rnase a  (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 rnase a
    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/rnase a/product/Qiagen
    Average 99 stars, based on 1127 article reviews
    Price from $9.99 to $1999.99
    rnase a - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) 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

    2) 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

    3) 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

    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 "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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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

    26) 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

    27) 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

    28) 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

    29) 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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    36) 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

    37) 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

    38) Product Images from "Intrinsic restriction activity by apolipoprotein B mRNA editing enzyme APOBEC1 against the mobility of autonomous retrotransposons"

    Article Title: Intrinsic restriction activity by apolipoprotein B mRNA editing enzyme APOBEC1 against the mobility of autonomous retrotransposons

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr124

    Formation of HMM A1 complexes. 293T cells were co-transfected with the expression plasmids for APOBEC family proteins and the L1 retrotransposition indicator construct pL1 RP -EGFP or pIRES-EGFP. After 24 h, the transfected cells were lysed and subjected to FPLC analysis. To test RNase sensitivity, the cell lysates were treated with RNase A at 37°C for 1 h, before fractionation. The each eluted fraction was subjected to western blot with HA antibody.
    Figure Legend Snippet: Formation of HMM A1 complexes. 293T cells were co-transfected with the expression plasmids for APOBEC family proteins and the L1 retrotransposition indicator construct pL1 RP -EGFP or pIRES-EGFP. After 24 h, the transfected cells were lysed and subjected to FPLC analysis. To test RNase sensitivity, the cell lysates were treated with RNase A at 37°C for 1 h, before fractionation. The each eluted fraction was subjected to western blot with HA antibody.

    Techniques Used: Transfection, Expressing, Construct, Fast Protein Liquid Chromatography, Fractionation, Western Blot

    39) Product Images from "A rigorous method to enrich for exosomes from brain tissue"

    Article Title: A rigorous method to enrich for exosomes from brain tissue

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2017.1348885

    Small RNA analysis of Fraction 2. (a) Bioanalyser analysis of small RNA associated with Fraction 2. Fraction 2 was treated with or without Triton-X 100 and or RNase A for 30 min, RNA was extracted and analysed on a small RNA chip which was submitted for bioanalysis. miRNA is between 4 and 22 nt in length. The peak visualized at 4 nt is the marker peak. (b + c) Analysis of RNase A-treated small RNA. Raw sequencing reads were aligned to the human genome (HG19) and mapped to miRBase V.20 and other small RNA from Ensembl Release 17 followed by normalization of raw reads to reads per million (RPM). The mean of reads per miRNA ( n = 3) was calculated. (b) The percentage of total reads mapped to non-coding small RNA and other RNA species identified by small RNA deep sequencing. (c) Left panel, unsupervised hierarchical clustering of highly abundant miRNA species ( > 10 RPM) identified in BH and F2 ( n = 3, all groups). Cluster 1 miRNAs are enriched in BH and cluster 2 miRNAs are enriched in F2. Right panel, unsupervised hierarchical clustering of the top 20 miRNA detected in F2. Red indicates high expression and blue indicates low expression.
    Figure Legend Snippet: Small RNA analysis of Fraction 2. (a) Bioanalyser analysis of small RNA associated with Fraction 2. Fraction 2 was treated with or without Triton-X 100 and or RNase A for 30 min, RNA was extracted and analysed on a small RNA chip which was submitted for bioanalysis. miRNA is between 4 and 22 nt in length. The peak visualized at 4 nt is the marker peak. (b + c) Analysis of RNase A-treated small RNA. Raw sequencing reads were aligned to the human genome (HG19) and mapped to miRBase V.20 and other small RNA from Ensembl Release 17 followed by normalization of raw reads to reads per million (RPM). The mean of reads per miRNA ( n = 3) was calculated. (b) The percentage of total reads mapped to non-coding small RNA and other RNA species identified by small RNA deep sequencing. (c) Left panel, unsupervised hierarchical clustering of highly abundant miRNA species ( > 10 RPM) identified in BH and F2 ( n = 3, all groups). Cluster 1 miRNAs are enriched in BH and cluster 2 miRNAs are enriched in F2. Right panel, unsupervised hierarchical clustering of the top 20 miRNA detected in F2. Red indicates high expression and blue indicates low expression.

    Techniques Used: Chromatin Immunoprecipitation, Marker, Sequencing, Expressing

    40) Product Images from "Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich dipeptides"

    Article Title: Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich dipeptides

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31096-z

    Poly-PR interacts with RNA. ( A ) Confocal imaging of HEK293 cells fixed/permeabilized and stained with FITC-(PR)20 in the absence of pretreatment of RNase A. ( B ) Colocalization analysis of signals from DAPI staining and FITC-(PR)20 in the HEK293 cells in the absence of pretreatment of RNase A. ( C ) Confocal imaging of HEK293 cells fixed/permeabilized and stained with FITC-(PR)20 in the presence of pretreatment of RNase A. ( D ) Colocalization analysis of signals from DAPI staining and FITC-(PR)20 in the HEK293 cells in the presence of pretreatment of RNase A. ( E ) Liquid-liquid phase separation of tetramethylrhodamine (TAMRA)-labeled random 15-mer RNA by FITC-(PR)20. ( F ) Agarose gel electrophoresis of human ribosomal RNA mixed with (PR)20 peptide at indicated concentration (μM). ( G ) Structure of RNA (base: adenine). ( H ) Crosslinking analysis of HA-(PR)20 peptide mixed with adenine, adenosine, ribose or ribose-phosphate in the presence or absence of RNA (poly-A). Arrowheads indicate crosslinked oligomer of HA-(PR)20 peptides. *polymerized HA-(PR)20.
    Figure Legend Snippet: Poly-PR interacts with RNA. ( A ) Confocal imaging of HEK293 cells fixed/permeabilized and stained with FITC-(PR)20 in the absence of pretreatment of RNase A. ( B ) Colocalization analysis of signals from DAPI staining and FITC-(PR)20 in the HEK293 cells in the absence of pretreatment of RNase A. ( C ) Confocal imaging of HEK293 cells fixed/permeabilized and stained with FITC-(PR)20 in the presence of pretreatment of RNase A. ( D ) Colocalization analysis of signals from DAPI staining and FITC-(PR)20 in the HEK293 cells in the presence of pretreatment of RNase A. ( E ) Liquid-liquid phase separation of tetramethylrhodamine (TAMRA)-labeled random 15-mer RNA by FITC-(PR)20. ( F ) Agarose gel electrophoresis of human ribosomal RNA mixed with (PR)20 peptide at indicated concentration (μM). ( G ) Structure of RNA (base: adenine). ( H ) Crosslinking analysis of HA-(PR)20 peptide mixed with adenine, adenosine, ribose or ribose-phosphate in the presence or absence of RNA (poly-A). Arrowheads indicate crosslinked oligomer of HA-(PR)20 peptides. *polymerized HA-(PR)20.

    Techniques Used: Imaging, Staining, Labeling, Agarose Gel Electrophoresis, Concentration Assay

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    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:

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    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.

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    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. ..

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    Qiagen rnaseq treg cells
    <t>Treg</t> cells show an activated phenotype and acquire a Th1-like profile during T. cruzi infection. (A) Plots of principal component analysis (PCA) for surface and intracellular markers of Treg cells from the spleens of Foxp3-EGFP mice at the indicated dpi (left panel) and the variables included in PC1 and PC2 (right panel). Each circle represents one animal. (B) Star plot displaying the expression of the indicated markers in Treg cells from non-infected (0 dpi, gray line) or infected (20 dpi, red line) mice. Each spoke of the star represents the geometric mean of fluorescence intensity for the indicated marker in a log2 scale from 1 to 16,384. The data length of a spoke is proportional to the MFI of expression of that marker in the corresponding sample. (C) Heat maps for the normalized expression scores (row mean values of 0 and variance of 1) of genes encoding suppression and/or activation markers in Treg cells (CD4 + CD25 + Foxp3-GFP + ) purified from the spleen of non-infected (0 dpi) or 22-days-infected (22 dpi) Foxp3-EGFP mice. (D) Gene set enrichment analysis of the Treg cell transcriptome for Th1, Th2 and Th17 signatures using selected gene sets. (E) Heat maps for the normalized expression scores (row mean values of 0 and variance of 1) of the selected genes used in (D) . <t>RNAseq</t> data are from one experiment with three biological replicates. The PCA plot, star plot and heat maps were created using InfoStat software, Microsoft Excel spreadsheet and Matrix2png online tool, respectively.
    Rnaseq Treg Cells, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen tex rnase pretreated mrna mirna
    The impact of <t>CIC-TEX</t> on EMT gene expression. a Tumor cells were seeded in soft agar containing 30 μg/ml TEX, where indicated; mean No of colonies±SD (5 replicates) and representative examples after 3wk of culture; b cell cycle progression (flow-cytometry, PI staining) of wt, CIC and kd cells cultured with/without CIC-TEX; mean % of cells (5 replicates) in G0, G1/S and G2/M; c IPA-based Reactome analysis of transcription factor-, stem cell-, EMT-, transcription-, and EMT-regulating genes that <t>mRNA</t> level is ≥2-fold up- or downregulated in CIC-TEX-treated v6kd and Tsp8kd cells (red), Tsp8kd cells (blue) or CD44v6kd cells (violet). d , e Reactome analysis after IPA coordination of <t>miRNA</t> with predicted mRNA targets (miRNA and targetscan databases) of ( d ) > 2-fold upregulated miRNA (framed) and ( e ) downregulated miRNA in CIC-TEX-treated kd cells affecting EMT-related genes in kd cells; in ( d ) mRNA pathways from upregulated miRNA towards EMT are included; for downregulated miRNA ( e ) only direct predicted mRNA targets are shown (color code as in c). f Flow-cytometry of EMT markers in A818.4 and kd cells with/without CIC-TEX-treatment (72 h); g confocal microscopy of kd cells with/without CIC-TEX-treatment stained for E- or N-cadherin and counterstained for v6 or Tsp8 (scale bar: 10 μm); h Flow-cytometry of ex vivo analyzed EMT markers in dispersed intrapancreatic v6kd tumors from mice with/without CIC-TEX-treatment; ( i ) Flow-cytometry of EMT-related transcription factors in A818.4 and kd cells with/without CIC-TEX-treatment (72 h); j Confocal microscopy of kd cells with/without CIC-TEX-treatment stained for EMT-related transcription factors NOTCH and Nanog and counterstained with anti-v6 or anti-Tsp8 (scale bar: 10 μm); f , h , i mean % stained cells±SD (3 assays/tumors); a , b , f , h , i significant differences between wt and kd cells: *, significant differences by CIC-TEX-treatment: s. (List of synonyms: Additional file 1 : Table S1). CIC-TEX partly rescue impaired anchorage-independent growth and accelerate kd cell cycle progression. DS, confirmed at the protein level, unraveled a strong impact of CIC-TEX on EMT-related transcription factors mostly in v6kd cells at the mRNA and miRNA level, the latter being particularly engaged in Wnt and NOTCH signaling
    Tex Rnase Pretreated Mrna Mirna, supplied by Qiagen, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rna sequencing rnaseq hwjsc spheroids
    <t>HPEKp/HWJSC</t> spheroid fusion transcriptomics and morphometric analysis. A: Schematic of initiation of fusion experiments with HWJSC/HPEKp spheroids in ultra low attachment round-bottom plates. B: <t>RNASeq</t> analysis of HPEKp/HWJSC spheroid (red) or HWJSC spheroid (blue) fusion samples at day 0, 1, 2, and 4 (two samples per time point and condition, except only one sample for HPEKp/HWJSC spheroids at day 4), presented on three principal component axes of RPKM values for all samples. C: Fluorescent z-slices showing the progression of fusion of spheroids in non-adherent culture in round bottom plates. Fluorescent micrographs represent day 0 (i), day 1 (ii), day 2 (iii), and day 4 (iv) after the initiation of fusion. White asterisks denote HPEKp in the seam between adjacent spheroids. Scale bar represents 200 μm.
    Rna Sequencing Rnaseq Hwjsc Spheroids, supplied by Qiagen, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen residual rnase
    Inactivation of ZIKV by Z2. ( a ) Immunofluorescence staining the ZIKV E protein expressed on 293T cells by the anti-E mAb 4G2 (green). Nuclei stained by 4,6-diamidino-2-phenylindole (blue). Scale bar, 100 μm. ( b ) Determination of the binding of Z2 with E protein expressed on 293T cells by flow cytometry. ( c ) Degradation of released genomic <t>RNA</t> of ZIKV mediated by Z2 in an <t>RNase</t> digestion assay. The primers were used to detect RNA sequences in viral genome coding PrM, E protein and Cap protein, respectively. ( d ) Degradation of released genomic RNA of ZIKV mediated by Z2-scr. ( e ) The separation of genomic RNA and E protein of ZIKV treated with Z2, Z2-scr, DMSO or Triton X-100 through a sucrose density gradient assay. Per cent of total E protein in each fraction was assessed by western blot and analysed by Image J software. Per cent of total RNA genome in each fraction was measured by RT–qPCR. ( f ) Inactivation of ZIKV by Z2. After incubation at room temperature for 2 h, ZIKV was separated from Z2 by PEG-8000 for the measurement of the residual infectivity. Data are means±s.d. of triplicate experiments.
    Residual Rnase, supplied by Qiagen, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Treg cells show an activated phenotype and acquire a Th1-like profile during T. cruzi infection. (A) Plots of principal component analysis (PCA) for surface and intracellular markers of Treg cells from the spleens of Foxp3-EGFP mice at the indicated dpi (left panel) and the variables included in PC1 and PC2 (right panel). Each circle represents one animal. (B) Star plot displaying the expression of the indicated markers in Treg cells from non-infected (0 dpi, gray line) or infected (20 dpi, red line) mice. Each spoke of the star represents the geometric mean of fluorescence intensity for the indicated marker in a log2 scale from 1 to 16,384. The data length of a spoke is proportional to the MFI of expression of that marker in the corresponding sample. (C) Heat maps for the normalized expression scores (row mean values of 0 and variance of 1) of genes encoding suppression and/or activation markers in Treg cells (CD4 + CD25 + Foxp3-GFP + ) purified from the spleen of non-infected (0 dpi) or 22-days-infected (22 dpi) Foxp3-EGFP mice. (D) Gene set enrichment analysis of the Treg cell transcriptome for Th1, Th2 and Th17 signatures using selected gene sets. (E) Heat maps for the normalized expression scores (row mean values of 0 and variance of 1) of the selected genes used in (D) . RNAseq data are from one experiment with three biological replicates. The PCA plot, star plot and heat maps were created using InfoStat software, Microsoft Excel spreadsheet and Matrix2png online tool, respectively.

    Journal: Frontiers in Immunology

    Article Title: Limited Foxp3+ Regulatory T Cells Response During Acute Trypanosoma cruzi Infection Is Required to Allow the Emergence of Robust Parasite-Specific CD8+ T Cell Immunity

    doi: 10.3389/fimmu.2018.02555

    Figure Lengend Snippet: Treg cells show an activated phenotype and acquire a Th1-like profile during T. cruzi infection. (A) Plots of principal component analysis (PCA) for surface and intracellular markers of Treg cells from the spleens of Foxp3-EGFP mice at the indicated dpi (left panel) and the variables included in PC1 and PC2 (right panel). Each circle represents one animal. (B) Star plot displaying the expression of the indicated markers in Treg cells from non-infected (0 dpi, gray line) or infected (20 dpi, red line) mice. Each spoke of the star represents the geometric mean of fluorescence intensity for the indicated marker in a log2 scale from 1 to 16,384. The data length of a spoke is proportional to the MFI of expression of that marker in the corresponding sample. (C) Heat maps for the normalized expression scores (row mean values of 0 and variance of 1) of genes encoding suppression and/or activation markers in Treg cells (CD4 + CD25 + Foxp3-GFP + ) purified from the spleen of non-infected (0 dpi) or 22-days-infected (22 dpi) Foxp3-EGFP mice. (D) Gene set enrichment analysis of the Treg cell transcriptome for Th1, Th2 and Th17 signatures using selected gene sets. (E) Heat maps for the normalized expression scores (row mean values of 0 and variance of 1) of the selected genes used in (D) . RNAseq data are from one experiment with three biological replicates. The PCA plot, star plot and heat maps were created using InfoStat software, Microsoft Excel spreadsheet and Matrix2png online tool, respectively.

    Article Snippet: RNAseq Treg cells were purified as described above from the spleens of non-infected and 22-days infected Foxp3-EGFP mice and immediately lysed with QIAzol reagent (Qiagen).

    Techniques: Infection, Mouse Assay, Expressing, Fluorescence, Marker, Activation Assay, Purification, Software

    The impact of CIC-TEX on EMT gene expression. a Tumor cells were seeded in soft agar containing 30 μg/ml TEX, where indicated; mean No of colonies±SD (5 replicates) and representative examples after 3wk of culture; b cell cycle progression (flow-cytometry, PI staining) of wt, CIC and kd cells cultured with/without CIC-TEX; mean % of cells (5 replicates) in G0, G1/S and G2/M; c IPA-based Reactome analysis of transcription factor-, stem cell-, EMT-, transcription-, and EMT-regulating genes that mRNA level is ≥2-fold up- or downregulated in CIC-TEX-treated v6kd and Tsp8kd cells (red), Tsp8kd cells (blue) or CD44v6kd cells (violet). d , e Reactome analysis after IPA coordination of miRNA with predicted mRNA targets (miRNA and targetscan databases) of ( d ) > 2-fold upregulated miRNA (framed) and ( e ) downregulated miRNA in CIC-TEX-treated kd cells affecting EMT-related genes in kd cells; in ( d ) mRNA pathways from upregulated miRNA towards EMT are included; for downregulated miRNA ( e ) only direct predicted mRNA targets are shown (color code as in c). f Flow-cytometry of EMT markers in A818.4 and kd cells with/without CIC-TEX-treatment (72 h); g confocal microscopy of kd cells with/without CIC-TEX-treatment stained for E- or N-cadherin and counterstained for v6 or Tsp8 (scale bar: 10 μm); h Flow-cytometry of ex vivo analyzed EMT markers in dispersed intrapancreatic v6kd tumors from mice with/without CIC-TEX-treatment; ( i ) Flow-cytometry of EMT-related transcription factors in A818.4 and kd cells with/without CIC-TEX-treatment (72 h); j Confocal microscopy of kd cells with/without CIC-TEX-treatment stained for EMT-related transcription factors NOTCH and Nanog and counterstained with anti-v6 or anti-Tsp8 (scale bar: 10 μm); f , h , i mean % stained cells±SD (3 assays/tumors); a , b , f , h , i significant differences between wt and kd cells: *, significant differences by CIC-TEX-treatment: s. (List of synonyms: Additional file 1 : Table S1). CIC-TEX partly rescue impaired anchorage-independent growth and accelerate kd cell cycle progression. DS, confirmed at the protein level, unraveled a strong impact of CIC-TEX on EMT-related transcription factors mostly in v6kd cells at the mRNA and miRNA level, the latter being particularly engaged in Wnt and NOTCH signaling

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Pancreatic cancer-initiating cell exosome message transfer into noncancer-initiating cells: the importance of CD44v6 in reprogramming

    doi: 10.1186/s13046-019-1129-8

    Figure Lengend Snippet: The impact of CIC-TEX on EMT gene expression. a Tumor cells were seeded in soft agar containing 30 μg/ml TEX, where indicated; mean No of colonies±SD (5 replicates) and representative examples after 3wk of culture; b cell cycle progression (flow-cytometry, PI staining) of wt, CIC and kd cells cultured with/without CIC-TEX; mean % of cells (5 replicates) in G0, G1/S and G2/M; c IPA-based Reactome analysis of transcription factor-, stem cell-, EMT-, transcription-, and EMT-regulating genes that mRNA level is ≥2-fold up- or downregulated in CIC-TEX-treated v6kd and Tsp8kd cells (red), Tsp8kd cells (blue) or CD44v6kd cells (violet). d , e Reactome analysis after IPA coordination of miRNA with predicted mRNA targets (miRNA and targetscan databases) of ( d ) > 2-fold upregulated miRNA (framed) and ( e ) downregulated miRNA in CIC-TEX-treated kd cells affecting EMT-related genes in kd cells; in ( d ) mRNA pathways from upregulated miRNA towards EMT are included; for downregulated miRNA ( e ) only direct predicted mRNA targets are shown (color code as in c). f Flow-cytometry of EMT markers in A818.4 and kd cells with/without CIC-TEX-treatment (72 h); g confocal microscopy of kd cells with/without CIC-TEX-treatment stained for E- or N-cadherin and counterstained for v6 or Tsp8 (scale bar: 10 μm); h Flow-cytometry of ex vivo analyzed EMT markers in dispersed intrapancreatic v6kd tumors from mice with/without CIC-TEX-treatment; ( i ) Flow-cytometry of EMT-related transcription factors in A818.4 and kd cells with/without CIC-TEX-treatment (72 h); j Confocal microscopy of kd cells with/without CIC-TEX-treatment stained for EMT-related transcription factors NOTCH and Nanog and counterstained with anti-v6 or anti-Tsp8 (scale bar: 10 μm); f , h , i mean % stained cells±SD (3 assays/tumors); a , b , f , h , i significant differences between wt and kd cells: *, significant differences by CIC-TEX-treatment: s. (List of synonyms: Additional file 1 : Table S1). CIC-TEX partly rescue impaired anchorage-independent growth and accelerate kd cell cycle progression. DS, confirmed at the protein level, unraveled a strong impact of CIC-TEX on EMT-related transcription factors mostly in v6kd cells at the mRNA and miRNA level, the latter being particularly engaged in Wnt and NOTCH signaling

    Article Snippet: mRNA and miRNA Cell and TEX (RNase-pretreated) mRNA/miRNA were extracted using miRNeasyMinikit following the supplier’s suggestion (Qiagen, Hildesheim, Germany).

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, Cell Culture, Indirect Immunoperoxidase Assay, Confocal Microscopy, Ex Vivo, Mouse Assay

    Correlation between CIC-TEX and CIC-TEX-induced changes in mRNA and miRNA profiles. a Numbers of ≥2-fold enriched mRNA (signal strength ≥1000) in CIC-TEX-treated v6kd and Tsp8kd cells sorted according to ≥2-fold higher versus comparable or lower recovery in CIC-TEX than kd cells; b numbers of miRNA ≥1.5-fold upregulated in CIC-TEX-treated v6kd and Tsp8kd cells sorted according to ≥1.5-fold higher versus comparable or lower recovery in CIC-TEX than kd cells; c Correlation between ≥1.5-fold up- or downregulated mRNA in CIC-TEX treated v6kd and Tsp8kd cells sorted according to reverse miRNA recovery in CIC-TEX-treated cells; d major activities (IPA-based Reactome analysis) of ≥2-fold up- or downregulated mRNA that are predicted targets of inversely recovered miRNA (miRNA database, target scan database) in both CIC-TEX-treated v6kd and Tsp8kd cell (List of synonyms: Additional file 1 : Table S1). mRNA and miRNA recovery are more strongly affected by CIC-TEX treatment in CD44v6kd than Tsp8kd cells. However, at the mRNA and the miRNA level coculture-induced changes rarely correlate with the CIC-TEX content. Instead, there is a strong correlation between coculture-induced up- or downregulated miRNA and reversely down- or upregulated mRNA in CIC-TEX-treated kd cells, indicating that not the CIC-TEX content, but CIC-TEX-induced target cell activation is dominating

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Pancreatic cancer-initiating cell exosome message transfer into noncancer-initiating cells: the importance of CD44v6 in reprogramming

    doi: 10.1186/s13046-019-1129-8

    Figure Lengend Snippet: Correlation between CIC-TEX and CIC-TEX-induced changes in mRNA and miRNA profiles. a Numbers of ≥2-fold enriched mRNA (signal strength ≥1000) in CIC-TEX-treated v6kd and Tsp8kd cells sorted according to ≥2-fold higher versus comparable or lower recovery in CIC-TEX than kd cells; b numbers of miRNA ≥1.5-fold upregulated in CIC-TEX-treated v6kd and Tsp8kd cells sorted according to ≥1.5-fold higher versus comparable or lower recovery in CIC-TEX than kd cells; c Correlation between ≥1.5-fold up- or downregulated mRNA in CIC-TEX treated v6kd and Tsp8kd cells sorted according to reverse miRNA recovery in CIC-TEX-treated cells; d major activities (IPA-based Reactome analysis) of ≥2-fold up- or downregulated mRNA that are predicted targets of inversely recovered miRNA (miRNA database, target scan database) in both CIC-TEX-treated v6kd and Tsp8kd cell (List of synonyms: Additional file 1 : Table S1). mRNA and miRNA recovery are more strongly affected by CIC-TEX treatment in CD44v6kd than Tsp8kd cells. However, at the mRNA and the miRNA level coculture-induced changes rarely correlate with the CIC-TEX content. Instead, there is a strong correlation between coculture-induced up- or downregulated miRNA and reversely down- or upregulated mRNA in CIC-TEX-treated kd cells, indicating that not the CIC-TEX content, but CIC-TEX-induced target cell activation is dominating

    Article Snippet: mRNA and miRNA Cell and TEX (RNase-pretreated) mRNA/miRNA were extracted using miRNeasyMinikit following the supplier’s suggestion (Qiagen, Hildesheim, Germany).

    Techniques: Indirect Immunoperoxidase Assay, Activation Assay

    CIC-TEX-initiated changes in RTK and downstream signaling molecules in CD44v6kd and Tspan8kd cells. a Signaling array of A818.4-v6kd cells cultured for 72 h with/without CIC-TEX. The relative signal strength was evaluated by ImageJ; significant differences by coculture with CIC-TEX: *. Flow-cytometry and WB analysis of ( b , c ) RTK expression in kd-TEX- or CIC-TEX-treated kd cells and ( d , e ) major pathway-engaged cytosolic signaling molecules; b , d mean % stained cells±SD (3 assays), significant differences by coculture with TEX: *; c , e representative examples and relative signal strength±SD of 3 independent experiments including p-values for kd cells compared to CIC-TEX-treated kd cells; f pathways from miRNA to RTK (IPA-based STRING analysis after predicted target mRNA selection by microrna.org and targetscan.org ) for ≥2-fold upregulated miRNA (framed) and ≥ 2-fold reduced mRNA recovery in CIC-TEX-treated v6kd or Tsp8kd cells compared to untreated kd cells; g IPA-based STRING analysis after predicted target mRNA selection by microrna.org and targetscan.org for ≥2-fold reduced miRNA in CIC-TEX-treated compared to untreated v6kd cells and of ≥2-fold upregulated predicted mRNA targets that are engaged in signal transduction. h Flow-cytometry analysis of ex vivo harvested intrapancreatic A818.4-v6kd cells from nude mice with/without weekly iv CIC-TEX treatment; mean % stained cells±SD (3 tumors), significant differences by CIC-TEX treatment: s; i Representative immunohistology examples of A818.4-v6kd and -Tsp8kd shock-frozen tumor sections with/without CIC-TEX treatment stained with the indicated antibodies (scale bar: 100 μm). (List of synonyms: Additional file 1 : Table S1). CIC-TEX treatment strongly affects RTK expression and downstream signaling molecules in vitro and in vivo. Changes in the recovery of mRNA engaged in signal transduction (Additional file 1 : Figure S2d, S2e) are accompanied at a noteworthy frequency by reversely altered miRNA expression in CIC-TEX-treated kd, predominantly v6kd cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Pancreatic cancer-initiating cell exosome message transfer into noncancer-initiating cells: the importance of CD44v6 in reprogramming

    doi: 10.1186/s13046-019-1129-8

    Figure Lengend Snippet: CIC-TEX-initiated changes in RTK and downstream signaling molecules in CD44v6kd and Tspan8kd cells. a Signaling array of A818.4-v6kd cells cultured for 72 h with/without CIC-TEX. The relative signal strength was evaluated by ImageJ; significant differences by coculture with CIC-TEX: *. Flow-cytometry and WB analysis of ( b , c ) RTK expression in kd-TEX- or CIC-TEX-treated kd cells and ( d , e ) major pathway-engaged cytosolic signaling molecules; b , d mean % stained cells±SD (3 assays), significant differences by coculture with TEX: *; c , e representative examples and relative signal strength±SD of 3 independent experiments including p-values for kd cells compared to CIC-TEX-treated kd cells; f pathways from miRNA to RTK (IPA-based STRING analysis after predicted target mRNA selection by microrna.org and targetscan.org ) for ≥2-fold upregulated miRNA (framed) and ≥ 2-fold reduced mRNA recovery in CIC-TEX-treated v6kd or Tsp8kd cells compared to untreated kd cells; g IPA-based STRING analysis after predicted target mRNA selection by microrna.org and targetscan.org for ≥2-fold reduced miRNA in CIC-TEX-treated compared to untreated v6kd cells and of ≥2-fold upregulated predicted mRNA targets that are engaged in signal transduction. h Flow-cytometry analysis of ex vivo harvested intrapancreatic A818.4-v6kd cells from nude mice with/without weekly iv CIC-TEX treatment; mean % stained cells±SD (3 tumors), significant differences by CIC-TEX treatment: s; i Representative immunohistology examples of A818.4-v6kd and -Tsp8kd shock-frozen tumor sections with/without CIC-TEX treatment stained with the indicated antibodies (scale bar: 100 μm). (List of synonyms: Additional file 1 : Table S1). CIC-TEX treatment strongly affects RTK expression and downstream signaling molecules in vitro and in vivo. Changes in the recovery of mRNA engaged in signal transduction (Additional file 1 : Figure S2d, S2e) are accompanied at a noteworthy frequency by reversely altered miRNA expression in CIC-TEX-treated kd, predominantly v6kd cells

    Article Snippet: mRNA and miRNA Cell and TEX (RNase-pretreated) mRNA/miRNA were extracted using miRNeasyMinikit following the supplier’s suggestion (Qiagen, Hildesheim, Germany).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Western Blot, Expressing, Staining, Indirect Immunoperoxidase Assay, Selection, Transduction, Ex Vivo, Mouse Assay, In Vitro, In Vivo

    HPEKp/HWJSC spheroid fusion transcriptomics and morphometric analysis. A: Schematic of initiation of fusion experiments with HWJSC/HPEKp spheroids in ultra low attachment round-bottom plates. B: RNASeq analysis of HPEKp/HWJSC spheroid (red) or HWJSC spheroid (blue) fusion samples at day 0, 1, 2, and 4 (two samples per time point and condition, except only one sample for HPEKp/HWJSC spheroids at day 4), presented on three principal component axes of RPKM values for all samples. C: Fluorescent z-slices showing the progression of fusion of spheroids in non-adherent culture in round bottom plates. Fluorescent micrographs represent day 0 (i), day 1 (ii), day 2 (iii), and day 4 (iv) after the initiation of fusion. White asterisks denote HPEKp in the seam between adjacent spheroids. Scale bar represents 200 μm.

    Journal: PLoS ONE

    Article Title: Engineering human cell spheroids to model embryonic tissue fusion in vitro

    doi: 10.1371/journal.pone.0184155

    Figure Lengend Snippet: HPEKp/HWJSC spheroid fusion transcriptomics and morphometric analysis. A: Schematic of initiation of fusion experiments with HWJSC/HPEKp spheroids in ultra low attachment round-bottom plates. B: RNASeq analysis of HPEKp/HWJSC spheroid (red) or HWJSC spheroid (blue) fusion samples at day 0, 1, 2, and 4 (two samples per time point and condition, except only one sample for HPEKp/HWJSC spheroids at day 4), presented on three principal component axes of RPKM values for all samples. C: Fluorescent z-slices showing the progression of fusion of spheroids in non-adherent culture in round bottom plates. Fluorescent micrographs represent day 0 (i), day 1 (ii), day 2 (iii), and day 4 (iv) after the initiation of fusion. White asterisks denote HPEKp in the seam between adjacent spheroids. Scale bar represents 200 μm.

    Article Snippet: Cultivating and harvesting HWJSC spheroids for RNA sequencing (RNASeq) HWJSC spheroids were prepared for RNAseq using RNeasy mini kit (Qiagen) following a modified version of the manufacturer’s protocol.

    Techniques:

    Inactivation of ZIKV by Z2. ( a ) Immunofluorescence staining the ZIKV E protein expressed on 293T cells by the anti-E mAb 4G2 (green). Nuclei stained by 4,6-diamidino-2-phenylindole (blue). Scale bar, 100 μm. ( b ) Determination of the binding of Z2 with E protein expressed on 293T cells by flow cytometry. ( c ) Degradation of released genomic RNA of ZIKV mediated by Z2 in an RNase digestion assay. The primers were used to detect RNA sequences in viral genome coding PrM, E protein and Cap protein, respectively. ( d ) Degradation of released genomic RNA of ZIKV mediated by Z2-scr. ( e ) The separation of genomic RNA and E protein of ZIKV treated with Z2, Z2-scr, DMSO or Triton X-100 through a sucrose density gradient assay. Per cent of total E protein in each fraction was assessed by western blot and analysed by Image J software. Per cent of total RNA genome in each fraction was measured by RT–qPCR. ( f ) Inactivation of ZIKV by Z2. After incubation at room temperature for 2 h, ZIKV was separated from Z2 by PEG-8000 for the measurement of the residual infectivity. Data are means±s.d. of triplicate experiments.

    Journal: Nature Communications

    Article Title: A peptide-based viral inactivator inhibits Zika virus infection in pregnant mice and fetuses

    doi: 10.1038/ncomms15672

    Figure Lengend Snippet: Inactivation of ZIKV by Z2. ( a ) Immunofluorescence staining the ZIKV E protein expressed on 293T cells by the anti-E mAb 4G2 (green). Nuclei stained by 4,6-diamidino-2-phenylindole (blue). Scale bar, 100 μm. ( b ) Determination of the binding of Z2 with E protein expressed on 293T cells by flow cytometry. ( c ) Degradation of released genomic RNA of ZIKV mediated by Z2 in an RNase digestion assay. The primers were used to detect RNA sequences in viral genome coding PrM, E protein and Cap protein, respectively. ( d ) Degradation of released genomic RNA of ZIKV mediated by Z2-scr. ( e ) The separation of genomic RNA and E protein of ZIKV treated with Z2, Z2-scr, DMSO or Triton X-100 through a sucrose density gradient assay. Per cent of total E protein in each fraction was assessed by western blot and analysed by Image J software. Per cent of total RNA genome in each fraction was measured by RT–qPCR. ( f ) Inactivation of ZIKV by Z2. After incubation at room temperature for 2 h, ZIKV was separated from Z2 by PEG-8000 for the measurement of the residual infectivity. Data are means±s.d. of triplicate experiments.

    Article Snippet: Briefly, about 1 × 103 plaque-forming units (p.f.u.) of ZIKV was incubated with Z2 or Z2-scr at room temperature for 2 h. The released genomic RNA from the treated ZIKV particles was then digested with micrococcal nuclease (New England BioLabs, MA) at 37 °C for 1 h. After inactivation of the residual RNase, the undigested genomic RNA in the intact viral particles was extracted using the Qiagen QIAamp Viral RNA Mini Kit (Valencia, CA) and reversed by using RT Reagent Kit (Takara Bio, Shiga, Japan).

    Techniques: Immunofluorescence, Staining, Binding Assay, Flow Cytometry, Cytometry, Western Blot, Software, Quantitative RT-PCR, Incubation, Infection