( > = 40) were picked, and the mRNA levels in total and immunoprecipitated RNA were quantified by RT-PCR. In the figure, the ratios of the amount of mRNA in the miR-122-transfected cells to that in the GL3-transfected cells are shown. Blue bars indicate the ratio for Ago2-associated RNA, while purple bars show that for total RNA. The left panel shows the clones picked from the miR-122-transfected cells, and the right panel shows the clones picked from the GL3-transfected cells (See Figure 6A). The clones are aligned in order of height of context score percentile from the left, with the ratio for GAPDH inserted as a standard.
Figure Legend Snippet: Changes in mRNA level in total and Ago2-associated RNA after miR-122 transfection . Seventeen clones with high score percentile for the miR-122 target site ( > = 40) were picked, and the mRNA levels in total and immunoprecipitated RNA were quantified by RT-PCR. In the figure, the ratios of the amount of mRNA in the miR-122-transfected cells to that in the GL3-transfected cells are shown. Blue bars indicate the ratio for Ago2-associated RNA, while purple bars show that for total RNA. The left panel shows the clones picked from the miR-122-transfected cells, and the right panel shows the clones picked from the GL3-transfected cells (See Figure 6A). The clones are aligned in order of height of context score percentile from the left, with the ratio for GAPDH inserted as a standard.

Techniques Used: Transfection, Clone Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Contents of miRNA and mRNA cDNA clones obtained from immunoprecipitates of miR-122- and GL3-transfected HepG2 cells . A: Contents of miRNA clones. 89 clones were analyzed for the miR-122-transfectant and 84 for the GL3-transfectant. Red bars show recovered miRNA clones originating from transfected nucleotides (upper figure; miR-122-transfected HepG2 cells, lower figure GL3-transfected HepG2 cells). B: Composition of RISC-associated polyA-containing RNA. The isolated cDNA clones were analyzed by BLAST search and classified into categories. The composition of each categorized RNA is shown (Upper figure: miR-122-transfected HepG2 cells, lower figure: GL3-transfected HepG2 cells). Alu RNA embedded mRNA is included in mRNA area. The percentages of Alu RNA embedded mRNA in the miR-122 and the GL3 transfectants were 10% and 19%, respectively.
Figure Legend Snippet: Contents of miRNA and mRNA cDNA clones obtained from immunoprecipitates of miR-122- and GL3-transfected HepG2 cells . A: Contents of miRNA clones. 89 clones were analyzed for the miR-122-transfectant and 84 for the GL3-transfectant. Red bars show recovered miRNA clones originating from transfected nucleotides (upper figure; miR-122-transfected HepG2 cells, lower figure GL3-transfected HepG2 cells). B: Composition of RISC-associated polyA-containing RNA. The isolated cDNA clones were analyzed by BLAST search and classified into categories. The composition of each categorized RNA is shown (Upper figure: miR-122-transfected HepG2 cells, lower figure: GL3-transfected HepG2 cells). Alu RNA embedded mRNA is included in mRNA area. The percentages of Alu RNA embedded mRNA in the miR-122 and the GL3 transfectants were 10% and 19%, respectively.

Techniques Used: Clone Assay, Transfection, Isolation

Selection of target mRNA candidate for miR-122 . A: List of cDNA clones with miR-122 seed match sequence in 3'UTR. All isolated clones were searched for the miR-122 target site by the miRNA target prediction program TargetScan 4.2, and the clones exhibiting context score percentiles (csp) are listed in the box. The left box shows the clones isolated from the miR-122-transfectant, and the right box shows those isolated from the GL3-transfectant. B: Enrichment of recovered cDNA clone containing miR-122 target site with high csp after miR-122 transfection. The numbers of cDNA clones with csp values for the miR-122 target site categorized as low (1–49), intermediate (50–79), and high (80–100) are shown separately. The blue bar shows the number of clones recovered from the miR-122 transfectant, while the purple bar shows those recovered from the GL3 transfectant.
Figure Legend Snippet: Selection of target mRNA candidate for miR-122 . A: List of cDNA clones with miR-122 seed match sequence in 3'UTR. All isolated clones were searched for the miR-122 target site by the miRNA target prediction program TargetScan 4.2, and the clones exhibiting context score percentiles (csp) are listed in the box. The left box shows the clones isolated from the miR-122-transfectant, and the right box shows those isolated from the GL3-transfectant. B: Enrichment of recovered cDNA clone containing miR-122 target site with high csp after miR-122 transfection. The numbers of cDNA clones with csp values for the miR-122 target site categorized as low (1–49), intermediate (50–79), and high (80–100) are shown separately. The blue bar shows the number of clones recovered from the miR-122 transfectant, while the purple bar shows those recovered from the GL3 transfectant.

Techniques Used: Selection, Clone Assay, Sequencing, Isolation, Transfection

35) Product Images from "Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles"

Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

Journal: Journal of Nanobiotechnology

doi: 10.1186/s12951-018-0401-y

Transfection of non-phagocytic cells, using mRNA:LPNs and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p
Figure Legend Snippet: Transfection of non-phagocytic cells, using mRNA:LPNs and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p

Techniques Used: Transfection, Flow Cytometry, Cytometry, Expressing

a1 Representative images depicted from live cell video after nine different time-points. The images are part of a video provided in Additional file 2 : Movie S1. DC2.4 cells were incubated with mRNA:FA-LPNs for a complete time duration of 4 h and with an interval of 3 min/image. Green dots on the images represent the fluorescence signal of labeled LPNs, while the cells signaling in red are the ones with successful mCherry expression. Scale bar = 100 µm. a2 Time-dependent change of the red fluorescence signal resulting from transfected cells and non-transfected, which are correlated with a3 green fluorescence signal of labeled nanoparticles. The tendency of each single cell being transfected is independent of the NPs uptake, as no significant difference between the uptake-behavior of transfected and non-transfected cells was observable. λ em = peak emission wavelength, MFI mean fluorescence intensity (mean ± SD, data from n = 32 fluorescent cells, n = 8 non-fluorescent cells, obtained from 4 independent videos)
Figure Legend Snippet: a1 Representative images depicted from live cell video after nine different time-points. The images are part of a video provided in Additional file 2 : Movie S1. DC2.4 cells were incubated with mRNA:FA-LPNs for a complete time duration of 4 h and with an interval of 3 min/image. Green dots on the images represent the fluorescence signal of labeled LPNs, while the cells signaling in red are the ones with successful mCherry expression. Scale bar = 100 µm. a2 Time-dependent change of the red fluorescence signal resulting from transfected cells and non-transfected, which are correlated with a3 green fluorescence signal of labeled nanoparticles. The tendency of each single cell being transfected is independent of the NPs uptake, as no significant difference between the uptake-behavior of transfected and non-transfected cells was observable. λ em = peak emission wavelength, MFI mean fluorescence intensity (mean ± SD, data from n = 32 fluorescent cells, n = 8 non-fluorescent cells, obtained from 4 independent videos)

Techniques Used: Incubation, Fluorescence, Labeling, Expressing, Transfection

Representative confocal images of DC2.4 cells transfected with both mRNA complexed NPs and by using jetPRIME ® as a positive control, naked mRNA as negative control. Transfection was analyzed with CLSM a 24 h and b 48 h post-transfection. Red fluorescence reveals cells successfully transfected with the nanoparticles while their morphology remains consistent with non-transfected cells (staining: green: cell membrane; blue: cell nucleus; scale bar: 50 μm)
Figure Legend Snippet: Representative confocal images of DC2.4 cells transfected with both mRNA complexed NPs and by using jetPRIME ® as a positive control, naked mRNA as negative control. Transfection was analyzed with CLSM a 24 h and b 48 h post-transfection. Red fluorescence reveals cells successfully transfected with the nanoparticles while their morphology remains consistent with non-transfected cells (staining: green: cell membrane; blue: cell nucleus; scale bar: 50 μm)

Techniques Used: Transfection, Positive Control, Negative Control, Confocal Laser Scanning Microscopy, Fluorescence, Staining

Quantification of cellular NP association for fluoresceinamine (FA) labeled blank ( a1 , a2 ) and mRNA complexed nanoparticles ( b1 , b2 ) tested in DC2.4 cells. NPs were incubated for 2 h and 4 h at different concentrations (20, 40 and 60 µg/mL corresponding to the weight ratios 1:10, 1:20 and 1:30 used for transfection). a1 , b1 Green NP fluorescent signal quantified by flow cytometry. Blank LPNs tend to show more uptake then blank CS-PLGA NPs, whereas for mRNA-loaded nanoparticles the opposite was observed. None of the samples showed a significant difference between 2 and 4 h incubation. a2 , b2 Representative graphs obtained for NP samples after 4 h incubation. N = 4, mean ± SD
Figure Legend Snippet: Quantification of cellular NP association for fluoresceinamine (FA) labeled blank ( a1 , a2 ) and mRNA complexed nanoparticles ( b1 , b2 ) tested in DC2.4 cells. NPs were incubated for 2 h and 4 h at different concentrations (20, 40 and 60 µg/mL corresponding to the weight ratios 1:10, 1:20 and 1:30 used for transfection). a1 , b1 Green NP fluorescent signal quantified by flow cytometry. Blank LPNs tend to show more uptake then blank CS-PLGA NPs, whereas for mRNA-loaded nanoparticles the opposite was observed. None of the samples showed a significant difference between 2 and 4 h incubation. a2 , b2 Representative graphs obtained for NP samples after 4 h incubation. N = 4, mean ± SD

Techniques Used: Labeling, Incubation, Transfection, Flow Cytometry, Cytometry

The kinetics of transfection for both mRNA-loaded NPs was quantified using flow cytometry, which indicated a significant difference between a1 mRNA:LPNs over a2 mRNA:CS-PLGA NPs, while mRNA:LPNs with a ratio of 1:20 and 1:30 elucidated a significant higher transfection rate over 1:10. a3 Control samples for transfection studies were JetPRIME ® as positive control and naked mRNA as negative control. (Note the enlarged y-axis, N = 4, mean ± SD.) a4 Representative graphs for evaluated time-points demonstrate a strong fluorescence shift for mRNA:LPNs with an increment in time and hence a higher transgene expression
Figure Legend Snippet: The kinetics of transfection for both mRNA-loaded NPs was quantified using flow cytometry, which indicated a significant difference between a1 mRNA:LPNs over a2 mRNA:CS-PLGA NPs, while mRNA:LPNs with a ratio of 1:20 and 1:30 elucidated a significant higher transfection rate over 1:10. a3 Control samples for transfection studies were JetPRIME ® as positive control and naked mRNA as negative control. (Note the enlarged y-axis, N = 4, mean ± SD.) a4 Representative graphs for evaluated time-points demonstrate a strong fluorescence shift for mRNA:LPNs with an increment in time and hence a higher transgene expression

Techniques Used: Transfection, Flow Cytometry, Cytometry, Positive Control, Negative Control, Fluorescence, Expressing

Schematic illustration of all nanoparticles used in this study. Both, LPNs and CS-PLGA NPs either complexed with mRNA or/and labeled with fluoresceinamine are used to quantify their uptake behavior and transfection efficiency in a dendritic cell line (DC2.4)
Figure Legend Snippet: Schematic illustration of all nanoparticles used in this study. Both, LPNs and CS-PLGA NPs either complexed with mRNA or/and labeled with fluoresceinamine are used to quantify their uptake behavior and transfection efficiency in a dendritic cell line (DC2.4)

Techniques Used: Labeling, Transfection

Gel retardation assay of mRNA complexed with different nanoparticles (NPs) at various ratios: a LPNs and b CS-PLGA NPs. Both images indicate mRNA binding to NPs and their appropriate release using heparin
Figure Legend Snippet: Gel retardation assay of mRNA complexed with different nanoparticles (NPs) at various ratios: a LPNs and b CS-PLGA NPs. Both images indicate mRNA binding to NPs and their appropriate release using heparin

Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay

Morphology of mRNA-loaded LPNs ( a1 , a2 ) and CS-PLGA NPs ( b1 , b2 ) visualized using SEM and TEM. a1 , b1 SEM images show a smooth, spherical morphology of the mRNA-loaded nanoparticles and a2 , b2 TEM images show the core–shell structure of the particles after staining with 0.5% (w/V) PTA
Figure Legend Snippet: Morphology of mRNA-loaded LPNs ( a1 , a2 ) and CS-PLGA NPs ( b1 , b2 ) visualized using SEM and TEM. a1 , b1 SEM images show a smooth, spherical morphology of the mRNA-loaded nanoparticles and a2 , b2 TEM images show the core–shell structure of the particles after staining with 0.5% (w/V) PTA

Techniques Used: Transmission Electron Microscopy, Staining

36) Product Images from "XPC Promotes Pluripotency of Human Dental Pulp Cells through Regulation of Oct-4/Sox2/c-Myc"

Article Title: XPC Promotes Pluripotency of Human Dental Pulp Cells through Regulation of Oct-4/Sox2/c-Myc

Journal: Stem Cells International

doi: 10.1155/2016/3454876

Effect of XPC on the expression of Oct-4, Sox2, and c-Myc in DPCs at various passages. Immunofluorescent staining revealed that XPC (a1), Oct-4 (a2), Sox2 (a3), and c-Myc (a4) were strongly expressed in DPCs with XPC overexpression at P7, mainly located in nucleus and moderately expressed in cytoplasm, whereas XPC (a5), Oct-4 (a6), Sox2 (a7), and c-Myc (a8) were barely detected in DPCs at P7 without transfection. Real-time PCR indicated that the mRNA expression of XPC, Oct-4, Sox2 , and c-Myc was upregulated significantly in DPCs with XPC overexpression at both P3 and P7 compared with vector groups ( ∗∗ p
Figure Legend Snippet: Effect of XPC on the expression of Oct-4, Sox2, and c-Myc in DPCs at various passages. Immunofluorescent staining revealed that XPC (a1), Oct-4 (a2), Sox2 (a3), and c-Myc (a4) were strongly expressed in DPCs with XPC overexpression at P7, mainly located in nucleus and moderately expressed in cytoplasm, whereas XPC (a5), Oct-4 (a6), Sox2 (a7), and c-Myc (a8) were barely detected in DPCs at P7 without transfection. Real-time PCR indicated that the mRNA expression of XPC, Oct-4, Sox2 , and c-Myc was upregulated significantly in DPCs with XPC overexpression at both P3 and P7 compared with vector groups ( ∗∗ p

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

Effect of XPC on the multilineage differentiation capability of DPCs after transfection. After 21 d of multilineage differentiation induction, immunofluorescent staining indicated that DSPP (a1), LPL (a2), and collagen type II (a3) were extensively expressed in DPCs at P7 with XPC overexpression, strongly expressed in the nucleus and moderately expressed in the cytoplasm of DPCs. However, DSPP (a4), LPL (a5), and collagen type II (a6) revealed weak expression in the nucleus and cytoplasm of DPCs at P7 without transfection. Real-time PCR indicated that mRNA expression level of odontogenic markers ( DMP1, DSPP ), adipogenic markers ( PPARγ2, LPL ), and chondrogenic markers ( collagen type II ) increased significantly in DPCs at P3 and P7 with XPC overexpression compared with vector groups (b1–b5).
Figure Legend Snippet: Effect of XPC on the multilineage differentiation capability of DPCs after transfection. After 21 d of multilineage differentiation induction, immunofluorescent staining indicated that DSPP (a1), LPL (a2), and collagen type II (a3) were extensively expressed in DPCs at P7 with XPC overexpression, strongly expressed in the nucleus and moderately expressed in the cytoplasm of DPCs. However, DSPP (a4), LPL (a5), and collagen type II (a6) revealed weak expression in the nucleus and cytoplasm of DPCs at P7 without transfection. Real-time PCR indicated that mRNA expression level of odontogenic markers ( DMP1, DSPP ), adipogenic markers ( PPARγ2, LPL ), and chondrogenic markers ( collagen type II ) increased significantly in DPCs at P3 and P7 with XPC overexpression compared with vector groups (b1–b5).

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

Expression of XPC, Oct-4 , Sox2, and c-Myc in DPCs at various passages. Real-time PCR showed that mRNA expression of XPC, Oct-4 , Sox2, and c-Myc was significantly higher in DPCs at passage 3 compared with passage 7 (a) ( ∗ p
Figure Legend Snippet: Expression of XPC, Oct-4 , Sox2, and c-Myc in DPCs at various passages. Real-time PCR showed that mRNA expression of XPC, Oct-4 , Sox2, and c-Myc was significantly higher in DPCs at passage 3 compared with passage 7 (a) ( ∗ p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Establishment of XPC overexpression DPCs model. Expression of pCDH-CMV-XPC-EF1-copGFP plasmid in HEK293T cells and DPCs at P2 (a1–a6). Strong green fluorescence was detected in HEK293T cells (a2, ×50) and DPCs (a5, ×100) after transfection. Combined figures of the bright field and fluorescence figure (a3, a6). Western blot and real-time PCR showed that the expression of XPC was enhanced in XPC+/DPCs at P2 at both protein and mRNA level compared with vector group, and XPC maintained high expression level in DPCs at P3 and P7 ( ∗∗ p
Figure Legend Snippet: Establishment of XPC overexpression DPCs model. Expression of pCDH-CMV-XPC-EF1-copGFP plasmid in HEK293T cells and DPCs at P2 (a1–a6). Strong green fluorescence was detected in HEK293T cells (a2, ×50) and DPCs (a5, ×100) after transfection. Combined figures of the bright field and fluorescence figure (a3, a6). Western blot and real-time PCR showed that the expression of XPC was enhanced in XPC+/DPCs at P2 at both protein and mRNA level compared with vector group, and XPC maintained high expression level in DPCs at P3 and P7 ( ∗∗ p

Techniques Used: Over Expression, Expressing, Plasmid Preparation, Fluorescence, Transfection, Western Blot, Real-time Polymerase Chain Reaction

37) Product Images from "Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus"

Article Title: Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus

Journal: Arthritis Research & Therapy

doi: 10.1186/ar3474

Relative expression of transcription factors BCL6 and ROR-y . The relative mRNA expression of BCL6 and ROR-y in unstimulated, sorted CD4 + T-cells was determined in SLE patients (SLE, n = 7) and healthy controls (HC, n = 4) ( A/B ). There was no significant difference in the relative expression between these two groups. Bars represent the mean values ± SD. In ( C ) a representative dot plot of unstimulated and stimulated PBMCs from a SLE patient a healthy control is shown. The percentages of BCL6 and ROR- γt + CD4 + T-cells in unstimulated (dots) and stimulated samples (squares) are given ( D ).
Figure Legend Snippet: Relative expression of transcription factors BCL6 and ROR-y . The relative mRNA expression of BCL6 and ROR-y in unstimulated, sorted CD4 + T-cells was determined in SLE patients (SLE, n = 7) and healthy controls (HC, n = 4) ( A/B ). There was no significant difference in the relative expression between these two groups. Bars represent the mean values ± SD. In ( C ) a representative dot plot of unstimulated and stimulated PBMCs from a SLE patient a healthy control is shown. The percentages of BCL6 and ROR- γt + CD4 + T-cells in unstimulated (dots) and stimulated samples (squares) are given ( D ).

Techniques Used: Expressing

38) Product Images from "Methylphenidate Exposure Induces Dopamine Neuron Loss and Activation of Microglia in the Basal Ganglia of Mice"

Article Title: Methylphenidate Exposure Induces Dopamine Neuron Loss and Activation of Microglia in the Basal Ganglia of Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033693

Acute and chronic administration of MPH alters gene expression in the substantia nigra (SN). (A) Heat map representation of gene expression changes following chronic administration of either 1 mg/kg MPH or 10 mg/kg MPH in the SN (n = 3). qPCR analysis demonstrating normalized fold-change expression of (B) bdnf , (C) gdnf , (D) dat1(slc6a3) , (E) vmat2(slc18a2) and (F) th mRNA in SN (n = 3). *p≤0.02 vs saline-controls (ctrl); **p≤0.02 vs saline-controls and 10 mg/kg MPH-acute dose; #p≤0.02 10 mg/kg MPH acute-dose vs saline-controls (ctrl). One-way ANOVA statistical test was performed to draw comparisons between the different groups followed by Bonferroni post-hoc tests.
Figure Legend Snippet: Acute and chronic administration of MPH alters gene expression in the substantia nigra (SN). (A) Heat map representation of gene expression changes following chronic administration of either 1 mg/kg MPH or 10 mg/kg MPH in the SN (n = 3). qPCR analysis demonstrating normalized fold-change expression of (B) bdnf , (C) gdnf , (D) dat1(slc6a3) , (E) vmat2(slc18a2) and (F) th mRNA in SN (n = 3). *p≤0.02 vs saline-controls (ctrl); **p≤0.02 vs saline-controls and 10 mg/kg MPH-acute dose; #p≤0.02 10 mg/kg MPH acute-dose vs saline-controls (ctrl). One-way ANOVA statistical test was performed to draw comparisons between the different groups followed by Bonferroni post-hoc tests.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

39) Product Images from "Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system"

Article Title: Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system

Journal: Experimental & Molecular Medicine

doi: 10.3858/emm.2012.44.5.037

Serum starvation induced the expression of the DAPK1 and BIM genes, which was prevented by culture with 2% PRP. The ccdPAs were seeded and incubated for 16 hr in DMEM/HAM containing 20% FBS, and the culture medium was then replaced with medium without serum (Serum (-)), or with 2% or 10% FBS, 2% PRP, or 2% PPP, followed by incubation for an additional 72 hr. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the mRNA expression levels of DAPK1 (A) and BIM (B). The quantification of the given genes was expressed as relative mRNA level compared with a control after normalization to 18S RNA. * P
Figure Legend Snippet: Serum starvation induced the expression of the DAPK1 and BIM genes, which was prevented by culture with 2% PRP. The ccdPAs were seeded and incubated for 16 hr in DMEM/HAM containing 20% FBS, and the culture medium was then replaced with medium without serum (Serum (-)), or with 2% or 10% FBS, 2% PRP, or 2% PPP, followed by incubation for an additional 72 hr. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the mRNA expression levels of DAPK1 (A) and BIM (B). The quantification of the given genes was expressed as relative mRNA level compared with a control after normalization to 18S RNA. * P

Techniques Used: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

40) Product Images from "Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease"

Article Title: Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease

Journal: Nature medicine

doi: 10.1038/nm.3842

PEPITEM induces the S1P release from endothelial cells which inhibits T cell migration ( a, b ) The effects of an S1PR antagonist (10 μM) on T cell migration in the presence or absence of ( a ) adiponectin (n=3–5) or ( b ) PEPITEM (n=3–7). ( c ) The effects on T cell transmigration of adding S1P to B cell depleted PBL n=3–6. ( d, e ) The effects of ( d ) SPHK1 specific inhibitor (5 μM) or ( e ) SPHK1/2 inhibitor (5 μM) on T cell transmigration in the presence of PEPITEM, n=3. ( f ) The expression of SPHK1 and SPHK2 mRNA in endothelial cells, n=7–8. ( g ) The effect of SPNS2 specific siRNA on T cell transmigration in the presence of PEPITEM, n=4. ( h, i ) The expression of S1PR1 on memory T cells (CD3 + CD45RO + ) on ( h ) stimulated endothelial cells (n=3) and ( i ) on plated ICAM-1 stimulated with CXCL10 (n=6). ( j ) The effects of S1P on the expression of the LFA-1 activation epitope (KIM127) on ICAM-1 adherent memory T cells (CD3 + CD45RO + ) stimulated with CXCL10, n=4. Data are mean±s.e.m and ( g - j ) normalized to control. * P ≤0.05, ** P ≤0.01, *** P ≤0.001 compared to untreated control by ANOVA and ( a-g ) Dunnett compared to ( a-b, d-e, g ) untreated control or ( c ) B cell depletion no adiponectin control or to ( f ) unstimulated (0) control or ( j ) Bonferroni post-test or ( h ) paired t-test on raw data or ( i ) Wilcoxon signed rank test.
Figure Legend Snippet: PEPITEM induces the S1P release from endothelial cells which inhibits T cell migration ( a, b ) The effects of an S1PR antagonist (10 μM) on T cell migration in the presence or absence of ( a ) adiponectin (n=3–5) or ( b ) PEPITEM (n=3–7). ( c ) The effects on T cell transmigration of adding S1P to B cell depleted PBL n=3–6. ( d, e ) The effects of ( d ) SPHK1 specific inhibitor (5 μM) or ( e ) SPHK1/2 inhibitor (5 μM) on T cell transmigration in the presence of PEPITEM, n=3. ( f ) The expression of SPHK1 and SPHK2 mRNA in endothelial cells, n=7–8. ( g ) The effect of SPNS2 specific siRNA on T cell transmigration in the presence of PEPITEM, n=4. ( h, i ) The expression of S1PR1 on memory T cells (CD3 + CD45RO + ) on ( h ) stimulated endothelial cells (n=3) and ( i ) on plated ICAM-1 stimulated with CXCL10 (n=6). ( j ) The effects of S1P on the expression of the LFA-1 activation epitope (KIM127) on ICAM-1 adherent memory T cells (CD3 + CD45RO + ) stimulated with CXCL10, n=4. Data are mean±s.e.m and ( g - j ) normalized to control. * P ≤0.05, ** P ≤0.01, *** P ≤0.001 compared to untreated control by ANOVA and ( a-g ) Dunnett compared to ( a-b, d-e, g ) untreated control or ( c ) B cell depletion no adiponectin control or to ( f ) unstimulated (0) control or ( j ) Bonferroni post-test or ( h ) paired t-test on raw data or ( i ) Wilcoxon signed rank test.

Techniques Used: Migration, Transmigration Assay, Expressing, Activation Assay

Related Articles

Electroporation:

Article Title: The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma
Article Snippet: .. RNA Production and Transfection The mRNA used for electroporation was generated and purified using the mMESSAGE mMACHINE T7 Ultra Transcription Kit (Life Technologies, Carlsbad, CA, USA) and the RNeasy Kit (Qiagen) according to the manufacturers’ instructions. .. 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.

Transfection:

Article Title: The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma
Article Snippet: .. RNA Production and Transfection The mRNA used for electroporation was generated and purified using the mMESSAGE mMACHINE T7 Ultra Transcription Kit (Life Technologies, Carlsbad, CA, USA) and the RNeasy Kit (Qiagen) according to the manufacturers’ instructions. .. 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.

esiRNA:

Article Title: Art27 Interacts with GATA4, FOG2 and NKX2.5 and Is a Novel Co-Repressor of Cardiac Genes
Article Snippet: .. Treatment of differentiated cells with Mission esiRNA against Art27 resulted in marked down-regulation (10-fold) of Art27 message relative to control esiRNA (against GFP) ( ). mRNA was harvested 36 h post-nucleofection and used for microarray analysis using Affymetrix Mouse Gene Arrays 2.0ST. ..

Positive Control:

Article Title: Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods
Article Snippet: .. The PCR cycles were as follows: for β-ACTIN , initial denaturation was performed at 94°C for 2.5 min, followed by 25 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 30 s, and a final extension at 72°C for 10 min. For the other genes, the cycles consisted of initial denaturation at 94°C for 2.5 min, followed by 30–40 cycles of 94°C for 30 s, 58–62°C for 30 s, 72°C for 30 s, and final extension at 72°C for 7 min. mRNA from human fetal kidney was used as a positive control for expression of OSR1 , PAX2 , LIM1 , WT1 , EYA1 , SALL1 , and CITED2 . mRNA from human adult ovaries was used as a positive control for SOX17 . mRNA from neural progenitor cells, induced from hiPSCs by SFEBq culture , was used as a positive control for SOX1 . mRNA from human umbilical vein endothelial cells (HUVECs) was used as a positive control for KDR . mRNA from human adult cervix was used as a positive control for SIX2 . mRNA from human adult kidney was used as a positive control for HOXD11 , FOXD1 , SALL4 , and HOXB7 . mRNA from human adult ovaries was used as a positive control for GATA4 and GATA6 . mRNA from human adult adrenal gland was used as a positive control for SF1 and HSD3β2 . mRNA from human adult testes was used as a positive control for DAX1 and LHX9 . qPCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems) and the SYBR Green PCR Master Mix (Takara). .. Denaturation was performed at 95°C for 30 s, followed by 45 cycles at 95°C for 5 s and at 60°C for 30 s. The threshold cycle method was used to analyze the data for the gene expression levels as recommended by the manufacturer, and was calibrated to the levels of the housekeeping gene, β-ACTIN .

Generated:

Article Title: The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma
Article Snippet: .. RNA Production and Transfection The mRNA used for electroporation was generated and purified using the mMESSAGE mMACHINE T7 Ultra Transcription Kit (Life Technologies, Carlsbad, CA, USA) and the RNeasy Kit (Qiagen) according to the manufacturers’ instructions. .. 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.

Article Title: EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis
Article Snippet: .. Treated mRNA was checked for complete removal of contaminating genomic DNA by PCR before proceeding to the reverse transcription step. cDNA from mRNA was generated using the Superscript III first-strand synthesis system (Invitrogen, Carlsbad, CA) and random hexamers. .. Three different primer pairs were used to amplify internal fragments of espI and eccD1, and the portions encompassing the coding sequence and intergenic regions between the two genes from the cDNA generated as described above.

Quantitative RT-PCR:

Article Title: Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability
Article Snippet: .. RT-qPCR quantification of RNA abundance 2–50 ng of mRNA (depending on sample) was used for reverse transcription using Superscript II [Life technologies] with random hexamer primers. cDNA was quantified on a StepOnePlus Real-Time PCR system using a SYBR green PCR mix [ThermoFisher] with gene specific primers ( ). .. High-throughput sequencing and RNAseq quantification 50–100 ng mRNA was used as input material to generate strand-specific sequencing libraries using the NEXTflex Rapid Directional Illumina RNA-Seq Library Prep Kit [BioO] according to the manufacturer’s instructions.

Purification:

Article Title: The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma
Article Snippet: .. RNA Production and Transfection The mRNA used for electroporation was generated and purified using the mMESSAGE mMACHINE T7 Ultra Transcription Kit (Life Technologies, Carlsbad, CA, USA) and the RNeasy Kit (Qiagen) according to the manufacturers’ instructions. .. 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.

SYBR Green Assay:

Article Title: Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods
Article Snippet: .. The PCR cycles were as follows: for β-ACTIN , initial denaturation was performed at 94°C for 2.5 min, followed by 25 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 30 s, and a final extension at 72°C for 10 min. For the other genes, the cycles consisted of initial denaturation at 94°C for 2.5 min, followed by 30–40 cycles of 94°C for 30 s, 58–62°C for 30 s, 72°C for 30 s, and final extension at 72°C for 7 min. mRNA from human fetal kidney was used as a positive control for expression of OSR1 , PAX2 , LIM1 , WT1 , EYA1 , SALL1 , and CITED2 . mRNA from human adult ovaries was used as a positive control for SOX17 . mRNA from neural progenitor cells, induced from hiPSCs by SFEBq culture , was used as a positive control for SOX1 . mRNA from human umbilical vein endothelial cells (HUVECs) was used as a positive control for KDR . mRNA from human adult cervix was used as a positive control for SIX2 . mRNA from human adult kidney was used as a positive control for HOXD11 , FOXD1 , SALL4 , and HOXB7 . mRNA from human adult ovaries was used as a positive control for GATA4 and GATA6 . mRNA from human adult adrenal gland was used as a positive control for SF1 and HSD3β2 . mRNA from human adult testes was used as a positive control for DAX1 and LHX9 . qPCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems) and the SYBR Green PCR Master Mix (Takara). .. Denaturation was performed at 95°C for 30 s, followed by 45 cycles at 95°C for 5 s and at 60°C for 30 s. The threshold cycle method was used to analyze the data for the gene expression levels as recommended by the manufacturer, and was calibrated to the levels of the housekeeping gene, β-ACTIN .

Article Title: Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability
Article Snippet: .. RT-qPCR quantification of RNA abundance 2–50 ng of mRNA (depending on sample) was used for reverse transcription using Superscript II [Life technologies] with random hexamer primers. cDNA was quantified on a StepOnePlus Real-Time PCR system using a SYBR green PCR mix [ThermoFisher] with gene specific primers ( ). .. High-throughput sequencing and RNAseq quantification 50–100 ng mRNA was used as input material to generate strand-specific sequencing libraries using the NEXTflex Rapid Directional Illumina RNA-Seq Library Prep Kit [BioO] according to the manufacturer’s instructions.

Microarray:

Article Title: Art27 Interacts with GATA4, FOG2 and NKX2.5 and Is a Novel Co-Repressor of Cardiac Genes
Article Snippet: .. Treatment of differentiated cells with Mission esiRNA against Art27 resulted in marked down-regulation (10-fold) of Art27 message relative to control esiRNA (against GFP) ( ). mRNA was harvested 36 h post-nucleofection and used for microarray analysis using Affymetrix Mouse Gene Arrays 2.0ST. ..

Random Hexamer Labeling:

Article Title: Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability
Article Snippet: .. RT-qPCR quantification of RNA abundance 2–50 ng of mRNA (depending on sample) was used for reverse transcription using Superscript II [Life technologies] with random hexamer primers. cDNA was quantified on a StepOnePlus Real-Time PCR system using a SYBR green PCR mix [ThermoFisher] with gene specific primers ( ). .. High-throughput sequencing and RNAseq quantification 50–100 ng mRNA was used as input material to generate strand-specific sequencing libraries using the NEXTflex Rapid Directional Illumina RNA-Seq Library Prep Kit [BioO] according to the manufacturer’s instructions.

Expressing:

Article Title: Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods
Article Snippet: .. The PCR cycles were as follows: for β-ACTIN , initial denaturation was performed at 94°C for 2.5 min, followed by 25 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 30 s, and a final extension at 72°C for 10 min. For the other genes, the cycles consisted of initial denaturation at 94°C for 2.5 min, followed by 30–40 cycles of 94°C for 30 s, 58–62°C for 30 s, 72°C for 30 s, and final extension at 72°C for 7 min. mRNA from human fetal kidney was used as a positive control for expression of OSR1 , PAX2 , LIM1 , WT1 , EYA1 , SALL1 , and CITED2 . mRNA from human adult ovaries was used as a positive control for SOX17 . mRNA from neural progenitor cells, induced from hiPSCs by SFEBq culture , was used as a positive control for SOX1 . mRNA from human umbilical vein endothelial cells (HUVECs) was used as a positive control for KDR . mRNA from human adult cervix was used as a positive control for SIX2 . mRNA from human adult kidney was used as a positive control for HOXD11 , FOXD1 , SALL4 , and HOXB7 . mRNA from human adult ovaries was used as a positive control for GATA4 and GATA6 . mRNA from human adult adrenal gland was used as a positive control for SF1 and HSD3β2 . mRNA from human adult testes was used as a positive control for DAX1 and LHX9 . qPCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems) and the SYBR Green PCR Master Mix (Takara). .. Denaturation was performed at 95°C for 30 s, followed by 45 cycles at 95°C for 5 s and at 60°C for 30 s. The threshold cycle method was used to analyze the data for the gene expression levels as recommended by the manufacturer, and was calibrated to the levels of the housekeeping gene, β-ACTIN .

Polymerase Chain Reaction:

Article Title: Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods
Article Snippet: .. The PCR cycles were as follows: for β-ACTIN , initial denaturation was performed at 94°C for 2.5 min, followed by 25 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 30 s, and a final extension at 72°C for 10 min. For the other genes, the cycles consisted of initial denaturation at 94°C for 2.5 min, followed by 30–40 cycles of 94°C for 30 s, 58–62°C for 30 s, 72°C for 30 s, and final extension at 72°C for 7 min. mRNA from human fetal kidney was used as a positive control for expression of OSR1 , PAX2 , LIM1 , WT1 , EYA1 , SALL1 , and CITED2 . mRNA from human adult ovaries was used as a positive control for SOX17 . mRNA from neural progenitor cells, induced from hiPSCs by SFEBq culture , was used as a positive control for SOX1 . mRNA from human umbilical vein endothelial cells (HUVECs) was used as a positive control for KDR . mRNA from human adult cervix was used as a positive control for SIX2 . mRNA from human adult kidney was used as a positive control for HOXD11 , FOXD1 , SALL4 , and HOXB7 . mRNA from human adult ovaries was used as a positive control for GATA4 and GATA6 . mRNA from human adult adrenal gland was used as a positive control for SF1 and HSD3β2 . mRNA from human adult testes was used as a positive control for DAX1 and LHX9 . qPCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems) and the SYBR Green PCR Master Mix (Takara). .. Denaturation was performed at 95°C for 30 s, followed by 45 cycles at 95°C for 5 s and at 60°C for 30 s. The threshold cycle method was used to analyze the data for the gene expression levels as recommended by the manufacturer, and was calibrated to the levels of the housekeeping gene, β-ACTIN .

Article Title: Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability
Article Snippet: .. RT-qPCR quantification of RNA abundance 2–50 ng of mRNA (depending on sample) was used for reverse transcription using Superscript II [Life technologies] with random hexamer primers. cDNA was quantified on a StepOnePlus Real-Time PCR system using a SYBR green PCR mix [ThermoFisher] with gene specific primers ( ). .. High-throughput sequencing and RNAseq quantification 50–100 ng mRNA was used as input material to generate strand-specific sequencing libraries using the NEXTflex Rapid Directional Illumina RNA-Seq Library Prep Kit [BioO] according to the manufacturer’s instructions.

Article Title: EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis
Article Snippet: .. Treated mRNA was checked for complete removal of contaminating genomic DNA by PCR before proceeding to the reverse transcription step. cDNA from mRNA was generated using the Superscript III first-strand synthesis system (Invitrogen, Carlsbad, CA) and random hexamers. .. Three different primer pairs were used to amplify internal fragments of espI and eccD1, and the portions encompassing the coding sequence and intergenic regions between the two genes from the cDNA generated as described above.

Real-time Polymerase Chain Reaction:

Article Title: G?12 Drives Invasion of Oral Squamous Cell Carcinoma through Up-Regulation of Proinflammatory Cytokines
Article Snippet: .. Quantitative real-time Reverse Transcription-PCR (qPCR) For reverse transcription of cellular mRNA to cDNA, an input of 2 µg of total RNA was used for the High-capacity cDNA Reverse transcription Kits (ABI Applied Biosystems, USA) according to the manufacturer's instructions. qPCR was performed on a Step One Real-time PCR system (ABI Applied Biosystems, USA) by using the KAPA SYBR FAST qPCR Kits (KAPA Biosystems, USA). .. The primer sequences of Gα12 used for qPCR were: forward-GTTTGTCGTCGTTGAGC, reverse-AGTAGTTTCACTCGCCC; for IL-8: forward-GGAGTGCTAAAGAACTTAGATG, reverse-TGGGGTCCAGACAGAG; for IL-6: forward-CAAAGATGTAGCCGCCC, reverse-GTTCAGGTTGTTTTCTGCC; for GAPDH: forward-CCTGCCAAATATGATGACATCAAG, reverse-ACCCTGTTGCTGTAGCCAAA.

Article Title: Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods
Article Snippet: .. The PCR cycles were as follows: for β-ACTIN , initial denaturation was performed at 94°C for 2.5 min, followed by 25 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 30 s, and a final extension at 72°C for 10 min. For the other genes, the cycles consisted of initial denaturation at 94°C for 2.5 min, followed by 30–40 cycles of 94°C for 30 s, 58–62°C for 30 s, 72°C for 30 s, and final extension at 72°C for 7 min. mRNA from human fetal kidney was used as a positive control for expression of OSR1 , PAX2 , LIM1 , WT1 , EYA1 , SALL1 , and CITED2 . mRNA from human adult ovaries was used as a positive control for SOX17 . mRNA from neural progenitor cells, induced from hiPSCs by SFEBq culture , was used as a positive control for SOX1 . mRNA from human umbilical vein endothelial cells (HUVECs) was used as a positive control for KDR . mRNA from human adult cervix was used as a positive control for SIX2 . mRNA from human adult kidney was used as a positive control for HOXD11 , FOXD1 , SALL4 , and HOXB7 . mRNA from human adult ovaries was used as a positive control for GATA4 and GATA6 . mRNA from human adult adrenal gland was used as a positive control for SF1 and HSD3β2 . mRNA from human adult testes was used as a positive control for DAX1 and LHX9 . qPCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems) and the SYBR Green PCR Master Mix (Takara). .. Denaturation was performed at 95°C for 30 s, followed by 45 cycles at 95°C for 5 s and at 60°C for 30 s. The threshold cycle method was used to analyze the data for the gene expression levels as recommended by the manufacturer, and was calibrated to the levels of the housekeeping gene, β-ACTIN .

Article Title: Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability
Article Snippet: .. RT-qPCR quantification of RNA abundance 2–50 ng of mRNA (depending on sample) was used for reverse transcription using Superscript II [Life technologies] with random hexamer primers. cDNA was quantified on a StepOnePlus Real-Time PCR system using a SYBR green PCR mix [ThermoFisher] with gene specific primers ( ). .. High-throughput sequencing and RNAseq quantification 50–100 ng mRNA was used as input material to generate strand-specific sequencing libraries using the NEXTflex Rapid Directional Illumina RNA-Seq Library Prep Kit [BioO] according to the manufacturer’s instructions.

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    Thermo Fisher mrna expression
    Cytokine production by CD4+ T cell clones derived from decidua of those experiencing successful pregnancy and URA and <t>mRNA</t> expression of cytokines and transcription factors in decidual biopsies of successful pregnancy. CD4+ T cell clones were generated from decidual biopsies, and peripheral blood was obtained from those experiencing successful pregnancy and those experiencing unexplained recurrent abortion (URA) (Experiment 1 in Section 4.3 ). IL-4, IL-13, IL-5, <t>IL-17A,</t> IL-17F, IL-22, and IFN-γ were measured in the supernatant of the CD4+ T cell clones by a multiplex bead-based assay. The statistical analysis was performed with the Wilcoxon test. The determination of mRNA level for IL-4, GATA-3, IL-17A, ROR-C, IL-22, AHR, T-bet, and IFN-γ in three biopsies of decidua from three pregnant women (with successful pregnancy) was performed by Quantigene 2.0.
    Mrna Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mrna abundance
    Hepatic TRAF2 is dispensable for glucose metabolism under normal conditions. A : TRAF2 <t>mRNA</t> abundance was measured in the liver by <t>qPCR</t> and normalized to 36B4 mRNA levels. WT-NC, wild-type mice fed a normal chow diet (14 weeks); WT-HFD, WT mice fed an HFD (14 weeks); ob/ob , leptin-deficient ob/ob mice (14 weeks); AU, arbitrary unit. B : Total RNAs were extracted from WT and HKO mice and used to measure TRAF2 transcription by RT-PCR. C : Genomic DNA was prepared from purified hepatocytes and Kupffer cells and subjected to PCR-based genotyping analysis. D : TRAF2 mRNA abundance was measured in purified hepatocytes and Kupffer cells by qPCR and normalized to 36B4 mRNA levels. E : Fasting (overnight) blood glucose levels in HKO and control male mice (23 weeks). F and G : GTTs ( d -glucose: 2 g/kg body wt) and ITTs (1 unit/kg body wt) were performed in HKO and control males at age 22–23 weeks. Data are mean ± SEM. * P
    Mrna Abundance, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher egfp mrna
    C-Lipo with reduced lipofectamine amount transfects cardiac fibroblasts efficiently without significant toxicity. Notes: ( A ) Cell viability of C-Lipo (0.5 μL of lipofectamine with 7 μg of CRPPR-R9 peptide per 1 μg <t>mRNA)</t> after daily transfections. Cardiac fibroblasts were transfected with C-Lipo and Lipo daily for 1 week and cell viabilities were measured on days 4 and 7. C-Lipo allows mRNA transfection in cardiac fibroblasts without significant toxicity for 1 week compared to Lipo, which causes significant toxicity. ( B ) and ( C ) Flow cytometry analyses of cardiac fibroblasts treated with C-Lipo and Lipo. C-Lipo shows comparable levels of transfection efficiency and higher mean <t>eGFP</t> fluorescence compared to Lipo. The results represent the mean ± SE, n=5. * P
    Egfp Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cytokine production by CD4+ T cell clones derived from decidua of those experiencing successful pregnancy and URA and mRNA expression of cytokines and transcription factors in decidual biopsies of successful pregnancy. CD4+ T cell clones were generated from decidual biopsies, and peripheral blood was obtained from those experiencing successful pregnancy and those experiencing unexplained recurrent abortion (URA) (Experiment 1 in Section 4.3 ). IL-4, IL-13, IL-5, IL-17A, IL-17F, IL-22, and IFN-γ were measured in the supernatant of the CD4+ T cell clones by a multiplex bead-based assay. The statistical analysis was performed with the Wilcoxon test. The determination of mRNA level for IL-4, GATA-3, IL-17A, ROR-C, IL-22, AHR, T-bet, and IFN-γ in three biopsies of decidua from three pregnant women (with successful pregnancy) was performed by Quantigene 2.0.

    Journal: International Journal of Molecular Sciences

    Article Title: Decidual Interleukin-22-Producing CD4+ T Cells (Th17/Th0/IL-22+ and Th17/Th2/IL-22+, Th2/IL-22+, Th0/IL-22+), Which Also Produce IL-4, Are Involved in the Success of Pregnancy

    doi: 10.3390/ijms20020428

    Figure Lengend Snippet: Cytokine production by CD4+ T cell clones derived from decidua of those experiencing successful pregnancy and URA and mRNA expression of cytokines and transcription factors in decidual biopsies of successful pregnancy. CD4+ T cell clones were generated from decidual biopsies, and peripheral blood was obtained from those experiencing successful pregnancy and those experiencing unexplained recurrent abortion (URA) (Experiment 1 in Section 4.3 ). IL-4, IL-13, IL-5, IL-17A, IL-17F, IL-22, and IFN-γ were measured in the supernatant of the CD4+ T cell clones by a multiplex bead-based assay. The statistical analysis was performed with the Wilcoxon test. The determination of mRNA level for IL-4, GATA-3, IL-17A, ROR-C, IL-22, AHR, T-bet, and IFN-γ in three biopsies of decidua from three pregnant women (with successful pregnancy) was performed by Quantigene 2.0.

    Article Snippet: Briefly, the mRNA expression of IL-4, IL-17A, IL-17F, IL-23R, IFN-γ, RORC, GATA3, AHR, IL-22, and Actb (high expression housekeeping gene) was measured using the QuantiGene multiplex assay (Thermo Fisher, Waltham, MA, USA).

    Techniques: Clone Assay, Derivative Assay, Expressing, Generated, Multiplex Assay, Bead-based Assay

    The determination of mRNA level for IL-4, GATA-3, IL-17A, ROR-C, IL-22, AHR, T-bet, and IFN -γ in Fallopian tube tissue taken at the embryo implantation site and distant from the implantation site of a woman suffering from ectopic pregnancy and in decidual biopsies from three women with successful pregnancy was performed by Quantigene 2.0.

    Journal: International Journal of Molecular Sciences

    Article Title: Decidual Interleukin-22-Producing CD4+ T Cells (Th17/Th0/IL-22+ and Th17/Th2/IL-22+, Th2/IL-22+, Th0/IL-22+), Which Also Produce IL-4, Are Involved in the Success of Pregnancy

    doi: 10.3390/ijms20020428

    Figure Lengend Snippet: The determination of mRNA level for IL-4, GATA-3, IL-17A, ROR-C, IL-22, AHR, T-bet, and IFN -γ in Fallopian tube tissue taken at the embryo implantation site and distant from the implantation site of a woman suffering from ectopic pregnancy and in decidual biopsies from three women with successful pregnancy was performed by Quantigene 2.0.

    Article Snippet: Briefly, the mRNA expression of IL-4, IL-17A, IL-17F, IL-23R, IFN-γ, RORC, GATA3, AHR, IL-22, and Actb (high expression housekeeping gene) was measured using the QuantiGene multiplex assay (Thermo Fisher, Waltham, MA, USA).

    Techniques:

    Th17/Th2/IL-22+ and Th17/Th0/IL-22+ CD4+ T cells are exclusively present at the implantation site of ectopic pregnancy. The percentages of Th0/IL-22+, Th2/IL-22+, Th17/Th0/IL-22+, Th17/Th2/IL-22+, and Th17/Th1/IL-22+ cells among the CD4+ T cell clones respectively derived from the implantation site of the embryo and distant from the implantation site in the same Fallopian tube of four women suffering from ectopic pregnancy were evaluated (Experiment 2 according to Section 4.3 ). The statistical analysis was performed with the chi-square test. The determination of mRNA level for IL-4, GATA-3, IL-17A, ROR-C, IL-22, AHR, T-bet, and IFN -γ in Fallopian tube tissue taken at the embryo implantation site and tissue sampled distant from the implantation site of an additional woman suffering from ectopic pregnancy was performed by Quantigene 2.0.

    Journal: International Journal of Molecular Sciences

    Article Title: Decidual Interleukin-22-Producing CD4+ T Cells (Th17/Th0/IL-22+ and Th17/Th2/IL-22+, Th2/IL-22+, Th0/IL-22+), Which Also Produce IL-4, Are Involved in the Success of Pregnancy

    doi: 10.3390/ijms20020428

    Figure Lengend Snippet: Th17/Th2/IL-22+ and Th17/Th0/IL-22+ CD4+ T cells are exclusively present at the implantation site of ectopic pregnancy. The percentages of Th0/IL-22+, Th2/IL-22+, Th17/Th0/IL-22+, Th17/Th2/IL-22+, and Th17/Th1/IL-22+ cells among the CD4+ T cell clones respectively derived from the implantation site of the embryo and distant from the implantation site in the same Fallopian tube of four women suffering from ectopic pregnancy were evaluated (Experiment 2 according to Section 4.3 ). The statistical analysis was performed with the chi-square test. The determination of mRNA level for IL-4, GATA-3, IL-17A, ROR-C, IL-22, AHR, T-bet, and IFN -γ in Fallopian tube tissue taken at the embryo implantation site and tissue sampled distant from the implantation site of an additional woman suffering from ectopic pregnancy was performed by Quantigene 2.0.

    Article Snippet: Briefly, the mRNA expression of IL-4, IL-17A, IL-17F, IL-23R, IFN-γ, RORC, GATA3, AHR, IL-22, and Actb (high expression housekeeping gene) was measured using the QuantiGene multiplex assay (Thermo Fisher, Waltham, MA, USA).

    Techniques: Clone Assay, Derivative Assay

    Effect of BMCH on mRNA expressions by RT-qPCR. ( A ) p53, ( B ) bax/bcl-2 ratio, and ( C ) caspase-3. Cells were pretreated with BMCH for 30 min followed by exposure of H 2 O 2 (600 μM) and incubation for 24 h. * p

    Journal: Marine Drugs

    Article Title: Amino Acid Composition, Antioxidant, and Cytoprotective Effect of Blue Mussel (Mytilus edulis) Hydrolysate through the Inhibition of Caspase-3 Activation in Oxidative Stress-Mediated Endothelial Cell Injury

    doi: 10.3390/md17020135

    Figure Lengend Snippet: Effect of BMCH on mRNA expressions by RT-qPCR. ( A ) p53, ( B ) bax/bcl-2 ratio, and ( C ) caspase-3. Cells were pretreated with BMCH for 30 min followed by exposure of H 2 O 2 (600 μM) and incubation for 24 h. * p

    Article Snippet: 4.6.8. mRNA Expression by Real Time-qPCR Total RNA was isolated using TRIzol reagent (Thermo scientific Co., Waltham, MA, USA).

    Techniques: Quantitative RT-PCR, Incubation

    Hepatic TRAF2 is dispensable for glucose metabolism under normal conditions. A : TRAF2 mRNA abundance was measured in the liver by qPCR and normalized to 36B4 mRNA levels. WT-NC, wild-type mice fed a normal chow diet (14 weeks); WT-HFD, WT mice fed an HFD (14 weeks); ob/ob , leptin-deficient ob/ob mice (14 weeks); AU, arbitrary unit. B : Total RNAs were extracted from WT and HKO mice and used to measure TRAF2 transcription by RT-PCR. C : Genomic DNA was prepared from purified hepatocytes and Kupffer cells and subjected to PCR-based genotyping analysis. D : TRAF2 mRNA abundance was measured in purified hepatocytes and Kupffer cells by qPCR and normalized to 36B4 mRNA levels. E : Fasting (overnight) blood glucose levels in HKO and control male mice (23 weeks). F and G : GTTs ( d -glucose: 2 g/kg body wt) and ITTs (1 unit/kg body wt) were performed in HKO and control males at age 22–23 weeks. Data are mean ± SEM. * P

    Journal: Diabetes

    Article Title: Hepatic TRAF2 Regulates Glucose Metabolism Through Enhancing Glucagon Responses

    doi: 10.2337/db11-0474

    Figure Lengend Snippet: Hepatic TRAF2 is dispensable for glucose metabolism under normal conditions. A : TRAF2 mRNA abundance was measured in the liver by qPCR and normalized to 36B4 mRNA levels. WT-NC, wild-type mice fed a normal chow diet (14 weeks); WT-HFD, WT mice fed an HFD (14 weeks); ob/ob , leptin-deficient ob/ob mice (14 weeks); AU, arbitrary unit. B : Total RNAs were extracted from WT and HKO mice and used to measure TRAF2 transcription by RT-PCR. C : Genomic DNA was prepared from purified hepatocytes and Kupffer cells and subjected to PCR-based genotyping analysis. D : TRAF2 mRNA abundance was measured in purified hepatocytes and Kupffer cells by qPCR and normalized to 36B4 mRNA levels. E : Fasting (overnight) blood glucose levels in HKO and control male mice (23 weeks). F and G : GTTs ( d -glucose: 2 g/kg body wt) and ITTs (1 unit/kg body wt) were performed in HKO and control males at age 22–23 weeks. Data are mean ± SEM. * P

    Article Snippet: The first-strand cDNAs were synthesized using random primers and Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). mRNA abundance was measured using ABsolute QPCR SYBR Mix (Thermo Fisher Scientific, Epsom, Surrey, U.K.) and Mx3000P real-time PCR system (Stratagene, La Jolla, CA).

    Techniques: Real-time Polymerase Chain Reaction, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Purification, Polymerase Chain Reaction

    Hepatocyte-specific deletion of TRAF2 suppresses the hepatic gluconeogenic program under HFD conditions. A : HKO and control (TRAF2 flox/flox : n = 13; albumin-Cre: n = 3) male mice were fed an HFD for 17 weeks. Mice were fasted for 16 h and intraperitoneally injected with sodium pyruvate (2 g/kg body wt). Blood glucose was monitored after injection, and AUCs were calculated. AU, arbitrary unit. B : Mice (7–8 weeks) were fed an HFD for 10–12 weeks and subjected to 2 H NMR analysis. 2 H NMR spectra of a MAG derived from plasma glucose (pooled from three animals per group) were presented. C : HKO and control males were fed an HFD for 18 weeks. Total liver RNAs were extracted and used to measure the mRNA abundance of the indicated genes by qPCR. The expression of these genes was normalized to the expression of 36B4. D : HKO and control males were fed an HFD for 18 weeks and fasted for 20–24 h. G6Pase activity was measured in liver microsomal fractions and normalized to microsomal protein levels. E : HKO and control males were fed an HFD for 18 weeks, and liver weight, glycogen contents, and triacylglycerol (TAG) levels were measured. Data are mean ± SEM. * P

    Journal: Diabetes

    Article Title: Hepatic TRAF2 Regulates Glucose Metabolism Through Enhancing Glucagon Responses

    doi: 10.2337/db11-0474

    Figure Lengend Snippet: Hepatocyte-specific deletion of TRAF2 suppresses the hepatic gluconeogenic program under HFD conditions. A : HKO and control (TRAF2 flox/flox : n = 13; albumin-Cre: n = 3) male mice were fed an HFD for 17 weeks. Mice were fasted for 16 h and intraperitoneally injected with sodium pyruvate (2 g/kg body wt). Blood glucose was monitored after injection, and AUCs were calculated. AU, arbitrary unit. B : Mice (7–8 weeks) were fed an HFD for 10–12 weeks and subjected to 2 H NMR analysis. 2 H NMR spectra of a MAG derived from plasma glucose (pooled from three animals per group) were presented. C : HKO and control males were fed an HFD for 18 weeks. Total liver RNAs were extracted and used to measure the mRNA abundance of the indicated genes by qPCR. The expression of these genes was normalized to the expression of 36B4. D : HKO and control males were fed an HFD for 18 weeks and fasted for 20–24 h. G6Pase activity was measured in liver microsomal fractions and normalized to microsomal protein levels. E : HKO and control males were fed an HFD for 18 weeks, and liver weight, glycogen contents, and triacylglycerol (TAG) levels were measured. Data are mean ± SEM. * P

    Article Snippet: The first-strand cDNAs were synthesized using random primers and Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). mRNA abundance was measured using ABsolute QPCR SYBR Mix (Thermo Fisher Scientific, Epsom, Surrey, U.K.) and Mx3000P real-time PCR system (Stratagene, La Jolla, CA).

    Techniques: Mouse Assay, Injection, Nuclear Magnetic Resonance, Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Activity Assay

    Hepatocyte-specific deletion of TRAF2 does not ameliorate liver inflammation levels under HFD conditions. HKO and control (Con) males (7–8 weeks) were fed an HFD for 18 weeks. A : Liver frozen sections were stained with anti-F4/80 antibody and DAPI. B : F4/80-positive cells were accounted and normalized to total cell numbers. AU, arbitrary unit. C : Total liver RNAs were extracted and used to measure the mRNA abundance of the indicated genes by qPCR. The expression of these genes was normalized to the expression of 36B4. Data are mean ± SEM. (A high-quality digital representation of this figure is available in the online issue.)

    Journal: Diabetes

    Article Title: Hepatic TRAF2 Regulates Glucose Metabolism Through Enhancing Glucagon Responses

    doi: 10.2337/db11-0474

    Figure Lengend Snippet: Hepatocyte-specific deletion of TRAF2 does not ameliorate liver inflammation levels under HFD conditions. HKO and control (Con) males (7–8 weeks) were fed an HFD for 18 weeks. A : Liver frozen sections were stained with anti-F4/80 antibody and DAPI. B : F4/80-positive cells were accounted and normalized to total cell numbers. AU, arbitrary unit. C : Total liver RNAs were extracted and used to measure the mRNA abundance of the indicated genes by qPCR. The expression of these genes was normalized to the expression of 36B4. Data are mean ± SEM. (A high-quality digital representation of this figure is available in the online issue.)

    Article Snippet: The first-strand cDNAs were synthesized using random primers and Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). mRNA abundance was measured using ABsolute QPCR SYBR Mix (Thermo Fisher Scientific, Epsom, Surrey, U.K.) and Mx3000P real-time PCR system (Stratagene, La Jolla, CA).

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

    TRAF2 directly promotes glucose counterregulation in hepatocytes. A : Primary hepatocyte cultures were isolated from C57BL/6 wild-type mice (10–11 weeks) and infected with GFP or TRAF2 adenoviruses. Cell extracts were prepared 16 h after infection and immunoblotted with antibodies against TRAF2 or tubulin. B : Primary hepatocytes were infected with GFP or TRAF2 adenoviruses and treated with glucagon 16 h after infection. HGP was measured 4 h after glucagon stimulation and normalized to total protein levels. n = 4. C : Primary hepatocytes were infected with GFP or TRAF2 adenoviruses and treated with or without glucagon 16 h after infection. Total RNAs were extracted 2 h after glucagon stimulation and used to measure the mRNA abundance of PEPCK and G6Pase by qPCR. The expression of PEPCK and G6Pase was normalized to 36B4 expression. D : Primary hepatocytes were infected with GFP or TRAF2 adenoviruses and treated with DB-cAMP (100 μmol/L for 2 h) 16 h after infection. Total RNAs were extracted and used to measure the mRNA abundance of PEPCK and G6Pase by qPCR. E : Primary hepatocytes were coinfected with CREB and GFP or TRAF2 adenoviruses. Sixteen hours after infection, the cells were treated with glucagon or DB-cAMP for 30 min. Cell extracts were immunoblotted with anti–phospho-CREB (pSer133) or anti-CREB antibodies. Data are mean ± SEM. * P

    Journal: Diabetes

    Article Title: Hepatic TRAF2 Regulates Glucose Metabolism Through Enhancing Glucagon Responses

    doi: 10.2337/db11-0474

    Figure Lengend Snippet: TRAF2 directly promotes glucose counterregulation in hepatocytes. A : Primary hepatocyte cultures were isolated from C57BL/6 wild-type mice (10–11 weeks) and infected with GFP or TRAF2 adenoviruses. Cell extracts were prepared 16 h after infection and immunoblotted with antibodies against TRAF2 or tubulin. B : Primary hepatocytes were infected with GFP or TRAF2 adenoviruses and treated with glucagon 16 h after infection. HGP was measured 4 h after glucagon stimulation and normalized to total protein levels. n = 4. C : Primary hepatocytes were infected with GFP or TRAF2 adenoviruses and treated with or without glucagon 16 h after infection. Total RNAs were extracted 2 h after glucagon stimulation and used to measure the mRNA abundance of PEPCK and G6Pase by qPCR. The expression of PEPCK and G6Pase was normalized to 36B4 expression. D : Primary hepatocytes were infected with GFP or TRAF2 adenoviruses and treated with DB-cAMP (100 μmol/L for 2 h) 16 h after infection. Total RNAs were extracted and used to measure the mRNA abundance of PEPCK and G6Pase by qPCR. E : Primary hepatocytes were coinfected with CREB and GFP or TRAF2 adenoviruses. Sixteen hours after infection, the cells were treated with glucagon or DB-cAMP for 30 min. Cell extracts were immunoblotted with anti–phospho-CREB (pSer133) or anti-CREB antibodies. Data are mean ± SEM. * P

    Article Snippet: The first-strand cDNAs were synthesized using random primers and Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). mRNA abundance was measured using ABsolute QPCR SYBR Mix (Thermo Fisher Scientific, Epsom, Surrey, U.K.) and Mx3000P real-time PCR system (Stratagene, La Jolla, CA).

    Techniques: Isolation, Mouse Assay, Infection, Real-time Polymerase Chain Reaction, Expressing

    C-Lipo with reduced lipofectamine amount transfects cardiac fibroblasts efficiently without significant toxicity. Notes: ( A ) Cell viability of C-Lipo (0.5 μL of lipofectamine with 7 μg of CRPPR-R9 peptide per 1 μg mRNA) after daily transfections. Cardiac fibroblasts were transfected with C-Lipo and Lipo daily for 1 week and cell viabilities were measured on days 4 and 7. C-Lipo allows mRNA transfection in cardiac fibroblasts without significant toxicity for 1 week compared to Lipo, which causes significant toxicity. ( B ) and ( C ) Flow cytometry analyses of cardiac fibroblasts treated with C-Lipo and Lipo. C-Lipo shows comparable levels of transfection efficiency and higher mean eGFP fluorescence compared to Lipo. The results represent the mean ± SE, n=5. * P

    Journal: International Journal of Nanomedicine

    Article Title: Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

    doi: 10.2147/IJN.S75124

    Figure Lengend Snippet: C-Lipo with reduced lipofectamine amount transfects cardiac fibroblasts efficiently without significant toxicity. Notes: ( A ) Cell viability of C-Lipo (0.5 μL of lipofectamine with 7 μg of CRPPR-R9 peptide per 1 μg mRNA) after daily transfections. Cardiac fibroblasts were transfected with C-Lipo and Lipo daily for 1 week and cell viabilities were measured on days 4 and 7. C-Lipo allows mRNA transfection in cardiac fibroblasts without significant toxicity for 1 week compared to Lipo, which causes significant toxicity. ( B ) and ( C ) Flow cytometry analyses of cardiac fibroblasts treated with C-Lipo and Lipo. C-Lipo shows comparable levels of transfection efficiency and higher mean eGFP fluorescence compared to Lipo. The results represent the mean ± SE, n=5. * P

    Article Snippet: Cellular uptake of Alexa594-mRNA via CRPPR-R9 in cardiac fibroblasts Alexa594 labeling was conducted on 10 μg of eGFP mRNA with Ulysis Alexa Fluor 594 nucleic acid labeling kit (Thermo Fisher Scientific) following the manufacturer’s manual.

    Techniques: Transfection, Flow Cytometry, Cytometry, Fluorescence

    CRPPR-R9 facilitates mRNA delivery in cardiac fibroblasts and CRPPR-R9/lipofectamine improves mRNA transfection efficiency. Notes: ( A ) Confocal images of cardiac fibroblasts treated with Alexa594-labeled mRNA/CRPPR-R9 complexes. mRNA and mRNA/CRPPR-R9 complexes were transfected into cardiac fibroblasts for 30 minutes. CRPPR-R9 induces facilitated mRNA uptake in cardiac fibroblasts. Alexa594-labeled mRNA and nucleus are shown in yellow and blue, respectively. Scale bar is 10 μm. ( B ) Transfection efficiency of eGFP mRNA on cardiac fibroblasts transfected by various combinations of CRPPR-R9 and lipofectamine and quantified by flow cytometry. CRPPR-R9 with lipofectamine increases eGFP mRNA transfection efficiency. ( C ) Mean eGFP fluorescence of cardiac fibroblasts quantified by flow cytometry. Mean eGFP fluorescence increases as transfection efficiency increases in CRPPR-R9 and lipofectamine transfection. The results represent the mean ± SE, n=4. * P

    Journal: International Journal of Nanomedicine

    Article Title: Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

    doi: 10.2147/IJN.S75124

    Figure Lengend Snippet: CRPPR-R9 facilitates mRNA delivery in cardiac fibroblasts and CRPPR-R9/lipofectamine improves mRNA transfection efficiency. Notes: ( A ) Confocal images of cardiac fibroblasts treated with Alexa594-labeled mRNA/CRPPR-R9 complexes. mRNA and mRNA/CRPPR-R9 complexes were transfected into cardiac fibroblasts for 30 minutes. CRPPR-R9 induces facilitated mRNA uptake in cardiac fibroblasts. Alexa594-labeled mRNA and nucleus are shown in yellow and blue, respectively. Scale bar is 10 μm. ( B ) Transfection efficiency of eGFP mRNA on cardiac fibroblasts transfected by various combinations of CRPPR-R9 and lipofectamine and quantified by flow cytometry. CRPPR-R9 with lipofectamine increases eGFP mRNA transfection efficiency. ( C ) Mean eGFP fluorescence of cardiac fibroblasts quantified by flow cytometry. Mean eGFP fluorescence increases as transfection efficiency increases in CRPPR-R9 and lipofectamine transfection. The results represent the mean ± SE, n=4. * P

    Article Snippet: Cellular uptake of Alexa594-mRNA via CRPPR-R9 in cardiac fibroblasts Alexa594 labeling was conducted on 10 μg of eGFP mRNA with Ulysis Alexa Fluor 594 nucleic acid labeling kit (Thermo Fisher Scientific) following the manufacturer’s manual.

    Techniques: Transfection, Labeling, Flow Cytometry, Cytometry, Fluorescence