rna  (Qiagen)

 
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    Name:
    RNase A
    Description:
    For RNase digestion during DNA preparation Kit contents Qiagen RNase A 17 500U 2 5mL 100mg mL Solution Endonuclease free Ready to use For RNase Digestion During DNA Preparation
    Catalog Number:
    19101
    Price:
    215
    Category:
    RNase A
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    Structured Review

    Qiagen rna
    RNase A
    For RNase digestion during DNA preparation Kit contents Qiagen RNase A 17 500U 2 5mL 100mg mL Solution Endonuclease free Ready to use For RNase Digestion During DNA Preparation
    https://www.bioz.com/result/rna/product/Qiagen
    Average 96 stars, based on 6041 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-07
    96/100 stars

    Images

    1) Product Images from "An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV"

    Article Title: An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

    Journal: Retrovirology

    doi: 10.1186/1742-4690-7-110

    Amplification of MLV-like viral sequences in Kit I . (A) One-step RT-PCR was conducted using Kit I with the indicated primer sets. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 35 (lanes 1, 3, 5 and 7) or 45 cycles (lanes 2, 4, 6 and 8) of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. Lanes 1, 2, 5 and 6: one-step RT-PCR with carrier RNA; Lanes 3, 4, 7 and 8: one-step RT-PCR without carrier RNA. Each reaction was carried out in duplicate. (B) One-step RT-PCR was conducted using Kit T (left panel) and Kit P (right panel) with primers 419F and 1154R with or without carrier RNA. The RT-PCR conditions using Kit T were as follows: reverse transcription at 50°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The RT-PCR conditions using Kit P were as follows: reverse transcription at 45°C for 45 minutes; activation at 95°C for 2 minutes; 45 cycles of the following steps: 95°C for 30 s, 57°C for 30 s, and 70°C for 45 s; and a final extension at 70°C for 5 minutes. (C) One-step RT-PCR was conducted with primers GAG-I-F and GAG-I-R using Kit I with or without RNaseA. Carrier RNA was not added to the reaction mixtures. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. (D) One-step RT-PCR was conducted using Kit I to amplify env region of the contaminants. One-step RT-PCR was carried out using two primer sets p-env1f and p-env1r (lane 1), and p-env3f and p-env5r (lane 2). The RT-PCR conditions were the same as in Figure 1C with the exception of the number of PCR cycles (60 cycles instead of 45 cycles). M: DNA size marker.
    Figure Legend Snippet: Amplification of MLV-like viral sequences in Kit I . (A) One-step RT-PCR was conducted using Kit I with the indicated primer sets. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 35 (lanes 1, 3, 5 and 7) or 45 cycles (lanes 2, 4, 6 and 8) of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. Lanes 1, 2, 5 and 6: one-step RT-PCR with carrier RNA; Lanes 3, 4, 7 and 8: one-step RT-PCR without carrier RNA. Each reaction was carried out in duplicate. (B) One-step RT-PCR was conducted using Kit T (left panel) and Kit P (right panel) with primers 419F and 1154R with or without carrier RNA. The RT-PCR conditions using Kit T were as follows: reverse transcription at 50°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The RT-PCR conditions using Kit P were as follows: reverse transcription at 45°C for 45 minutes; activation at 95°C for 2 minutes; 45 cycles of the following steps: 95°C for 30 s, 57°C for 30 s, and 70°C for 45 s; and a final extension at 70°C for 5 minutes. (C) One-step RT-PCR was conducted with primers GAG-I-F and GAG-I-R using Kit I with or without RNaseA. Carrier RNA was not added to the reaction mixtures. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. (D) One-step RT-PCR was conducted using Kit I to amplify env region of the contaminants. One-step RT-PCR was carried out using two primer sets p-env1f and p-env1r (lane 1), and p-env3f and p-env5r (lane 2). The RT-PCR conditions were the same as in Figure 1C with the exception of the number of PCR cycles (60 cycles instead of 45 cycles). M: DNA size marker.

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Polymerase Chain Reaction, Marker

    2) Product Images from "The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module"

    Article Title: The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx852

    Mutation of key lysine residues reduces  in vitro  and cellular RNA-binding. ( A ) Residue amides in FKBP25 (1–74)  that are strongly and moderately affected by titration with dsRNA-10 are coloured in orange and light orange, respectively. Orientations of the domain in the image are the same as in Figure   5 . Chemical shift perturbation details are in   Supplementary Figure S8A . ( B ) NMR spectra following titration of dsRNA-10 into 100 μM  15 N-labeled FKBP25 (1–74)  with the K22M/K23M or K48/K52A mutations. Colours as in Figure   5 . ( C  and  D ) Electrophoretic mobility shift assays with varying concentrations of full-length FKBP25 (K22M/K23M) as in Figure   4A  and   B . ( E ) Western blot analysis of FLAG-tagged FKBP25 constructs (wild-type  and the K22M/K23M mutant) relative to endogenous FKBP25 (empty vector control) in HEK 293 cells. Antibodies against α-tubulin, FLAG-tag and FKBP25 correspond to the loading control, detection of FKBP25 constructs, and detection of both endogenous and FKBP25 constructs, respectively. ( F ) FLAG-affinity capture of cells expressing an empty vector control, wild-type FKBP25, or the K22M/K23M mutant with analysis by western blot using antibodies against the FKBP25-interacting proteins Parp1, nucleolin and RPS6. FKBP25 construct expression verified by antibodies against the FLAG tag. ( G ) RNA cross-linking IP (CLIP) experiment with wild-type FKBP25 or the K22M/K23M mutant in HEK293 cells, with DNAse pre-treatment coupled with variable amounts of RNase A.
    Figure Legend Snippet: Mutation of key lysine residues reduces in vitro and cellular RNA-binding. ( A ) Residue amides in FKBP25 (1–74) that are strongly and moderately affected by titration with dsRNA-10 are coloured in orange and light orange, respectively. Orientations of the domain in the image are the same as in Figure 5 . Chemical shift perturbation details are in Supplementary Figure S8A . ( B ) NMR spectra following titration of dsRNA-10 into 100 μM 15 N-labeled FKBP25 (1–74) with the K22M/K23M or K48/K52A mutations. Colours as in Figure 5 . ( C and D ) Electrophoretic mobility shift assays with varying concentrations of full-length FKBP25 (K22M/K23M) as in Figure 4A and  B . ( E ) Western blot analysis of FLAG-tagged FKBP25 constructs (wild-type and the K22M/K23M mutant) relative to endogenous FKBP25 (empty vector control) in HEK 293 cells. Antibodies against α-tubulin, FLAG-tag and FKBP25 correspond to the loading control, detection of FKBP25 constructs, and detection of both endogenous and FKBP25 constructs, respectively. ( F ) FLAG-affinity capture of cells expressing an empty vector control, wild-type FKBP25, or the K22M/K23M mutant with analysis by western blot using antibodies against the FKBP25-interacting proteins Parp1, nucleolin and RPS6. FKBP25 construct expression verified by antibodies against the FLAG tag. ( G ) RNA cross-linking IP (CLIP) experiment with wild-type FKBP25 or the K22M/K23M mutant in HEK293 cells, with DNAse pre-treatment coupled with variable amounts of RNase A.

    Techniques Used: Mutagenesis, In Vitro, RNA Binding Assay, Titration, Nuclear Magnetic Resonance, Labeling, Electrophoretic Mobility Shift Assay, Western Blot, Construct, Plasmid Preparation, FLAG-tag, Expressing, Cross-linking Immunoprecipitation

    3) Product Images from "Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood"

    Article Title: Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood

    Journal: Journal of Extracellular Vesicles

    doi: 10.3402/jev.v3.23743

    Work flow of study design and sample processing. Whole blood from 3 different individuals was collected by venepuncture into each tube using a Multi-fly and processed to analyse intracellular, cell-free and exosomal miRNA. Asterisks indicate the point of RNaseA treatment (100 ng/ml, 37°C for 10 minutes) to investigate RNA degradation in these samples. The workflow outlines the sample collection and preparation from 1 individual. The number of tubes collected from each volunteer was: 2×PAXgene 2.5 ml tubes, 3×Sarstedt S-Monovette serum-gel 7.5 ml tubes and 3×Sarstedt S-Monovette EDTA 7.5 ml tubes. Upon centrifugation of the Sarstedt S-Monovette EDTA tubes, approximately 10 ml of plasma was obtained across 3 Sarstedt S-Monovette tubes which are then separately aliquoted into Lo-Bind DNA tubes (4×1 ml, 2×1.2 ml tubes) for RNA analysis and deep sequencing. The remaining plasma was aliquoted for Western immunoblotting (WB, 1.2 ml), transmission electron microscopy (EM, 1.2 ml) and qNano (1 ml) analysis. For the RNA work involving RNaseA treatment, samples were allocated for an untreated control and RNaseA treatment: 2×1.2 ml for the ultracentrifugation exosomal RNA isolation (Plasma UC), 2×1 ml for the Norgen Biotek exosomal RNA isolation (Plasma NG), and 2×1 ml aliquot was reserved for cell-free plasma RNA isolation. The collection process and sample allocation are repeated for serum collection. Exosomes isolated from serum via the ultracentrifuge are denoted as Serum UC. Exosomal RNA isolated by the Norgen Biotek Kit are denoted as Serum NG. As for the 2×PAXgene tubes, RNA is isolated from 2.5 ml of whole blood per tube and isolated as recommended by the manufacturers protocol. One tube was treated with RNaseA and one was left untreated.
    Figure Legend Snippet: Work flow of study design and sample processing. Whole blood from 3 different individuals was collected by venepuncture into each tube using a Multi-fly and processed to analyse intracellular, cell-free and exosomal miRNA. Asterisks indicate the point of RNaseA treatment (100 ng/ml, 37°C for 10 minutes) to investigate RNA degradation in these samples. The workflow outlines the sample collection and preparation from 1 individual. The number of tubes collected from each volunteer was: 2×PAXgene 2.5 ml tubes, 3×Sarstedt S-Monovette serum-gel 7.5 ml tubes and 3×Sarstedt S-Monovette EDTA 7.5 ml tubes. Upon centrifugation of the Sarstedt S-Monovette EDTA tubes, approximately 10 ml of plasma was obtained across 3 Sarstedt S-Monovette tubes which are then separately aliquoted into Lo-Bind DNA tubes (4×1 ml, 2×1.2 ml tubes) for RNA analysis and deep sequencing. The remaining plasma was aliquoted for Western immunoblotting (WB, 1.2 ml), transmission electron microscopy (EM, 1.2 ml) and qNano (1 ml) analysis. For the RNA work involving RNaseA treatment, samples were allocated for an untreated control and RNaseA treatment: 2×1.2 ml for the ultracentrifugation exosomal RNA isolation (Plasma UC), 2×1 ml for the Norgen Biotek exosomal RNA isolation (Plasma NG), and 2×1 ml aliquot was reserved for cell-free plasma RNA isolation. The collection process and sample allocation are repeated for serum collection. Exosomes isolated from serum via the ultracentrifuge are denoted as Serum UC. Exosomal RNA isolated by the Norgen Biotek Kit are denoted as Serum NG. As for the 2×PAXgene tubes, RNA is isolated from 2.5 ml of whole blood per tube and isolated as recommended by the manufacturers protocol. One tube was treated with RNaseA and one was left untreated.

    Techniques Used: Flow Cytometry, Centrifugation, Sequencing, Western Blot, Transmission Assay, Electron Microscopy, Isolation

    Small RNA profiles extracted from intracellular, cell-free and exosomal isolation from blood before and after RNaseA treatment. RNA was extracted from samples and run on a Small RNA Bioanalyser assay. Experiments shown here are representative of samples collected from 1 volunteer.
    Figure Legend Snippet: Small RNA profiles extracted from intracellular, cell-free and exosomal isolation from blood before and after RNaseA treatment. RNA was extracted from samples and run on a Small RNA Bioanalyser assay. Experiments shown here are representative of samples collected from 1 volunteer.

    Techniques Used: Isolation

    4) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00428

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
    Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

    RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
    Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Techniques Used: Activation Assay

    RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
    Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Techniques Used: Labeling, Injection, Mouse Assay

    RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
    Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

    Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
    Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

    Techniques Used:

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Staining

    Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Immunostaining, Staining

    RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used:

    RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
    Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Techniques Used: Immunostaining, Marker, Staining

    5) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00428

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
    Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

    RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
    Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Techniques Used: Activation Assay

    RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
    Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Techniques Used: Labeling, Injection, Mouse Assay

    RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
    Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

    Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
    Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

    Techniques Used:

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Staining

    Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Immunostaining, Staining

    RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used:

    RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
    Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Techniques Used: Immunostaining, Marker, Staining

    6) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00428

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
    Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

    RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
    Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Techniques Used: Activation Assay

    RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
    Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Techniques Used: Labeling, Injection, Mouse Assay

    RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
    Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

    Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
    Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

    Techniques Used:

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Staining

    Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Immunostaining, Staining

    RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used:

    RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
    Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Techniques Used: Immunostaining, Marker, Staining

    7) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00428

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
    Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

    RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
    Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Techniques Used: Activation Assay

    RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
    Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Techniques Used: Labeling, Injection, Mouse Assay

    RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
    Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

    Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
    Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

    Techniques Used:

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Staining

    Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Immunostaining, Staining

    RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used:

    RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
    Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Techniques Used: Immunostaining, Marker, Staining

    8) Product Images from "A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy"

    Article Title: A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1334

    CARM1 can be co-immunoprecipitated with UPF1 and the interaction between UPF1 and the β-Globin T39 mutant (MT) decreases with CARM1 knockdown. ( A ) Total cell lysates were prepared from MN-1 cells and subjected to immunoprecipitation with an IgG CTRL or UPF1 antibodies. Immunoprecipitated proteins were then analysed by western blot using antibodies against UPF1 and CARM1. ( B ) UPF1 immunoprecipitation experiments were performed with or without (w/o) pretreatment of the cell lysate with RNase A (1 μg/ml) for 30 min at 37°C. ( C ) Then, the CARM1/UPF1 ratios in response to the RNase A treatments were assessed. Values shown in the bar graph are means +/− SEM ( n = 3). ( D ) The MT reporter was transiently transfected either into the MN-1 pGIPZ CTRL or the MN-1 shCARM1 cell line. RT-PCR analysis was performed using primers specific for the MT mRNA or pre-mRNA, on total RNA extracted from the CTRL (left panel) or shCARM1 (right panel). ( E ) β-Globin mRNA levels, shown here as percent bound to UPF1, were normalized to overall immunoprecipated UPF1 levels and Gapdh mRNA was used as a loading control. Data are means +/− SEM ( n = 3).
    Figure Legend Snippet: CARM1 can be co-immunoprecipitated with UPF1 and the interaction between UPF1 and the β-Globin T39 mutant (MT) decreases with CARM1 knockdown. ( A ) Total cell lysates were prepared from MN-1 cells and subjected to immunoprecipitation with an IgG CTRL or UPF1 antibodies. Immunoprecipitated proteins were then analysed by western blot using antibodies against UPF1 and CARM1. ( B ) UPF1 immunoprecipitation experiments were performed with or without (w/o) pretreatment of the cell lysate with RNase A (1 μg/ml) for 30 min at 37°C. ( C ) Then, the CARM1/UPF1 ratios in response to the RNase A treatments were assessed. Values shown in the bar graph are means +/− SEM ( n = 3). ( D ) The MT reporter was transiently transfected either into the MN-1 pGIPZ CTRL or the MN-1 shCARM1 cell line. RT-PCR analysis was performed using primers specific for the MT mRNA or pre-mRNA, on total RNA extracted from the CTRL (left panel) or shCARM1 (right panel). ( E ) β-Globin mRNA levels, shown here as percent bound to UPF1, were normalized to overall immunoprecipated UPF1 levels and Gapdh mRNA was used as a loading control. Data are means +/− SEM ( n = 3).

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

    9) Product Images from "Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation"

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx362

    Deamination assay of HEK293T cell lysate expressing the wild-type A3B and mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A. ( E ) The amount of A3B and mutants in the 293T cells lysate are normalized to the similar levels, and confirmed by western blot. (F, G) EMSA assay of MBP-A3B-CD1m and MBP-A3B-CD1-4Y mutant with 30 nt ssDNA (F) and 50nt RNA ( G ).
    Figure Legend Snippet: Deamination assay of HEK293T cell lysate expressing the wild-type A3B and mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A. ( E ) The amount of A3B and mutants in the 293T cells lysate are normalized to the similar levels, and confirmed by western blot. (F, G) EMSA assay of MBP-A3B-CD1m and MBP-A3B-CD1-4Y mutant with 30 nt ssDNA (F) and 50nt RNA ( G ).

    Techniques Used: Expressing, Western Blot, Mutagenesis

    Analysis of the oligomeric status of the wild-type A3B. ( A ) Western blot of FPLC fractions of HEK293T cell lysate expressing A3B and A3G under no RNase A and with RNase A conditions. α-tubulin is an endogenous control. The fraction shift of A3B under with RNase A condition is due to the slightly variation of FPLC, as shown in Supplementary Figure S5A . ( B ) Western blot of FPLC fractions from MDA-MB231 cells lysate, showing the endogenous A3B. ( C ) The deamination activity of A3B FPLC fractions from A.
    Figure Legend Snippet: Analysis of the oligomeric status of the wild-type A3B. ( A ) Western blot of FPLC fractions of HEK293T cell lysate expressing A3B and A3G under no RNase A and with RNase A conditions. α-tubulin is an endogenous control. The fraction shift of A3B under with RNase A condition is due to the slightly variation of FPLC, as shown in Supplementary Figure S5A . ( B ) Western blot of FPLC fractions from MDA-MB231 cells lysate, showing the endogenous A3B. ( C ) The deamination activity of A3B FPLC fractions from A.

    Techniques Used: Western Blot, Fast Protein Liquid Chromatography, Expressing, Multiple Displacement Amplification, Activity Assay

    Deamination assay of HEK293T cell lysate expressing patch 1 and 2 mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A and without RNase A. ( E ) The expression of A3B and mutants in the 293T cells lysate are at similar levels, confirmed by western blot.
    Figure Legend Snippet: Deamination assay of HEK293T cell lysate expressing patch 1 and 2 mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A and without RNase A. ( E ) The expression of A3B and mutants in the 293T cells lysate are at similar levels, confirmed by western blot.

    Techniques Used: Expressing, Western Blot

    10) Product Images from "Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation"

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx362

    Deamination assay of HEK293T cell lysate expressing the wild-type A3B and mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A. ( E ) The amount of A3B and mutants in the 293T cells lysate are normalized to the similar levels, and confirmed by western blot. (F, G) EMSA assay of MBP-A3B-CD1m and MBP-A3B-CD1-4Y mutant with 30 nt ssDNA (F) and 50nt RNA ( G ).
    Figure Legend Snippet: Deamination assay of HEK293T cell lysate expressing the wild-type A3B and mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A. ( E ) The amount of A3B and mutants in the 293T cells lysate are normalized to the similar levels, and confirmed by western blot. (F, G) EMSA assay of MBP-A3B-CD1m and MBP-A3B-CD1-4Y mutant with 30 nt ssDNA (F) and 50nt RNA ( G ).

    Techniques Used: Expressing, Western Blot, Mutagenesis

    Analysis of the oligomeric status of the wild-type A3B. ( A ) Western blot of FPLC fractions of HEK293T cell lysate expressing A3B and A3G under no RNase A and with RNase A conditions. α-tubulin is an endogenous control. The fraction shift of A3B under with RNase A condition is due to the slightly variation of FPLC, as shown in Supplementary Figure S5A . ( B ) Western blot of FPLC fractions from MDA-MB231 cells lysate, showing the endogenous A3B. ( C ) The deamination activity of A3B FPLC fractions from A.
    Figure Legend Snippet: Analysis of the oligomeric status of the wild-type A3B. ( A ) Western blot of FPLC fractions of HEK293T cell lysate expressing A3B and A3G under no RNase A and with RNase A conditions. α-tubulin is an endogenous control. The fraction shift of A3B under with RNase A condition is due to the slightly variation of FPLC, as shown in Supplementary Figure S5A . ( B ) Western blot of FPLC fractions from MDA-MB231 cells lysate, showing the endogenous A3B. ( C ) The deamination activity of A3B FPLC fractions from A.

    Techniques Used: Western Blot, Fast Protein Liquid Chromatography, Expressing, Multiple Displacement Amplification, Activity Assay

    Deamination assay of HEK293T cell lysate expressing patch 1 and 2 mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A and without RNase A. ( E ) The expression of A3B and mutants in the 293T cells lysate are at similar levels, confirmed by western blot.
    Figure Legend Snippet: Deamination assay of HEK293T cell lysate expressing patch 1 and 2 mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A and without RNase A. ( E ) The expression of A3B and mutants in the 293T cells lysate are at similar levels, confirmed by western blot.

    Techniques Used: Expressing, Western Blot

    11) Product Images from "Circular RNA TTN Acts As a miR-432 Sponge to Facilitate Proliferation and Differentiation of Myoblasts via the IGF2/PI3K/AKT Signaling Pathway"

    Article Title: Circular RNA TTN Acts As a miR-432 Sponge to Facilitate Proliferation and Differentiation of Myoblasts via the IGF2/PI3K/AKT Signaling Pathway

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.10.019

    Characterization of Bovine circTTN (A) Schematic view illustrating the design of primers for circTTN . A divergent primer was also used in quantitative real-time PCR. (B) A convergent primer and divergent primer were used to confirm the circular nature of circTTN . (C) The circular junction of circTTN was identified by using a divergent primer on Sanger sequencing. (D) RNase R detected the presence of circTTN . (E) The expression of circTTN and TTN mRNA in myoblasts treated with RNase R was determined by quantitative real-time PCR. (F) Quantitative real-time PCR for the abundance of circTTN and TTN mRNA in bovine primary myoblasts treated with Actinomycin D at the indicated time points. (G) RNA-FISH assay was performed to determine circTTN subcellular localization. Blue indicates nuclei stained with DAPI; green indicates the RNA probe that recognizes circTTN . Scale bar, 50 μm. (H) The expression of circTTN in the cytoplasm and nuclear was detected by semiquantitative PCR. (I) The expression of circTTN in myoblasts differentiated for −2, −1, 0, 1, 3, and 5 days is shown. Data are presented as means ± SEM for three individuals.
    Figure Legend Snippet: Characterization of Bovine circTTN (A) Schematic view illustrating the design of primers for circTTN . A divergent primer was also used in quantitative real-time PCR. (B) A convergent primer and divergent primer were used to confirm the circular nature of circTTN . (C) The circular junction of circTTN was identified by using a divergent primer on Sanger sequencing. (D) RNase R detected the presence of circTTN . (E) The expression of circTTN and TTN mRNA in myoblasts treated with RNase R was determined by quantitative real-time PCR. (F) Quantitative real-time PCR for the abundance of circTTN and TTN mRNA in bovine primary myoblasts treated with Actinomycin D at the indicated time points. (G) RNA-FISH assay was performed to determine circTTN subcellular localization. Blue indicates nuclei stained with DAPI; green indicates the RNA probe that recognizes circTTN . Scale bar, 50 μm. (H) The expression of circTTN in the cytoplasm and nuclear was detected by semiquantitative PCR. (I) The expression of circTTN in myoblasts differentiated for −2, −1, 0, 1, 3, and 5 days is shown. Data are presented as means ± SEM for three individuals.

    Techniques Used: Real-time Polymerase Chain Reaction, Sequencing, Expressing, Fluorescence In Situ Hybridization, Staining, Polymerase Chain Reaction

    12) Product Images from "Production, Purification, and Capsid Stability of Rhinovirus C Types"

    Article Title: Production, Purification, and Capsid Stability of Rhinovirus C Types

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2015.02.019

    Response of RV-C41 to physical stress HeLa cell lysates containing newly synthesized A16 or C41 were subjected to conditions intended to induce potential capsid disassembly. The assays measured gain/loss of vRNA PCR signal, following treatment with RNAse A. To assay osmotic stress factors, (A), lysates were incubated for 1h, with shaking at RT in PBS containing 0, 1, 3, 10, or 30% sucrose (w/v, n=3). To assay thermal stress (B), lysates were incubated for 1h at 4, 37, 46, 55, or 65°C (n=5). To assay pH stress (C), lysates were diluted into citrate-phosphate buffers with the indicated pH, and then incubated for 1h with shaking (n=3). *, **, ***  p -value
    Figure Legend Snippet: Response of RV-C41 to physical stress HeLa cell lysates containing newly synthesized A16 or C41 were subjected to conditions intended to induce potential capsid disassembly. The assays measured gain/loss of vRNA PCR signal, following treatment with RNAse A. To assay osmotic stress factors, (A), lysates were incubated for 1h, with shaking at RT in PBS containing 0, 1, 3, 10, or 30% sucrose (w/v, n=3). To assay thermal stress (B), lysates were incubated for 1h at 4, 37, 46, 55, or 65°C (n=5). To assay pH stress (C), lysates were diluted into citrate-phosphate buffers with the indicated pH, and then incubated for 1h with shaking (n=3). *, **, *** p -value

    Techniques Used: Synthesized, Polymerase Chain Reaction, Incubation

    13) Product Images from "Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein"

    Article Title: Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein

    Journal: Journal of Virology

    doi:

    Stimulation of coupled joining by various nucleic acid binding proteins. (A) Products generated in the presence of 35 nM purified integrase and the indicated viral proteins. Expected structures of integration products are shown beside the gel. Maximum concentrations for each (right-most lane in each titration) are as follows: NC, 8 μg/ml (1 μM); MA, 16 μg/ml (0.94 μM); Rev, 4 μg/ml (0.2 μM); Tat, 4 μg/ml (0.28 μM); RT, 4 μg/ml (78 nM). Each protein was diluted 1:10 and 1:100 in the two left lanes. (B) Products generated in the presence of 35 nM purified integrase and the indicated cellular DNA binding proteins. Maximum concentrations for each (right-most lane in each titration) are as follows: NC, 8 μg/ml (1 μM); HMG I(Y), 16 μg/ml (1.4 μM); HMG-1, 4 μg/ml (0.16 μM); HMG-2, 8 μg/ml (0.64 μM); histone H1, 8 μg/ml (0.4 μM); Hu, 2.4 μg/ml (0.13 μM); BAF, 2 μg/ml (0.2 μM); RNase A, 4 μg/ml (0.3 μM); polylysine, 4 μg/ml (4 μM). Each protein was diluted 1:10 and 1:100 in the two left lanes.
    Figure Legend Snippet: Stimulation of coupled joining by various nucleic acid binding proteins. (A) Products generated in the presence of 35 nM purified integrase and the indicated viral proteins. Expected structures of integration products are shown beside the gel. Maximum concentrations for each (right-most lane in each titration) are as follows: NC, 8 μg/ml (1 μM); MA, 16 μg/ml (0.94 μM); Rev, 4 μg/ml (0.2 μM); Tat, 4 μg/ml (0.28 μM); RT, 4 μg/ml (78 nM). Each protein was diluted 1:10 and 1:100 in the two left lanes. (B) Products generated in the presence of 35 nM purified integrase and the indicated cellular DNA binding proteins. Maximum concentrations for each (right-most lane in each titration) are as follows: NC, 8 μg/ml (1 μM); HMG I(Y), 16 μg/ml (1.4 μM); HMG-1, 4 μg/ml (0.16 μM); HMG-2, 8 μg/ml (0.64 μM); histone H1, 8 μg/ml (0.4 μM); Hu, 2.4 μg/ml (0.13 μM); BAF, 2 μg/ml (0.2 μM); RNase A, 4 μg/ml (0.3 μM); polylysine, 4 μg/ml (4 μM). Each protein was diluted 1:10 and 1:100 in the two left lanes.

    Techniques Used: Binding Assay, Generated, Purification, Titration, DNA Binding Assay

    14) Product Images from "Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation"

    Article Title: Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation

    Journal: Nature Communications

    doi: 10.1038/ncomms8815

    Presence of ssDNA at condensin binding sites. ( a ) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). ( b ) RNase treatment of condensin-bound DNA fragments. RNase A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. ( n =2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.
    Figure Legend Snippet: Presence of ssDNA at condensin binding sites. ( a ) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). ( b ) RNase treatment of condensin-bound DNA fragments. RNase A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. ( n =2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.

    Techniques Used: Binding Assay, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    15) Product Images from "Exosome can prevent RNase from degrading microRNA in feces"

    Article Title: Exosome can prevent RNase from degrading microRNA in feces

    Journal: Journal of Gastrointestinal Oncology

    doi: 10.3978/j.issn.2078-6891.2011.015

    Degradation of naked RNA using RNase. (A) Electropherogram of total RNA treated with RNase. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 4°C and 37°C. Two peaks, 18S and 28S ribosomal RNA (rRNA), are observed in total RNA without RNase treatment. (B) Relative quantification (RQ) of each miRNA treated with RNase at 4°C. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 4°C. RQ of each miRNA is normalized by 18S rRNA (C) RQ of each miRNA treated with RNase at 37°C. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 37°C. RQ of each miRNA is normalized by 18S rRNA.
    Figure Legend Snippet: Degradation of naked RNA using RNase. (A) Electropherogram of total RNA treated with RNase. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 4°C and 37°C. Two peaks, 18S and 28S ribosomal RNA (rRNA), are observed in total RNA without RNase treatment. (B) Relative quantification (RQ) of each miRNA treated with RNase at 4°C. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 4°C. RQ of each miRNA is normalized by 18S rRNA (C) RQ of each miRNA treated with RNase at 37°C. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 37°C. RQ of each miRNA is normalized by 18S rRNA.

    Techniques Used:

    RQ of each miRNA in fecal samples treated with RNase. (A) RQ of each miRNA in cellular miRNA treated with RNase. Exfoliated colonocytes are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each miRNA is compared with that of a no-treatment group. (B) RQ of each miRNA in exosomal miRNA treated with RNase. Exosomes are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each group is compared with that of a no-treatment group. (C) RQ of each miRNA in fecal RNA treated with RNase. Fecal samples are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each group is compared with that of a no-treatment group. Mean ± SD.
    Figure Legend Snippet: RQ of each miRNA in fecal samples treated with RNase. (A) RQ of each miRNA in cellular miRNA treated with RNase. Exfoliated colonocytes are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each miRNA is compared with that of a no-treatment group. (B) RQ of each miRNA in exosomal miRNA treated with RNase. Exosomes are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each group is compared with that of a no-treatment group. (C) RQ of each miRNA in fecal RNA treated with RNase. Fecal samples are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each group is compared with that of a no-treatment group. Mean ± SD.

    Techniques Used:

    16) Product Images from "The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts"

    Article Title: The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005761

    RNase treatment strongly reduces MLE signal at the integration site of the plasmid. (A) Left panel, MLE staining of polytene chromosomes from male larvae expressing a dsRNA targeting Hrb87F after induction with Actin5C-GAL4 . The incubation of the salivary glands in RNase A (RNase A) perturbs MLE localization at the integration site of the plasmid while in the absence of RNase A (ctrl) the MLE signal is still highly enriched. The white arrows indicate the plasmid integration site. In the right panel is a detail of the region marked by the arrows. (B) Quantitative analysis of fluorescence levels. MLE signal at the integration site of the plasmid, expressed in terms of corrected total band fluorescence (CTBF), is significantly reduced after RNase A treatment (p value
    Figure Legend Snippet: RNase treatment strongly reduces MLE signal at the integration site of the plasmid. (A) Left panel, MLE staining of polytene chromosomes from male larvae expressing a dsRNA targeting Hrb87F after induction with Actin5C-GAL4 . The incubation of the salivary glands in RNase A (RNase A) perturbs MLE localization at the integration site of the plasmid while in the absence of RNase A (ctrl) the MLE signal is still highly enriched. The white arrows indicate the plasmid integration site. In the right panel is a detail of the region marked by the arrows. (B) Quantitative analysis of fluorescence levels. MLE signal at the integration site of the plasmid, expressed in terms of corrected total band fluorescence (CTBF), is significantly reduced after RNase A treatment (p value

    Techniques Used: Plasmid Preparation, Staining, Expressing, Incubation, Fluorescence, Significance Assay

    17) Product Images from "Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells"

    Article Title: Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw312

    PKR binding to HCV 5′NTR is out-competed by HCV IRES domain IIId. ( A ) PKR binds to HCV 5′NTR. One picomole of Cy5-labeled HCV 5′NTR was incubated with increasing concentrations of purified PKR (0.75, 1.5, 3 and 6 pmol) (lanes 1–4). The samples were UV-irradiated, then treated with RNase A and resolved by SDS-PAGE. Crosslinked RNA–protein complexes were detected using a Typhoon scanner. ( B ) PKR competes specifically for domain IIId of the IRES region. Purified PKR was crosslinked with Cy5-labeled 5′NTR in the absence (lane 1) or presence (lanes 3–5) of unlabeled competitor RNA corresponding to domains I+II, IIIabc, IIId and IV. ( C ) PKR specifically binds to domain IIId of HCV IRES. Internally 32 P-labeled in-vitro -transcribed RNA fragments corresponding to domains I+II, IIIabc, IIId and IV were crosslinked to PKR, treated with RNase A and resolved by SDS-PAGE. Only domain IIId crosslinked with PKR (lane 3). ( D ) Stau1 and PKR compete for the same binding site on HCV 5′NTR IIId region. We first incubated a fixed concentration (1 pmol) of Stau1 or PKR with 1 pmol in-vitro -transcribed Cy5-labeled domain IIId RNA on ice for 15 min and then supplemented with increasing concentration (1–3 pmol) of the competitor (PKR or Stau1) protein. After 20-min incubation on ice, the mixture was photocrosslinked, treated with RNase-A and resolved by SDS-PAGE. Lanes 1 and 6, respectively, represent binding of Stau1 and PKR to HCV IRES domain IIId RNA in the absence of competitor protein. Lanes 2–3 represents the competition of Stau1 binding to domain IIId RNA with PKR; Lanes 7–9 represent the competition of PKR binding to domain IIId RNA with Stau1.
    Figure Legend Snippet: PKR binding to HCV 5′NTR is out-competed by HCV IRES domain IIId. ( A ) PKR binds to HCV 5′NTR. One picomole of Cy5-labeled HCV 5′NTR was incubated with increasing concentrations of purified PKR (0.75, 1.5, 3 and 6 pmol) (lanes 1–4). The samples were UV-irradiated, then treated with RNase A and resolved by SDS-PAGE. Crosslinked RNA–protein complexes were detected using a Typhoon scanner. ( B ) PKR competes specifically for domain IIId of the IRES region. Purified PKR was crosslinked with Cy5-labeled 5′NTR in the absence (lane 1) or presence (lanes 3–5) of unlabeled competitor RNA corresponding to domains I+II, IIIabc, IIId and IV. ( C ) PKR specifically binds to domain IIId of HCV IRES. Internally 32 P-labeled in-vitro -transcribed RNA fragments corresponding to domains I+II, IIIabc, IIId and IV were crosslinked to PKR, treated with RNase A and resolved by SDS-PAGE. Only domain IIId crosslinked with PKR (lane 3). ( D ) Stau1 and PKR compete for the same binding site on HCV 5′NTR IIId region. We first incubated a fixed concentration (1 pmol) of Stau1 or PKR with 1 pmol in-vitro -transcribed Cy5-labeled domain IIId RNA on ice for 15 min and then supplemented with increasing concentration (1–3 pmol) of the competitor (PKR or Stau1) protein. After 20-min incubation on ice, the mixture was photocrosslinked, treated with RNase-A and resolved by SDS-PAGE. Lanes 1 and 6, respectively, represent binding of Stau1 and PKR to HCV IRES domain IIId RNA in the absence of competitor protein. Lanes 2–3 represents the competition of Stau1 binding to domain IIId RNA with PKR; Lanes 7–9 represent the competition of PKR binding to domain IIId RNA with Stau1.

    Techniques Used: Binding Assay, Labeling, Incubation, Purification, Irradiation, SDS Page, In Vitro, Concentration Assay

    18) Product Images from "Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells"

    Article Title: Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw312

    PKR binding to HCV 5′NTR is out-competed by HCV IRES domain IIId. ( A ) PKR binds to HCV 5′NTR. One picomole of Cy5-labeled HCV 5′NTR was incubated with increasing concentrations of purified PKR (0.75, 1.5, 3 and 6 pmol) (lanes 1–4). The samples were UV-irradiated, then treated with RNase A and resolved by SDS-PAGE. Crosslinked RNA–protein complexes were detected using a Typhoon scanner. ( B ) PKR competes specifically for domain IIId of the IRES region. Purified PKR was crosslinked with Cy5-labeled 5′NTR in the absence (lane 1) or presence (lanes 3–5) of unlabeled competitor RNA corresponding to domains I+II, IIIabc, IIId and IV. ( C ) PKR specifically binds to domain IIId of HCV IRES. Internally 32 P-labeled in-vitro -transcribed RNA fragments corresponding to domains I+II, IIIabc, IIId and IV were crosslinked to PKR, treated with RNase A and resolved by SDS-PAGE. Only domain IIId crosslinked with PKR (lane 3). ( D ) Stau1 and PKR compete for the same binding site on HCV 5′NTR IIId region. We first incubated a fixed concentration (1 pmol) of Stau1 or PKR with 1 pmol in-vitro -transcribed Cy5-labeled domain IIId RNA on ice for 15 min and then supplemented with increasing concentration (1–3 pmol) of the competitor (PKR or Stau1) protein. After 20-min incubation on ice, the mixture was photocrosslinked, treated with RNase-A and resolved by SDS-PAGE. Lanes 1 and 6, respectively, represent binding of Stau1 and PKR to HCV IRES domain IIId RNA in the absence of competitor protein. Lanes 2–3 represents the competition of Stau1 binding to domain IIId RNA with PKR; Lanes 7–9 represent the competition of PKR binding to domain IIId RNA with Stau1.
    Figure Legend Snippet: PKR binding to HCV 5′NTR is out-competed by HCV IRES domain IIId. ( A ) PKR binds to HCV 5′NTR. One picomole of Cy5-labeled HCV 5′NTR was incubated with increasing concentrations of purified PKR (0.75, 1.5, 3 and 6 pmol) (lanes 1–4). The samples were UV-irradiated, then treated with RNase A and resolved by SDS-PAGE. Crosslinked RNA–protein complexes were detected using a Typhoon scanner. ( B ) PKR competes specifically for domain IIId of the IRES region. Purified PKR was crosslinked with Cy5-labeled 5′NTR in the absence (lane 1) or presence (lanes 3–5) of unlabeled competitor RNA corresponding to domains I+II, IIIabc, IIId and IV. ( C ) PKR specifically binds to domain IIId of HCV IRES. Internally 32 P-labeled in-vitro -transcribed RNA fragments corresponding to domains I+II, IIIabc, IIId and IV were crosslinked to PKR, treated with RNase A and resolved by SDS-PAGE. Only domain IIId crosslinked with PKR (lane 3). ( D ) Stau1 and PKR compete for the same binding site on HCV 5′NTR IIId region. We first incubated a fixed concentration (1 pmol) of Stau1 or PKR with 1 pmol in-vitro -transcribed Cy5-labeled domain IIId RNA on ice for 15 min and then supplemented with increasing concentration (1–3 pmol) of the competitor (PKR or Stau1) protein. After 20-min incubation on ice, the mixture was photocrosslinked, treated with RNase-A and resolved by SDS-PAGE. Lanes 1 and 6, respectively, represent binding of Stau1 and PKR to HCV IRES domain IIId RNA in the absence of competitor protein. Lanes 2–3 represents the competition of Stau1 binding to domain IIId RNA with PKR; Lanes 7–9 represent the competition of PKR binding to domain IIId RNA with Stau1.

    Techniques Used: Binding Assay, Labeling, Incubation, Purification, Irradiation, SDS Page, In Vitro, Concentration Assay

    19) Product Images from "Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β"

    Article Title: Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.90

    IFN- β is derived from activated hMSCs, following coculture with MDA cells. ( a ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Values are mean±S.D. for triplicate of the assay. ( b ) Real-time RT-PCR for IFN- β from hMSCs treated with apoptotic MDA (MB-231) cells (Apot cells). Apoptotic MDA cells were treated with either RNase (R) or DNase (D). Values are mean±S.D. for triplicate of the assay. ( c ) Quantitative RT-PCR for IFN- β from hMSCs treated with TNF- α and either poly(I:C) or poly(dA:dT) (1 μ g/ml). Values are mean±S.D. for triplicate of the assay. ( d ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Before coculture, hMSCs were treated with siRNA for AIM2 (siAIM2 hMSCs) or IFIH1 (siIFIH1 hMSCs). Scrambled siRNA (siSCR hMSCs) were used as a control. Values are mean±S.D. for triplicate of the assay. ( e ) Quantitative RT-PCR assays for AIM2, IFIH1 and IFN- β in hMSCs, following coculture with Hcc38, MCF-7 and BT20 cells for 24 h. CC, hMSCs+cancer cells; CCT, hMSCs+cancer cells+TNF- α . Values are mean±S.D. for triplicate of the assay. ( f ) Western blot assay for TRAIL expression in BT20, Hcc38 and MCF-7 cells from control (C), TNF- α (T; 20 ng/ml) treatment or coculture with act hMSCs (MDA+hMSCs+TNF- α ; CCT)
    Figure Legend Snippet: IFN- β is derived from activated hMSCs, following coculture with MDA cells. ( a ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Values are mean±S.D. for triplicate of the assay. ( b ) Real-time RT-PCR for IFN- β from hMSCs treated with apoptotic MDA (MB-231) cells (Apot cells). Apoptotic MDA cells were treated with either RNase (R) or DNase (D). Values are mean±S.D. for triplicate of the assay. ( c ) Quantitative RT-PCR for IFN- β from hMSCs treated with TNF- α and either poly(I:C) or poly(dA:dT) (1 μ g/ml). Values are mean±S.D. for triplicate of the assay. ( d ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Before coculture, hMSCs were treated with siRNA for AIM2 (siAIM2 hMSCs) or IFIH1 (siIFIH1 hMSCs). Scrambled siRNA (siSCR hMSCs) were used as a control. Values are mean±S.D. for triplicate of the assay. ( e ) Quantitative RT-PCR assays for AIM2, IFIH1 and IFN- β in hMSCs, following coculture with Hcc38, MCF-7 and BT20 cells for 24 h. CC, hMSCs+cancer cells; CCT, hMSCs+cancer cells+TNF- α . Values are mean±S.D. for triplicate of the assay. ( f ) Western blot assay for TRAIL expression in BT20, Hcc38 and MCF-7 cells from control (C), TNF- α (T; 20 ng/ml) treatment or coculture with act hMSCs (MDA+hMSCs+TNF- α ; CCT)

    Techniques Used: Derivative Assay, Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot, Expressing, Activated Clotting Time Assay

    20) Product Images from "Evidence in Support of RNA-Mediated Inhibition of Phosphatidylserine-Dependent HIV-1 Gag Membrane Binding in Cells"

    Article Title: Evidence in Support of RNA-Mediated Inhibition of Phosphatidylserine-Dependent HIV-1 Gag Membrane Binding in Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.00075-13

    tRNA below intracellular levels inhibits Gag binding to liposomes containing PS but not PI(4,5)P 2 . (A) [ 35 S]-labeled Gag synthesized using rabbit reticulocyte lysates was treated with 400 ng of RNase A at 37°C for 20 min. RNase A was blocked using
    Figure Legend Snippet: tRNA below intracellular levels inhibits Gag binding to liposomes containing PS but not PI(4,5)P 2 . (A) [ 35 S]-labeled Gag synthesized using rabbit reticulocyte lysates was treated with 400 ng of RNase A at 37°C for 20 min. RNase A was blocked using

    Techniques Used: Binding Assay, Labeling, Synthesized

    21) Product Images from "Evidence in Support of RNA-Mediated Inhibition of Phosphatidylserine-Dependent HIV-1 Gag Membrane Binding in Cells"

    Article Title: Evidence in Support of RNA-Mediated Inhibition of Phosphatidylserine-Dependent HIV-1 Gag Membrane Binding in Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.00075-13

    tRNA below intracellular levels inhibits Gag binding to liposomes containing PS but not PI(4,5)P 2 . (A) [ 35 S]-labeled Gag synthesized using rabbit reticulocyte lysates was treated with 400 ng of RNase A at 37°C for 20 min. RNase A was blocked using
    Figure Legend Snippet: tRNA below intracellular levels inhibits Gag binding to liposomes containing PS but not PI(4,5)P 2 . (A) [ 35 S]-labeled Gag synthesized using rabbit reticulocyte lysates was treated with 400 ng of RNase A at 37°C for 20 min. RNase A was blocked using

    Techniques Used: Binding Assay, Labeling, Synthesized

    22) Product Images from "Activation-Induced Deaminase, AID, is catalytically active as a monomer on single-stranded DNA"

    Article Title: Activation-Induced Deaminase, AID, is catalytically active as a monomer on single-stranded DNA

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2007.08.002

    RNAse A is required for deamination, but pre-incubation does not alter the rate of deamination. 10 nM of L-oligo was incubated with 500 nM of AID protein without any RNaseA (triangles) or with 400 ng RNaseA added either with the oligo (diamonds) or 30 minutes before the additon of oligo (squares).
    Figure Legend Snippet: RNAse A is required for deamination, but pre-incubation does not alter the rate of deamination. 10 nM of L-oligo was incubated with 500 nM of AID protein without any RNaseA (triangles) or with 400 ng RNaseA added either with the oligo (diamonds) or 30 minutes before the additon of oligo (squares).

    Techniques Used: Incubation

    23) Product Images from "Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H"

    Article Title: Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21955-0

    Positively charged patches are important for subcellular localization and deaminase activity of A3H. ( A ) A3H structure showing the positively charged residues mutated in three patch mutants (patch 1–3). ( B ) Cell fractionation analysis of A3H and various mutants, showing the distribution between nucleus and cytosol in HEK293T cells. Transfected 293T cells expressing wild-type A3H hap I, hap II, and various hap II mutants were fractionated into whole cell (WC), cytoplasmic (Cyto) and nuclear (Nuc) fractions. A3B (mostly nucleus) and A3G (both cytoplasm and nucleus) were also used as controls. FLAG-A3H proteins in each fraction were analyzed by Western blot. ( C ) The deaminase assay of selected A3H mutants using the cell lysates of transfected HEK293T cells with or without RNase A treatment. The deaminase reaction was performed with cell lysate range of 0–6 μg (total protein amount, 2-fold dilutions from 6 μg) and 300 nM ssDNA.
    Figure Legend Snippet: Positively charged patches are important for subcellular localization and deaminase activity of A3H. ( A ) A3H structure showing the positively charged residues mutated in three patch mutants (patch 1–3). ( B ) Cell fractionation analysis of A3H and various mutants, showing the distribution between nucleus and cytosol in HEK293T cells. Transfected 293T cells expressing wild-type A3H hap I, hap II, and various hap II mutants were fractionated into whole cell (WC), cytoplasmic (Cyto) and nuclear (Nuc) fractions. A3B (mostly nucleus) and A3G (both cytoplasm and nucleus) were also used as controls. FLAG-A3H proteins in each fraction were analyzed by Western blot. ( C ) The deaminase assay of selected A3H mutants using the cell lysates of transfected HEK293T cells with or without RNase A treatment. The deaminase reaction was performed with cell lysate range of 0–6 μg (total protein amount, 2-fold dilutions from 6 μg) and 300 nM ssDNA.

    Techniques Used: Activity Assay, Cell Fractionation, Transfection, Expressing, Western Blot

    Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After RNase A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.
    Figure Legend Snippet: Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After RNase A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.

    Techniques Used: Inhibition, Activity Assay, Molecular Weight, Expressing, Size-exclusion Chromatography, Western Blot

    Protein purification and the overall structure of A3H. ( A ) SEC elution profiles of MBP-A3H dimeric and monomeric mutants on Superdex 200. A3H m1 forms a stable dimer after extensive RNase A treatment (blue). The purified m1 dimer can dissociate to monomer and free RNA after RNase A treatment followed by 1.5 M or higher salt buffer (black). The RNA-bound m1 dimer was disrupted by two sets of mutations on loop 7: H114A (m1+H114A) or W115A/C116S (m1+W115A/C116S), and clean monomers were purified from m1+H114A (light blue) m1+W115A/C116S (green). ( B ) MALS of MBP-fused m1+W115A/C116S mutant, showing the clean monomeric form. The expected molecular mass of a monomer is 63.2 kDa. ( C,D ) Crystal structure of A3H m1+W115A/C116S monomer mutant ( C ) and the superimposition of the A3H (green) with A3A (PDB: 4XXO, yellow), A3B-CD2 (PDB: 5CQI, salmon) and AID (PDB: 5W0R, purple) ( D ), with secondary structures indicated (Supplementary Figure S2A ). The long helix 6 (h6), break of β5, and the long loop 1 of A3H can be visualized in panels C and D (Supplementary Figure S3A , B ).
    Figure Legend Snippet: Protein purification and the overall structure of A3H. ( A ) SEC elution profiles of MBP-A3H dimeric and monomeric mutants on Superdex 200. A3H m1 forms a stable dimer after extensive RNase A treatment (blue). The purified m1 dimer can dissociate to monomer and free RNA after RNase A treatment followed by 1.5 M or higher salt buffer (black). The RNA-bound m1 dimer was disrupted by two sets of mutations on loop 7: H114A (m1+H114A) or W115A/C116S (m1+W115A/C116S), and clean monomers were purified from m1+H114A (light blue) m1+W115A/C116S (green). ( B ) MALS of MBP-fused m1+W115A/C116S mutant, showing the clean monomeric form. The expected molecular mass of a monomer is 63.2 kDa. ( C,D ) Crystal structure of A3H m1+W115A/C116S monomer mutant ( C ) and the superimposition of the A3H (green) with A3A (PDB: 4XXO, yellow), A3B-CD2 (PDB: 5CQI, salmon) and AID (PDB: 5W0R, purple) ( D ), with secondary structures indicated (Supplementary Figure S2A ). The long helix 6 (h6), break of β5, and the long loop 1 of A3H can be visualized in panels C and D (Supplementary Figure S3A , B ).

    Techniques Used: Protein Purification, Size-exclusion Chromatography, Purification, Mutagenesis

    24) Product Images from "Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H"

    Article Title: Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21955-0

    Positively charged patches are important for subcellular localization and deaminase activity of A3H. ( A ) A3H structure showing the positively charged residues mutated in three patch mutants (patch 1–3). ( B ) Cell fractionation analysis of A3H and various mutants, showing the distribution between nucleus and cytosol in HEK293T cells. Transfected 293T cells expressing wild-type A3H hap I, hap II, and various hap II mutants were fractionated into whole cell (WC), cytoplasmic (Cyto) and nuclear (Nuc) fractions. A3B (mostly nucleus) and A3G (both cytoplasm and nucleus) were also used as controls. FLAG-A3H proteins in each fraction were analyzed by Western blot. ( C ) The deaminase assay of selected A3H mutants using the cell lysates of transfected HEK293T cells with or without RNase A treatment. The deaminase reaction was performed with cell lysate range of 0–6 μg (total protein amount, 2-fold dilutions from 6 μg) and 300 nM ssDNA.
    Figure Legend Snippet: Positively charged patches are important for subcellular localization and deaminase activity of A3H. ( A ) A3H structure showing the positively charged residues mutated in three patch mutants (patch 1–3). ( B ) Cell fractionation analysis of A3H and various mutants, showing the distribution between nucleus and cytosol in HEK293T cells. Transfected 293T cells expressing wild-type A3H hap I, hap II, and various hap II mutants were fractionated into whole cell (WC), cytoplasmic (Cyto) and nuclear (Nuc) fractions. A3B (mostly nucleus) and A3G (both cytoplasm and nucleus) were also used as controls. FLAG-A3H proteins in each fraction were analyzed by Western blot. ( C ) The deaminase assay of selected A3H mutants using the cell lysates of transfected HEK293T cells with or without RNase A treatment. The deaminase reaction was performed with cell lysate range of 0–6 μg (total protein amount, 2-fold dilutions from 6 μg) and 300 nM ssDNA.

    Techniques Used: Activity Assay, Cell Fractionation, Transfection, Expressing, Western Blot

    Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After RNase A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.
    Figure Legend Snippet: Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After RNase A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.

    Techniques Used: Inhibition, Activity Assay, Molecular Weight, Expressing, Size-exclusion Chromatography, Western Blot

    Protein purification and the overall structure of A3H. ( A ) SEC elution profiles of MBP-A3H dimeric and monomeric mutants on Superdex 200. A3H m1 forms a stable dimer after extensive RNase A treatment (blue). The purified m1 dimer can dissociate to monomer and free RNA after RNase A treatment followed by 1.5 M or higher salt buffer (black). The RNA-bound m1 dimer was disrupted by two sets of mutations on loop 7: H114A (m1+H114A) or W115A/C116S (m1+W115A/C116S), and clean monomers were purified from m1+H114A (light blue) m1+W115A/C116S (green). ( B ) MALS of MBP-fused m1+W115A/C116S mutant, showing the clean monomeric form. The expected molecular mass of a monomer is 63.2 kDa. ( C,D ) Crystal structure of A3H m1+W115A/C116S monomer mutant ( C ) and the superimposition of the A3H (green) with A3A (PDB: 4XXO, yellow), A3B-CD2 (PDB: 5CQI, salmon) and AID (PDB: 5W0R, purple) ( D ), with secondary structures indicated (Supplementary Figure S2A ). The long helix 6 (h6), break of β5, and the long loop 1 of A3H can be visualized in panels C and D (Supplementary Figure S3A , B ).
    Figure Legend Snippet: Protein purification and the overall structure of A3H. ( A ) SEC elution profiles of MBP-A3H dimeric and monomeric mutants on Superdex 200. A3H m1 forms a stable dimer after extensive RNase A treatment (blue). The purified m1 dimer can dissociate to monomer and free RNA after RNase A treatment followed by 1.5 M or higher salt buffer (black). The RNA-bound m1 dimer was disrupted by two sets of mutations on loop 7: H114A (m1+H114A) or W115A/C116S (m1+W115A/C116S), and clean monomers were purified from m1+H114A (light blue) m1+W115A/C116S (green). ( B ) MALS of MBP-fused m1+W115A/C116S mutant, showing the clean monomeric form. The expected molecular mass of a monomer is 63.2 kDa. ( C,D ) Crystal structure of A3H m1+W115A/C116S monomer mutant ( C ) and the superimposition of the A3H (green) with A3A (PDB: 4XXO, yellow), A3B-CD2 (PDB: 5CQI, salmon) and AID (PDB: 5W0R, purple) ( D ), with secondary structures indicated (Supplementary Figure S2A ). The long helix 6 (h6), break of β5, and the long loop 1 of A3H can be visualized in panels C and D (Supplementary Figure S3A , B ).

    Techniques Used: Protein Purification, Size-exclusion Chromatography, Purification, Mutagenesis

    25) Product Images from "Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses"

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses

    Journal: Nature medicine

    doi: 10.1038/s41591-018-0116-5

    SPARCS expression is inducible and triggers positive feedback amplification. ( a ) Isotype control versus PD-L1 or CD44 surface expression on H69AR cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n= 3 biological replicates). ( b ) Schematic of IFNγ pulse treatment (200 ng/mL) of H69 or H69AR cells. ( c ) qRT-PCR of MLT1C49 in H69 and H69AR cells ± 200 ng/mL IFNγ pulse – 24 h chase. Mean ± s.e.m of n=3 biological replicates shown (Two-way ANOVA; Sidak’s multiple comparisons tests). ( d ) ATAC-seq insertion tracks of H69 and H69AR cells around TRIM22 , TRIM38 and PD-L1 . Differentially accessible regions indicated with arrows. ( e ) Immunoblot of EZH2 and β-actin in H69, H69M and H69AR cells. ( f ) Log-2 fold change cytokine/chemokine differences between EZH2i treated H69 cells after IFNγ pulse, EZH2i treated cells, and IFNγ pulsed H69 cells relative to untreated control cells. *same as H69M-PD-L1 high cytokine profile in Fig. 1f . ( g ) Log-2 fold change comparison of IFNγ induced expression of SPARCS ERVs in EZH2i treated H69 cells versus H69AR cells. ( h ) qRT-PCR of 36B4 control, MLT1C49 , MLT1J and MLT1A in H69AR cells + 10 min IFNγ pulse - 24 h chase. RNA was treated with RNase A and immunoprecipitated with anti-dsRNA J2 antibody, values normalized against beta-actin. Mean ± s.e.m of n=3 biological replicates shown (Unpaired two-tailed Student’s t test). ( i ) Immunoblot of pTBK1, TBK1, pIRF3, IRF3, pSTAT1, STAT1 and β-actin levels in H69 and H69AR cells ± 200 ng/mL IFNγ 10 min pulse - 24 h chase. ( j ) qRT-PCR of MLT1C49 , IFN-β and CXCL10 in H69AR cells ± 10 min IFN-γ pulse - 24 h chase. Mean ± s.e.m of n=3 biological replicates shown (Unpaired two-tailed Student’s t test). ( k ) qRT-PCR and ELISA of CXCL10 in sgCTRL and sgMAVS-H69AR cells 72 h following Poly(I:C) transfection. Mean ± s.e.m of n=2 biological replicates shown (Unpaired two-tailed Student’s t test). ( l ) Log-2 fold change cytokine/chemokine differences in CM between CRISPR-H69AR cells after 10 min IFNγ 10 ng/mL pulse relative to sgCTRL cells (Scramble). ( m ) CXCL10 ELISA in Scramble, STING KO, MAVS KO and dKO H69AR CM following 10 min IFNγ 10 ng/mL pulse and chase for 3 days. Mean ± s.e.m of n=3 biological replicates shown (Unpaired two-tailed Student’s t test). ( n ) Photograph of representative excised tumors from sgCTRL and sgMAVS H69AR cells and tumor volumes measurements after 38 days of injection. Each data point represents mean ± s.e.m. tumor volumes (n=6 in sgCTRL group and n=6 in sgMAVS group; Two-way ANOVA; Sidak’s multiple comparisons tests). *p
    Figure Legend Snippet: SPARCS expression is inducible and triggers positive feedback amplification. ( a ) Isotype control versus PD-L1 or CD44 surface expression on H69AR cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n= 3 biological replicates). ( b ) Schematic of IFNγ pulse treatment (200 ng/mL) of H69 or H69AR cells. ( c ) qRT-PCR of MLT1C49 in H69 and H69AR cells ± 200 ng/mL IFNγ pulse – 24 h chase. Mean ± s.e.m of n=3 biological replicates shown (Two-way ANOVA; Sidak’s multiple comparisons tests). ( d ) ATAC-seq insertion tracks of H69 and H69AR cells around TRIM22 , TRIM38 and PD-L1 . Differentially accessible regions indicated with arrows. ( e ) Immunoblot of EZH2 and β-actin in H69, H69M and H69AR cells. ( f ) Log-2 fold change cytokine/chemokine differences between EZH2i treated H69 cells after IFNγ pulse, EZH2i treated cells, and IFNγ pulsed H69 cells relative to untreated control cells. *same as H69M-PD-L1 high cytokine profile in Fig. 1f . ( g ) Log-2 fold change comparison of IFNγ induced expression of SPARCS ERVs in EZH2i treated H69 cells versus H69AR cells. ( h ) qRT-PCR of 36B4 control, MLT1C49 , MLT1J and MLT1A in H69AR cells + 10 min IFNγ pulse - 24 h chase. RNA was treated with RNase A and immunoprecipitated with anti-dsRNA J2 antibody, values normalized against beta-actin. Mean ± s.e.m of n=3 biological replicates shown (Unpaired two-tailed Student’s t test). ( i ) Immunoblot of pTBK1, TBK1, pIRF3, IRF3, pSTAT1, STAT1 and β-actin levels in H69 and H69AR cells ± 200 ng/mL IFNγ 10 min pulse - 24 h chase. ( j ) qRT-PCR of MLT1C49 , IFN-β and CXCL10 in H69AR cells ± 10 min IFN-γ pulse - 24 h chase. Mean ± s.e.m of n=3 biological replicates shown (Unpaired two-tailed Student’s t test). ( k ) qRT-PCR and ELISA of CXCL10 in sgCTRL and sgMAVS-H69AR cells 72 h following Poly(I:C) transfection. Mean ± s.e.m of n=2 biological replicates shown (Unpaired two-tailed Student’s t test). ( l ) Log-2 fold change cytokine/chemokine differences in CM between CRISPR-H69AR cells after 10 min IFNγ 10 ng/mL pulse relative to sgCTRL cells (Scramble). ( m ) CXCL10 ELISA in Scramble, STING KO, MAVS KO and dKO H69AR CM following 10 min IFNγ 10 ng/mL pulse and chase for 3 days. Mean ± s.e.m of n=3 biological replicates shown (Unpaired two-tailed Student’s t test). ( n ) Photograph of representative excised tumors from sgCTRL and sgMAVS H69AR cells and tumor volumes measurements after 38 days of injection. Each data point represents mean ± s.e.m. tumor volumes (n=6 in sgCTRL group and n=6 in sgMAVS group; Two-way ANOVA; Sidak’s multiple comparisons tests). *p

    Techniques Used: Expressing, Amplification, Quantitative RT-PCR, Immunoprecipitation, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Transfection, CRISPR, Injection

    26) Product Images from "Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype"

    Article Title: Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08989-2

    MΦ uptake of miR-375 as a non-exosome entity. a , b Primary human MΦ were cocultured with MCF-7 cells for 24 h. a One hour before and during the coculture period, MΦ were treated either with vehicle, cytochalasin B, nocodazole, and b carbenoxolone. MiR-375 abundance was quantified via qPCR and normalized to untreated MΦ or untreated coculture MΦ, respectively ( n ≥ 3). c MiR-375 was measured by qPCR in the supernatants of MΦ, viable MCF-7 cells (MCF-7), STS-treated apoptotic MCF-7 cells (ap MCF-7), media, and normalized to untreated MΦ. Synthetic cel-miR-39a was used as spike-in control ( n ≥ 2). d VCM and ACM of ER+ (EFM-192A, MCF-7, T47D) and ER− (MDA-MB-468, MDA-MB-231, SKBR3, HCC1937) breast carcinoma cells, mammary epithelial cells (MCF-10A), and primary mammary epithelial cells (HMEC) were analyzed for the abundance of miR-375 ( n = 3). e MiR-375 level was measured by qPCR in MΦ cocultured with STS-treated apoptotic MCF-7 cells for 4 h. MCF-7 cells were removed from cocultures and MΦ were further cultivated for 20 h (24 h time point). Data are normalized to control MΦ ( n = 5). f MΦ were treated with STS as a control, or 1:1 diluted supernatants of viable (VCM) or apoptotic (ACM) MCF-7 cells for 30 min. Cells were washed and further cultured in MΦ media for 4 and 24 h, and miR-375 abundance was measured and normalized to untreated MΦ ( n ≥ 5). g ACM was incubated with control or 50 µg/mL RNase A at 37 °C for indicated time. Before RNA isolation, cel-miR-39a was added as a normalization control. MiR-375 abundance was quantified by qPCR and normalized to control ACM ( n ≥ 3). h MΦ were incubated for 30 min with either MCF-7 control ACM or RNase A-treated ACM. Cells were washed and cultured for another 24 h in MΦ media. MiR-375 level was determined and normalized to untreated MΦ ( n = 5). Data of a – h are mean ± SEM and p -values were calculated using one-sample t -test. * p
    Figure Legend Snippet: MΦ uptake of miR-375 as a non-exosome entity. a , b Primary human MΦ were cocultured with MCF-7 cells for 24 h. a One hour before and during the coculture period, MΦ were treated either with vehicle, cytochalasin B, nocodazole, and b carbenoxolone. MiR-375 abundance was quantified via qPCR and normalized to untreated MΦ or untreated coculture MΦ, respectively ( n ≥ 3). c MiR-375 was measured by qPCR in the supernatants of MΦ, viable MCF-7 cells (MCF-7), STS-treated apoptotic MCF-7 cells (ap MCF-7), media, and normalized to untreated MΦ. Synthetic cel-miR-39a was used as spike-in control ( n ≥ 2). d VCM and ACM of ER+ (EFM-192A, MCF-7, T47D) and ER− (MDA-MB-468, MDA-MB-231, SKBR3, HCC1937) breast carcinoma cells, mammary epithelial cells (MCF-10A), and primary mammary epithelial cells (HMEC) were analyzed for the abundance of miR-375 ( n = 3). e MiR-375 level was measured by qPCR in MΦ cocultured with STS-treated apoptotic MCF-7 cells for 4 h. MCF-7 cells were removed from cocultures and MΦ were further cultivated for 20 h (24 h time point). Data are normalized to control MΦ ( n = 5). f MΦ were treated with STS as a control, or 1:1 diluted supernatants of viable (VCM) or apoptotic (ACM) MCF-7 cells for 30 min. Cells were washed and further cultured in MΦ media for 4 and 24 h, and miR-375 abundance was measured and normalized to untreated MΦ ( n ≥ 5). g ACM was incubated with control or 50 µg/mL RNase A at 37 °C for indicated time. Before RNA isolation, cel-miR-39a was added as a normalization control. MiR-375 abundance was quantified by qPCR and normalized to control ACM ( n ≥ 3). h MΦ were incubated for 30 min with either MCF-7 control ACM or RNase A-treated ACM. Cells were washed and cultured for another 24 h in MΦ media. MiR-375 level was determined and normalized to untreated MΦ ( n = 5). Data of a – h are mean ± SEM and p -values were calculated using one-sample t -test. * p

    Techniques Used: Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Cell Culture, Incubation, Isolation

    27) Product Images from "E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis"

    Article Title: E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.1.220-231.2002

    Sub-G 1 assay shows that E5 protects HFKs from UV B-irradiation-induced apoptosis. LXSN-infected and L(16E5)SN-infected keratinocytes were grown in complete K-SFM and irradiated with 0 and 400 J of UV B per m 2 , and 16 h later the cells were fixed with 70% ethanol at 4°C overnight. Cells were then washed with 5 ml of PBS and resuspended in 1 ml of PBS containing 50 μg of PI per ml and 100 μg of RNase A per ml. FACS analysis was performed with a Becton Dickinson FACScan to evaluate the sub-G 1 ratio. The results of a representative experiment are shown.
    Figure Legend Snippet: Sub-G 1 assay shows that E5 protects HFKs from UV B-irradiation-induced apoptosis. LXSN-infected and L(16E5)SN-infected keratinocytes were grown in complete K-SFM and irradiated with 0 and 400 J of UV B per m 2 , and 16 h later the cells were fixed with 70% ethanol at 4°C overnight. Cells were then washed with 5 ml of PBS and resuspended in 1 ml of PBS containing 50 μg of PI per ml and 100 μg of RNase A per ml. FACS analysis was performed with a Becton Dickinson FACScan to evaluate the sub-G 1 ratio. The results of a representative experiment are shown.

    Techniques Used: Irradiation, Infection, FACS

    28) Product Images from "A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1"

    Article Title: A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1

    Journal: Genes & Development

    doi: 10.1101/gad.289769.116

    Defining the minimum DNA fragment size required to activate an IFN response and determining the size of IR-induced cytosolic DNA fragments. ( A ) Purified DNA samples obtained from irradiated and nonirradiated MCF7 whole-cell lysate (W) and nuclear (N) and cytosolic (C) fractions labeled on the 5′ end with γ- 32 P-dATP. All samples were treated with RNase A, and the S1 lane was additionally treated with S1 nuclease prior to labeling before analysis on a 6% nondenaturing polyacrylamide gel. ( B ) Western blotting of p-STAT1 (Y701) and total STAT1 for MCF7 whole-cell extracts 24 h after transfection with irradiated and nonirradiated 21-mer. ( C – E ) Western blotting of p-STAT1 at Y701 and total STAT1 for whole-cell extract prepared from MCF7 cells transfected with IR-damaged and undamaged 25-mer, 60-mer, and 100-mer oligonucleotides.
    Figure Legend Snippet: Defining the minimum DNA fragment size required to activate an IFN response and determining the size of IR-induced cytosolic DNA fragments. ( A ) Purified DNA samples obtained from irradiated and nonirradiated MCF7 whole-cell lysate (W) and nuclear (N) and cytosolic (C) fractions labeled on the 5′ end with γ- 32 P-dATP. All samples were treated with RNase A, and the S1 lane was additionally treated with S1 nuclease prior to labeling before analysis on a 6% nondenaturing polyacrylamide gel. ( B ) Western blotting of p-STAT1 (Y701) and total STAT1 for MCF7 whole-cell extracts 24 h after transfection with irradiated and nonirradiated 21-mer. ( C – E ) Western blotting of p-STAT1 at Y701 and total STAT1 for whole-cell extract prepared from MCF7 cells transfected with IR-damaged and undamaged 25-mer, 60-mer, and 100-mer oligonucleotides.

    Techniques Used: Purification, Irradiation, Labeling, Western Blot, Transfection

    29) Product Images from "Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition"

    Article Title: Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition

    Journal: Nucleic Acids Research

    doi:

    UV cross-linking analysis. 32 P-radiolabeled human DHFR mRNA (100 000 c.p.m.; 3.8 fmol) was incubated with 42.6 pmol of wild-type or mutant human His-Tag DHFR protein as described in the Materials and Methods. After UV cross-linking, the reaction mixture was incubated with RNase A to digest the unprotected RNAs. The UV cross-linked complexes were then resolved on SDS–12.5% PAGE. Lane 1, probe only; lane 2, His-Tag DHFR protein without UV cross-linking; lane 3, His-Tag DHFR protein; lane 4, C6A; lane 5, E30A; lane 6, GST.
    Figure Legend Snippet: UV cross-linking analysis. 32 P-radiolabeled human DHFR mRNA (100 000 c.p.m.; 3.8 fmol) was incubated with 42.6 pmol of wild-type or mutant human His-Tag DHFR protein as described in the Materials and Methods. After UV cross-linking, the reaction mixture was incubated with RNase A to digest the unprotected RNAs. The UV cross-linked complexes were then resolved on SDS–12.5% PAGE. Lane 1, probe only; lane 2, His-Tag DHFR protein without UV cross-linking; lane 3, His-Tag DHFR protein; lane 4, C6A; lane 5, E30A; lane 6, GST.

    Techniques Used: Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis

     Isolation of bound RNAs from DHFR RNP complexes.  32 P-radiolabeled human DHFR mRNA (3 000 000 c.p.m.; 114 fmol) was incubated with wild-type, His-Tag human DHFR protein (127.8 pmol), followed by digestion with RNase T1 and RNase A, and the addition of heparin. RNAs bound to the DHFR protein were eluted and resolved on a 15% polyacrylamide–8 M urea gel as described in the Materials and Methods. Lane 1, RNA markers; lane 2, RNAs isolated from the DHFR RNP complex.
    Figure Legend Snippet: Isolation of bound RNAs from DHFR RNP complexes. 32 P-radiolabeled human DHFR mRNA (3 000 000 c.p.m.; 114 fmol) was incubated with wild-type, His-Tag human DHFR protein (127.8 pmol), followed by digestion with RNase T1 and RNase A, and the addition of heparin. RNAs bound to the DHFR protein were eluted and resolved on a 15% polyacrylamide–8 M urea gel as described in the Materials and Methods. Lane 1, RNA markers; lane 2, RNAs isolated from the DHFR RNP complex.

    Techniques Used: Isolation, Incubation

    30) Product Images from "Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb"

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki997

    Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with RNase A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.
    Figure Legend Snippet: Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with RNase A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.

    Techniques Used: FLAG-tag, Immunoprecipitation, SDS Page, Western Blot, Binding Assay, Mutagenesis, Fluorescence

    The C-terminal domain and 7SK snRNA mediate the oligomerization of HEXIM1. ( A ) Schematic diagram of Hex1 proteins used. The signs at their N-termini depict the respective tags. ( B ) HEXIM1 forms oligomers. The x.Hex1 and f.Hex1 proteins were either expressed alone (lanes 4 and 1, 2, 3, 8, respectively) or f.Hex1 was co-expressed with x.Hex1 in HeLa cells (lanes 5–7 and 9) as indicated. Lysates were co-immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates of x.Hex1 were identified as presented on the upper western blot (WB). The middle and lower WB contain 10% of input proteins for immunoprecipitations (IP). Wild-type and mutant HEXIM1 proteins are identified by arrows. ( C ) 7SK snRNA and the C-terminal domain of HEXIM1 mediate the oligomerization of HEXIM1. x.Hex1 was expressed alone (lanes 1 and 2) or with the indicated f.Hex1 proteins (lanes 3–8). IP were performed as in (B) and were treated with RNase A where indicated.
    Figure Legend Snippet: The C-terminal domain and 7SK snRNA mediate the oligomerization of HEXIM1. ( A ) Schematic diagram of Hex1 proteins used. The signs at their N-termini depict the respective tags. ( B ) HEXIM1 forms oligomers. The x.Hex1 and f.Hex1 proteins were either expressed alone (lanes 4 and 1, 2, 3, 8, respectively) or f.Hex1 was co-expressed with x.Hex1 in HeLa cells (lanes 5–7 and 9) as indicated. Lysates were co-immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates of x.Hex1 were identified as presented on the upper western blot (WB). The middle and lower WB contain 10% of input proteins for immunoprecipitations (IP). Wild-type and mutant HEXIM1 proteins are identified by arrows. ( C ) 7SK snRNA and the C-terminal domain of HEXIM1 mediate the oligomerization of HEXIM1. x.Hex1 was expressed alone (lanes 1 and 2) or with the indicated f.Hex1 proteins (lanes 3–8). IP were performed as in (B) and were treated with RNase A where indicated.

    Techniques Used: Immunoprecipitation, Western Blot, Mutagenesis

    Oligomerization of HEXIM1 via its BR or CR2 is required for the inhibition of transcription. ( A ) Schematic diagram of Hex1 proteins used. The BR, CR1 and CR2 regions participating in the oligomerization are depicted. The wild-type and the mutated residues of the BR are depicted above and below the diagram, respectively. The mutated BR is indicated by asterisk. The schematic picture represents the f.Hex1 and mutant f.Hex1(1–314), f.Hex1mBR and f.Hex1mBR(1–315) proteins used. ( B ) HEXIM1 without the BR and the CR2 does not oligomerize. The x.Hex1 and f.Hex1 proteins were co-expressed as depicted. Lysates were treated with RNase A where noted and IP was performed as described. Upper panel represents WB with the immunoprecipitated x.Hex1 proteins, whereas the middle and lower panels show 10% input of proteins used for IP. ( C ) HEXIM1 without the BR and the CR2 does not inhibit P-TEFb. Bars represent CAT data obtained by co-transfection of HeLa cells with pG6TAR (0.3 µg), Gal.CycT1 (1 µg) and indicated f.Hex1 plasmids (2.7 µg). The lower panel presents the expression of f.Hex1 proteins.
    Figure Legend Snippet: Oligomerization of HEXIM1 via its BR or CR2 is required for the inhibition of transcription. ( A ) Schematic diagram of Hex1 proteins used. The BR, CR1 and CR2 regions participating in the oligomerization are depicted. The wild-type and the mutated residues of the BR are depicted above and below the diagram, respectively. The mutated BR is indicated by asterisk. The schematic picture represents the f.Hex1 and mutant f.Hex1(1–314), f.Hex1mBR and f.Hex1mBR(1–315) proteins used. ( B ) HEXIM1 without the BR and the CR2 does not oligomerize. The x.Hex1 and f.Hex1 proteins were co-expressed as depicted. Lysates were treated with RNase A where noted and IP was performed as described. Upper panel represents WB with the immunoprecipitated x.Hex1 proteins, whereas the middle and lower panels show 10% input of proteins used for IP. ( C ) HEXIM1 without the BR and the CR2 does not inhibit P-TEFb. Bars represent CAT data obtained by co-transfection of HeLa cells with pG6TAR (0.3 µg), Gal.CycT1 (1 µg) and indicated f.Hex1 plasmids (2.7 µg). The lower panel presents the expression of f.Hex1 proteins.

    Techniques Used: Inhibition, Mutagenesis, Western Blot, Immunoprecipitation, Cotransfection, Expressing

    31) Product Images from "Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR"

    Article Title: Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-21

    RT-PCR in the analyses of naturally occurring endogenous sense-antisense RNA pair expression . (a) Schematic of the cardiac MHC (MYH) gene locus and its transcription products. The upper strand transcribes the cardiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1]. (b) : representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase H - enzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p). (c) Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts. (d) Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods. (e) Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme.
    Figure Legend Snippet: RT-PCR in the analyses of naturally occurring endogenous sense-antisense RNA pair expression . (a) Schematic of the cardiac MHC (MYH) gene locus and its transcription products. The upper strand transcribes the cardiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1]. (b) : representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase H - enzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p). (c) Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts. (d) Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods. (e) Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Serial Dilution, Purification, Amplification

    Testing different RT enzyme properties and conditions . (a) In a two-step RT-PCR system, 2 μg total RNA from NC and PTU treated hearts were reverse transcribed in 20 μl reactions in absence (-p) or presence (+p) of RT primers. The RT primer targeted the MYH7 sense RNA, and the PCR primer set amplified a 284 bp product corresponding to the 3' end of the MYH7 gene. PCR used 1 μl cDNA and was carried out for either 28 or 30 cycles. Shown are results from using two different RT enzymes that differed by their RNase H properties. RNase H - and RNase H + . For each enzyme, the RT reactions were carried out under two different temperatures: 44°C or 50°C for 30 minutes/ea. (b) RT-PCR targeting the antisense MYH7 RNA in total RNA mixes of known proportions of sense and antisense RNA. RNA template contained either only sense MYH7 RNA, or a mix of sense and antisense MYH7 RNA corresponding to 99 to1 or 90 to 10 sense to antisense ratios (S:AS). Soleus total RNA was used as a source of the sense MYH7 RNA in absence of antisense. Whereas, T3-treated heart total RNA was used as a source of the antisense MYH7 RNA without co-expression of the sense. Mixes of soleus and T3 treated heart RNA were used to achieve the noted S:AS amounts in 2 μg of total RNA per 20 μl reactions. Reverse transcriptions were carried out in absence of RT primers (-P), in presence of the forward primer (+F) targeting the antisense, and in presence of a non specific primer corresponding to the 3' untranslated region of the human MYH4 mRNA sequence (+N). RT reactions used RNase H - RT (Invitrogen), performed at 44°C or at 50°C for 30 min. PCR was carried out on 1 μcDNA for 28 cycles targeting the 3' end of the MYH7 gene. See Additional file 4 for primers information.
    Figure Legend Snippet: Testing different RT enzyme properties and conditions . (a) In a two-step RT-PCR system, 2 μg total RNA from NC and PTU treated hearts were reverse transcribed in 20 μl reactions in absence (-p) or presence (+p) of RT primers. The RT primer targeted the MYH7 sense RNA, and the PCR primer set amplified a 284 bp product corresponding to the 3' end of the MYH7 gene. PCR used 1 μl cDNA and was carried out for either 28 or 30 cycles. Shown are results from using two different RT enzymes that differed by their RNase H properties. RNase H - and RNase H + . For each enzyme, the RT reactions were carried out under two different temperatures: 44°C or 50°C for 30 minutes/ea. (b) RT-PCR targeting the antisense MYH7 RNA in total RNA mixes of known proportions of sense and antisense RNA. RNA template contained either only sense MYH7 RNA, or a mix of sense and antisense MYH7 RNA corresponding to 99 to1 or 90 to 10 sense to antisense ratios (S:AS). Soleus total RNA was used as a source of the sense MYH7 RNA in absence of antisense. Whereas, T3-treated heart total RNA was used as a source of the antisense MYH7 RNA without co-expression of the sense. Mixes of soleus and T3 treated heart RNA were used to achieve the noted S:AS amounts in 2 μg of total RNA per 20 μl reactions. Reverse transcriptions were carried out in absence of RT primers (-P), in presence of the forward primer (+F) targeting the antisense, and in presence of a non specific primer corresponding to the 3' untranslated region of the human MYH4 mRNA sequence (+N). RT reactions used RNase H - RT (Invitrogen), performed at 44°C or at 50°C for 30 min. PCR was carried out on 1 μcDNA for 28 cycles targeting the 3' end of the MYH7 gene. See Additional file 4 for primers information.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Expressing, Sequencing

    32) Product Images from "High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells"

    Article Title: High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-158

    Schematic of RNAi screen with analysis of fumarate levels throughout the screen. (A) Schematic of FH synthetic lethality screening strategy. Briefly, the screen involves three steps: (i) cell transduction with a library of shRNA agents; (ii) splitting the culture and silencing FH in one subculture; (iii) quantifying shRNA agent abundance by means of barcode sequencing. The expression of different shRNAs can have lethal (red), synthetic lethal (yellow), synthetic rescue (blue), or no effect (green) on the proliferation of each cell during the screen. (B) Residual FH mRNA levels at indicated times post siRNA transfection. (C) Residual FH protein level at indicated times post siRNA transfection.
    Figure Legend Snippet: Schematic of RNAi screen with analysis of fumarate levels throughout the screen. (A) Schematic of FH synthetic lethality screening strategy. Briefly, the screen involves three steps: (i) cell transduction with a library of shRNA agents; (ii) splitting the culture and silencing FH in one subculture; (iii) quantifying shRNA agent abundance by means of barcode sequencing. The expression of different shRNAs can have lethal (red), synthetic lethal (yellow), synthetic rescue (blue), or no effect (green) on the proliferation of each cell during the screen. (B) Residual FH mRNA levels at indicated times post siRNA transfection. (C) Residual FH protein level at indicated times post siRNA transfection.

    Techniques Used: Transduction, shRNA, Sequencing, Expressing, Transfection

    33) Product Images from "Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein"

    Article Title: Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1338221

    E2F1 and pRB heterodimerize at the E2F1 promoter in association with a decrease in pRB phosphorylation. (A) ChIP analysis of pRB and E2F1 bound to the E2F1 promoter after 16h treatment of cells with 5nME2 ± 5 µM iAs expressed as % InPut E2F1. Representative experiment, repeated twice, Error Bars = SEM from qRT-PCR triplicate analysis. (B) Western blot analysis of pRB and phosphor-T373 pRB across the cell cycle in cells treated with 5nM E2 ± 5 µM iAs. GAPDH is a loading control. Dots to the left indicate mobility changes in pRB. (C) Quantification of western blot shown in (B) with phosphor-T373 in each lane normalized to the amount of pRB expressed in the corresponding lane (ratio T373/pRB). Blot was hybridized with antibody to pRB, stripped and re-hybridized with antibody to T373. GAPDH is a loading control. (D) Quantification of western blot (not shown) incubated with antibody to phosphor-S608-pRB normalized to the amount of pRB expressed in the corresponding lane as in (B)(ratio S608/pRB). Blot was hybridized to antibody to pRB, stripped and re-hybridized with antibody to S608-pRB. GAPDH is a loading control. Both (C) and (D) experiments were repeated 3 times.
    Figure Legend Snippet: E2F1 and pRB heterodimerize at the E2F1 promoter in association with a decrease in pRB phosphorylation. (A) ChIP analysis of pRB and E2F1 bound to the E2F1 promoter after 16h treatment of cells with 5nME2 ± 5 µM iAs expressed as % InPut E2F1. Representative experiment, repeated twice, Error Bars = SEM from qRT-PCR triplicate analysis. (B) Western blot analysis of pRB and phosphor-T373 pRB across the cell cycle in cells treated with 5nM E2 ± 5 µM iAs. GAPDH is a loading control. Dots to the left indicate mobility changes in pRB. (C) Quantification of western blot shown in (B) with phosphor-T373 in each lane normalized to the amount of pRB expressed in the corresponding lane (ratio T373/pRB). Blot was hybridized with antibody to pRB, stripped and re-hybridized with antibody to T373. GAPDH is a loading control. (D) Quantification of western blot (not shown) incubated with antibody to phosphor-S608-pRB normalized to the amount of pRB expressed in the corresponding lane as in (B)(ratio S608/pRB). Blot was hybridized to antibody to pRB, stripped and re-hybridized with antibody to S608-pRB. GAPDH is a loading control. Both (C) and (D) experiments were repeated 3 times.

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

    34) Product Images from "Adenosine leakage from perforin-burst extracellular vesicles inhibits perforin secretion by cytotoxic T-lymphocytes"

    Article Title: Adenosine leakage from perforin-burst extracellular vesicles inhibits perforin secretion by cytotoxic T-lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0231430

    Measurement of the EV destruction by perforin. (A) EVs from vehicle-treated D3H2LN cells (vehicle EVs). (B) EVs from IFN-γ-treated D3H2LN cells (IFN-γ EVs). Both types of EV were treated with or without perforin, and miRNAs were degraded by RNase A (right panels). MiRNA degradation was measured by real-time PCR; cel-miR-39 was used as a spike-in control. P
    Figure Legend Snippet: Measurement of the EV destruction by perforin. (A) EVs from vehicle-treated D3H2LN cells (vehicle EVs). (B) EVs from IFN-γ-treated D3H2LN cells (IFN-γ EVs). Both types of EV were treated with or without perforin, and miRNAs were degraded by RNase A (right panels). MiRNA degradation was measured by real-time PCR; cel-miR-39 was used as a spike-in control. P

    Techniques Used: Real-time Polymerase Chain Reaction

    35) Product Images from "Adenosine leakage from perforin-burst extracellular vesicles inhibits perforin secretion by cytotoxic T-lymphocytes"

    Article Title: Adenosine leakage from perforin-burst extracellular vesicles inhibits perforin secretion by cytotoxic T-lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0231430

    Measurement of the EV destruction by perforin. (A) EVs from vehicle-treated D3H2LN cells (vehicle EVs). (B) EVs from IFN-γ-treated D3H2LN cells (IFN-γ EVs). Both types of EV were treated with or without perforin, and miRNAs were degraded by RNase A (right panels). MiRNA degradation was measured by real-time PCR; cel-miR-39 was used as a spike-in control. P
    Figure Legend Snippet: Measurement of the EV destruction by perforin. (A) EVs from vehicle-treated D3H2LN cells (vehicle EVs). (B) EVs from IFN-γ-treated D3H2LN cells (IFN-γ EVs). Both types of EV were treated with or without perforin, and miRNAs were degraded by RNase A (right panels). MiRNA degradation was measured by real-time PCR; cel-miR-39 was used as a spike-in control. P

    Techniques Used: Real-time Polymerase Chain Reaction

    36) Product Images from "circRNA Profiling Reveals an Abundant circFUT10 that Promotes Adipocyte Proliferation and Inhibits Adipocyte Differentiation via Sponging let-7"

    Article Title: circRNA Profiling Reveals an Abundant circFUT10 that Promotes Adipocyte Proliferation and Inhibits Adipocyte Differentiation via Sponging let-7

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2020.03.011

    Expression of circFUT10 in Calf and Adult Cattle Adipose Tissue (A) The expression of eight circRNAs whose maternal genes were associated with fat development in our sequencing results. Validation of the expression of these eight circRNAs in three calf and adult fat tissues using qPCR is shown. (B) Schematic view illustrating the design of primers for circFUT10 used in qPCR and Sanger sequencing of the junction of back-spliced products. (C) circFUT10 exhibits obvious resistance to RNase R digestion. (D–F) Oil Red O staining (D) and qPCR analysis of adipogenesis markers PPARγ (E) and C/EBPβ (F) confirm the identity of bovine preadipocytes; quantitative analysis of oil Red O staining was performed using ImageJ. (G) The expression dynamics of circFUT10 during adipocyte differentiation were determined by qPCR, the expression of circFUT10 was normalized to 1 at day 0 of differentiation, and the expression of other days of differentiation was corrected according to this standard. (H) The expression level of FUT10 gene was measured when circFUT10 was overexpressed in bovine fat cells. Values are mean ± SEM for three biological replicates. ∗p
    Figure Legend Snippet: Expression of circFUT10 in Calf and Adult Cattle Adipose Tissue (A) The expression of eight circRNAs whose maternal genes were associated with fat development in our sequencing results. Validation of the expression of these eight circRNAs in three calf and adult fat tissues using qPCR is shown. (B) Schematic view illustrating the design of primers for circFUT10 used in qPCR and Sanger sequencing of the junction of back-spliced products. (C) circFUT10 exhibits obvious resistance to RNase R digestion. (D–F) Oil Red O staining (D) and qPCR analysis of adipogenesis markers PPARγ (E) and C/EBPβ (F) confirm the identity of bovine preadipocytes; quantitative analysis of oil Red O staining was performed using ImageJ. (G) The expression dynamics of circFUT10 during adipocyte differentiation were determined by qPCR, the expression of circFUT10 was normalized to 1 at day 0 of differentiation, and the expression of other days of differentiation was corrected according to this standard. (H) The expression level of FUT10 gene was measured when circFUT10 was overexpressed in bovine fat cells. Values are mean ± SEM for three biological replicates. ∗p

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

    37) Product Images from "Exosome-mediated transfer of lncRNA RP11-838N2.4 promotes erlotinib resistance in non-small cell lung cancer"

    Article Title: Exosome-mediated transfer of lncRNA RP11-838N2.4 promotes erlotinib resistance in non-small cell lung cancer

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4412

    Serum exosomal lncRNA RP11-838N2.4 is associated with erlotinib resistance in patients with non-small cell lung cancer (NSCLC). (A) RT-qPCR analysis of lncRNA RP11-838N2.4 in patients responding or not responding to erlotinib treatment. (B-D) The exosomal lncRNA RP11-838N2.4 expression level was not significantly influenced by (B) the exposure time, (C) RNase A digestion or (D) pH values. (E) Receiver operating characteristic (ROC) curve analysis of the diagnostic value of exosomal lncRNA RP11-838N2.4 in patients with NSCLC receiving erlotinib treatment. Arrow indicates the position of cut-off value (0.09). (F) RT-qPCR revealed that the proportion of patients that exhibited resistance to erlotinib therapy was significantly higher in the high lncRNA RP11-838N2.4 expression groups than in the low expression group.
    Figure Legend Snippet: Serum exosomal lncRNA RP11-838N2.4 is associated with erlotinib resistance in patients with non-small cell lung cancer (NSCLC). (A) RT-qPCR analysis of lncRNA RP11-838N2.4 in patients responding or not responding to erlotinib treatment. (B-D) The exosomal lncRNA RP11-838N2.4 expression level was not significantly influenced by (B) the exposure time, (C) RNase A digestion or (D) pH values. (E) Receiver operating characteristic (ROC) curve analysis of the diagnostic value of exosomal lncRNA RP11-838N2.4 in patients with NSCLC receiving erlotinib treatment. Arrow indicates the position of cut-off value (0.09). (F) RT-qPCR revealed that the proportion of patients that exhibited resistance to erlotinib therapy was significantly higher in the high lncRNA RP11-838N2.4 expression groups than in the low expression group.

    Techniques Used: Quantitative RT-PCR, Expressing, Diagnostic Assay

    38) Product Images from "Synthetic Scrapie Infectivity: Interaction between Recombinant PrP and Scrapie Brain-Derived RNA"

    Article Title: Synthetic Scrapie Infectivity: Interaction between Recombinant PrP and Scrapie Brain-Derived RNA

    Journal: Virulence

    doi: 10.4161/21505594.2014.989795

    In-vitro amplification of PrP Sc  is fully reconstituted by the addition of synthetic polyA RNA. Serial PMCA (5 rounds) was performed with nuclease-treated PrP Sc  seeds purified from 263K scrapie hamster brains and differently pretreated normal hamster brain homogenates (NBHs) as substrates. NBHs were digested with ( A ) Benzonase, ( B ) Benzonase subsequently spiked with PolyA RNA (200 μg/ml), or ( C ) RNAse A. The amount of original seeding material corresponded to an extract from 1 × 10 −6  g 263K scrapie hamster brain homogenate. After each PMCA round, samples were diluted 1:5 in the corresponding substrates. P: PK digested 263K scrapie hamster brain homogenate containing 5 × 10 −7  g of 263K hamster brain tissue, which served as a western blot positive control.
    Figure Legend Snippet: In-vitro amplification of PrP Sc is fully reconstituted by the addition of synthetic polyA RNA. Serial PMCA (5 rounds) was performed with nuclease-treated PrP Sc seeds purified from 263K scrapie hamster brains and differently pretreated normal hamster brain homogenates (NBHs) as substrates. NBHs were digested with ( A ) Benzonase, ( B ) Benzonase subsequently spiked with PolyA RNA (200 μg/ml), or ( C ) RNAse A. The amount of original seeding material corresponded to an extract from 1 × 10 −6 g 263K scrapie hamster brain homogenate. After each PMCA round, samples were diluted 1:5 in the corresponding substrates. P: PK digested 263K scrapie hamster brain homogenate containing 5 × 10 −7 g of 263K hamster brain tissue, which served as a western blot positive control.

    Techniques Used: In Vitro, Amplification, Purification, Western Blot, Positive Control

    39) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00428

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
    Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

    RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
    Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Techniques Used: Activation Assay

    RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
    Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Techniques Used: Labeling, Injection, Mouse Assay

    RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
    Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

    Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
    Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

    Techniques Used:

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Staining

    Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Immunostaining, Staining

    RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used:

    RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
    Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Techniques Used: Immunostaining, Marker, Staining

    40) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00428

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
    Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

    RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
    Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Techniques Used: Activation Assay

    RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
    Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Techniques Used: Labeling, Injection, Mouse Assay

    RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
    Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

    Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
    Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

    Techniques Used:

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Staining

    Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Immunostaining, Staining

    RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used:

    RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
    Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Techniques Used: Immunostaining, Marker, Staining

    Related Articles

    Lysis:

    Article Title: A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy
    Article Snippet: .. For RNase A treatment (Qiagen, 19101), the cell pellets were first incubated with lysis buffer. .. Then, RNase A (1 μg/ml) was added to supernatants and the samples were incubated for 1 h at 37°C prior to realize an immunoprecipitation with an UPF1 antibody.

    Incubation:

    Article Title: A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy
    Article Snippet: .. For RNase A treatment (Qiagen, 19101), the cell pellets were first incubated with lysis buffer. .. Then, RNase A (1 μg/ml) was added to supernatants and the samples were incubated for 1 h at 37°C prior to realize an immunoprecipitation with an UPF1 antibody.

    other:

    Article Title: The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module
    Article Snippet: This localization is efficiently disrupted with treatment by RNase A, whereas UBF, which is incorporated into nucleolar chromatin, remains intact.

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway
    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation
    Article Snippet: Deamination assays of mutants that neutralized the positively charged regions on CD1 under the conditions with and without RNase A, allowed us to distinguish different interactions with ssDNA and RNA.

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway
    Article Snippet: Thus, RNase A also promotes the growth of neurospheres.

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway
    Article Snippet: These data suggest that RNase A-induced NPCs are able to migrate to various brain regions and they further support the role of RNase A in promoting NPC proliferation and maintenance.

    Next-Generation Sequencing:

    Article Title: Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood
    Article Snippet: .. Exosomal small RNA including miRNA are protected against RNaseA treatment To compare the profiles of miRNA found in different blood components, whole blood, cell-free blood (serum and plasma) and exosomes were collected and prepared from 3 age-matched healthy individuals according to the study design depicted in for NGS. ..

    Cell Surface Receptor Assay:

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway
    Article Snippet: .. EGFR is the common cell surface receptor of RNase A and angiogenin to trigger proliferation of pancreatic cancer cells (Wang et al., ). .. Bovine RNase A binds to EGFR and transmits EGFR downstream signaling, including ERK activation, in various types of cancer cells (Wang et al., ).

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    Qiagen rnase a
    Mutation of key lysine residues reduces  in vitro  and cellular RNA-binding. ( A ) Residue amides in FKBP25 (1–74)  that are strongly and moderately affected by titration with dsRNA-10 are coloured in orange and light orange, respectively. Orientations of the domain in the image are the same as in Figure   5 . Chemical shift perturbation details are in   Supplementary Figure S8A . ( B ) NMR spectra following titration of dsRNA-10 into 100 μM  15 N-labeled FKBP25 (1–74)  with the K22M/K23M or K48/K52A mutations. Colours as in Figure   5 . ( C  and  D ) Electrophoretic mobility shift assays with varying concentrations of full-length FKBP25 (K22M/K23M) as in Figure   4A  and   B . ( E ) Western blot analysis of FLAG-tagged FKBP25 constructs (wild-type  and the K22M/K23M mutant) relative to endogenous FKBP25 (empty vector control) in HEK 293 cells. Antibodies against α-tubulin, FLAG-tag and FKBP25 correspond to the loading control, detection of FKBP25 constructs, and detection of both endogenous and FKBP25 constructs, respectively. ( F ) FLAG-affinity capture of cells expressing an empty vector control, wild-type FKBP25, or the K22M/K23M mutant with analysis by western blot using antibodies against the FKBP25-interacting proteins Parp1, nucleolin and RPS6. FKBP25 construct expression verified by antibodies against the FLAG tag. ( G ) RNA cross-linking IP (CLIP) experiment with wild-type FKBP25 or the K22M/K23M mutant in HEK293 cells, with DNAse pre-treatment coupled with variable amounts of RNase A.
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    Mutation of key lysine residues reduces  in vitro  and cellular RNA-binding. ( A ) Residue amides in FKBP25 (1–74)  that are strongly and moderately affected by titration with dsRNA-10 are coloured in orange and light orange, respectively. Orientations of the domain in the image are the same as in Figure   5 . Chemical shift perturbation details are in   Supplementary Figure S8A . ( B ) NMR spectra following titration of dsRNA-10 into 100 μM  15 N-labeled FKBP25 (1–74)  with the K22M/K23M or K48/K52A mutations. Colours as in Figure   5 . ( C  and  D ) Electrophoretic mobility shift assays with varying concentrations of full-length FKBP25 (K22M/K23M) as in Figure   4A  and   B . ( E ) Western blot analysis of FLAG-tagged FKBP25 constructs (wild-type  and the K22M/K23M mutant) relative to endogenous FKBP25 (empty vector control) in HEK 293 cells. Antibodies against α-tubulin, FLAG-tag and FKBP25 correspond to the loading control, detection of FKBP25 constructs, and detection of both endogenous and FKBP25 constructs, respectively. ( F ) FLAG-affinity capture of cells expressing an empty vector control, wild-type FKBP25, or the K22M/K23M mutant with analysis by western blot using antibodies against the FKBP25-interacting proteins Parp1, nucleolin and RPS6. FKBP25 construct expression verified by antibodies against the FLAG tag. ( G ) RNA cross-linking IP (CLIP) experiment with wild-type FKBP25 or the K22M/K23M mutant in HEK293 cells, with DNAse pre-treatment coupled with variable amounts of RNase A.

    Journal: Nucleic Acids Research

    Article Title: The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

    doi: 10.1093/nar/gkx852

    Figure Lengend Snippet: Mutation of key lysine residues reduces in vitro and cellular RNA-binding. ( A ) Residue amides in FKBP25 (1–74) that are strongly and moderately affected by titration with dsRNA-10 are coloured in orange and light orange, respectively. Orientations of the domain in the image are the same as in Figure 5 . Chemical shift perturbation details are in Supplementary Figure S8A . ( B ) NMR spectra following titration of dsRNA-10 into 100 μM 15 N-labeled FKBP25 (1–74) with the K22M/K23M or K48/K52A mutations. Colours as in Figure 5 . ( C and D ) Electrophoretic mobility shift assays with varying concentrations of full-length FKBP25 (K22M/K23M) as in Figure 4A and  B . ( E ) Western blot analysis of FLAG-tagged FKBP25 constructs (wild-type and the K22M/K23M mutant) relative to endogenous FKBP25 (empty vector control) in HEK 293 cells. Antibodies against α-tubulin, FLAG-tag and FKBP25 correspond to the loading control, detection of FKBP25 constructs, and detection of both endogenous and FKBP25 constructs, respectively. ( F ) FLAG-affinity capture of cells expressing an empty vector control, wild-type FKBP25, or the K22M/K23M mutant with analysis by western blot using antibodies against the FKBP25-interacting proteins Parp1, nucleolin and RPS6. FKBP25 construct expression verified by antibodies against the FLAG tag. ( G ) RNA cross-linking IP (CLIP) experiment with wild-type FKBP25 or the K22M/K23M mutant in HEK293 cells, with DNAse pre-treatment coupled with variable amounts of RNase A.

    Article Snippet: This localization is efficiently disrupted with treatment by RNase A, whereas UBF, which is incorporated into nucleolar chromatin, remains intact.

    Techniques: Mutagenesis, In Vitro, RNA Binding Assay, Titration, Nuclear Magnetic Resonance, Labeling, Electrophoretic Mobility Shift Assay, Western Blot, Construct, Plasmid Preparation, FLAG-tag, Expressing, Cross-linking Immunoprecipitation

    Work flow of study design and sample processing. Whole blood from 3 different individuals was collected by venepuncture into each tube using a Multi-fly and processed to analyse intracellular, cell-free and exosomal miRNA. Asterisks indicate the point of RNaseA treatment (100 ng/ml, 37°C for 10 minutes) to investigate RNA degradation in these samples. The workflow outlines the sample collection and preparation from 1 individual. The number of tubes collected from each volunteer was: 2×PAXgene 2.5 ml tubes, 3×Sarstedt S-Monovette serum-gel 7.5 ml tubes and 3×Sarstedt S-Monovette EDTA 7.5 ml tubes. Upon centrifugation of the Sarstedt S-Monovette EDTA tubes, approximately 10 ml of plasma was obtained across 3 Sarstedt S-Monovette tubes which are then separately aliquoted into Lo-Bind DNA tubes (4×1 ml, 2×1.2 ml tubes) for RNA analysis and deep sequencing. The remaining plasma was aliquoted for Western immunoblotting (WB, 1.2 ml), transmission electron microscopy (EM, 1.2 ml) and qNano (1 ml) analysis. For the RNA work involving RNaseA treatment, samples were allocated for an untreated control and RNaseA treatment: 2×1.2 ml for the ultracentrifugation exosomal RNA isolation (Plasma UC), 2×1 ml for the Norgen Biotek exosomal RNA isolation (Plasma NG), and 2×1 ml aliquot was reserved for cell-free plasma RNA isolation. The collection process and sample allocation are repeated for serum collection. Exosomes isolated from serum via the ultracentrifuge are denoted as Serum UC. Exosomal RNA isolated by the Norgen Biotek Kit are denoted as Serum NG. As for the 2×PAXgene tubes, RNA is isolated from 2.5 ml of whole blood per tube and isolated as recommended by the manufacturers protocol. One tube was treated with RNaseA and one was left untreated.

    Journal: Journal of Extracellular Vesicles

    Article Title: Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood

    doi: 10.3402/jev.v3.23743

    Figure Lengend Snippet: Work flow of study design and sample processing. Whole blood from 3 different individuals was collected by venepuncture into each tube using a Multi-fly and processed to analyse intracellular, cell-free and exosomal miRNA. Asterisks indicate the point of RNaseA treatment (100 ng/ml, 37°C for 10 minutes) to investigate RNA degradation in these samples. The workflow outlines the sample collection and preparation from 1 individual. The number of tubes collected from each volunteer was: 2×PAXgene 2.5 ml tubes, 3×Sarstedt S-Monovette serum-gel 7.5 ml tubes and 3×Sarstedt S-Monovette EDTA 7.5 ml tubes. Upon centrifugation of the Sarstedt S-Monovette EDTA tubes, approximately 10 ml of plasma was obtained across 3 Sarstedt S-Monovette tubes which are then separately aliquoted into Lo-Bind DNA tubes (4×1 ml, 2×1.2 ml tubes) for RNA analysis and deep sequencing. The remaining plasma was aliquoted for Western immunoblotting (WB, 1.2 ml), transmission electron microscopy (EM, 1.2 ml) and qNano (1 ml) analysis. For the RNA work involving RNaseA treatment, samples were allocated for an untreated control and RNaseA treatment: 2×1.2 ml for the ultracentrifugation exosomal RNA isolation (Plasma UC), 2×1 ml for the Norgen Biotek exosomal RNA isolation (Plasma NG), and 2×1 ml aliquot was reserved for cell-free plasma RNA isolation. The collection process and sample allocation are repeated for serum collection. Exosomes isolated from serum via the ultracentrifuge are denoted as Serum UC. Exosomal RNA isolated by the Norgen Biotek Kit are denoted as Serum NG. As for the 2×PAXgene tubes, RNA is isolated from 2.5 ml of whole blood per tube and isolated as recommended by the manufacturers protocol. One tube was treated with RNaseA and one was left untreated.

    Article Snippet: Exosomal small RNA including miRNA are protected against RNaseA treatment To compare the profiles of miRNA found in different blood components, whole blood, cell-free blood (serum and plasma) and exosomes were collected and prepared from 3 age-matched healthy individuals according to the study design depicted in for NGS.

    Techniques: Flow Cytometry, Centrifugation, Sequencing, Western Blot, Transmission Assay, Electron Microscopy, Isolation

    Small RNA profiles extracted from intracellular, cell-free and exosomal isolation from blood before and after RNaseA treatment. RNA was extracted from samples and run on a Small RNA Bioanalyser assay. Experiments shown here are representative of samples collected from 1 volunteer.

    Journal: Journal of Extracellular Vesicles

    Article Title: Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood

    doi: 10.3402/jev.v3.23743

    Figure Lengend Snippet: Small RNA profiles extracted from intracellular, cell-free and exosomal isolation from blood before and after RNaseA treatment. RNA was extracted from samples and run on a Small RNA Bioanalyser assay. Experiments shown here are representative of samples collected from 1 volunteer.

    Article Snippet: Exosomal small RNA including miRNA are protected against RNaseA treatment To compare the profiles of miRNA found in different blood components, whole blood, cell-free blood (serum and plasma) and exosomes were collected and prepared from 3 age-matched healthy individuals according to the study design depicted in for NGS.

    Techniques: Isolation

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques: Acetylene Reduction Assay, Staining, Microscopy

    RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques: Activation Assay

    RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques: Labeling, Injection, Mouse Assay

    RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

    Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques:

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques: Staining

    Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques: Immunostaining, Staining

    RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques:

    RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Article Snippet: We then examined the effect of RNase A on neurosphere cultures.

    Techniques: Immunostaining, Marker, Staining