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Addgene inc retroviral vector backbone pmig
Retroviral Vector Backbone Pmig, supplied by Addgene inc, 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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retroviral vector backbone pmig - by Bioz Stars, 2024-06
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Addgene inc pmscv thy1 1 retroviral backbone
Pmscv Thy1 1 Retroviral Backbone, supplied by Addgene inc, 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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Addgene inc pmscv puro retroviral backbone
Pmscv Puro Retroviral Backbone, supplied by Addgene inc, 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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Addgene inc retroviral backbone psir dsred express2
(A) Schematic processes of T reg cell lineage commitment. mCpG, methylated CpG. Off rate indicates unstable Foxp3 expression. (B) Experimental procedures for assaying the stability of Foxp3 expression in iT reg cells. (C) The stability and levels of Foxp3 expression in mock- and ASC-pretreated iT reg cells. Technical replicates are shown. Data represent 2 experiments. (D and E) Quantification of Foxp3 transcription by EU pulse labeling and RT-qPCR. Triplicates are shown. Data represent 2 experiments. (F and G) Foxp3 expression in mock- and ASC-pretreated iT reg cells grown in media containing titrated JQ1 for an additional 4 d. Data represent 3 experiments. (H–J) CD4 Tn cells isolated from Tet1 - 3 fl/fl mice were cultured in T reg -induction medium. <t>Retroviral</t> Cre was transduced at d 1 and PBS or ASC was added from d 3 to d 5. Cells were then grown in media containing DMSO (with IL-2), aIL-2, or JQ1 (with IL-2) with TCR agonists for an additional 3 d before analyzing Foxp3 + cells (H, I). Total live cells were also used to quantify Foxp3 and Gapdh mRNA with RT-qPCR (J). Data show means + SEMs of triplicates and represent 2 experiments. Unpaired, two-tailed t test. See also .
Retroviral Backbone Psir Dsred Express2, supplied by Addgene inc, 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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retroviral backbone psir dsred express2 - by Bioz Stars, 2024-06
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1) Product Images from "Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation"

Article Title: Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation

Journal: Cell reports

doi: 10.1016/j.celrep.2021.110124

(A) Schematic processes of T reg cell lineage commitment. mCpG, methylated CpG. Off rate indicates unstable Foxp3 expression. (B) Experimental procedures for assaying the stability of Foxp3 expression in iT reg cells. (C) The stability and levels of Foxp3 expression in mock- and ASC-pretreated iT reg cells. Technical replicates are shown. Data represent 2 experiments. (D and E) Quantification of Foxp3 transcription by EU pulse labeling and RT-qPCR. Triplicates are shown. Data represent 2 experiments. (F and G) Foxp3 expression in mock- and ASC-pretreated iT reg cells grown in media containing titrated JQ1 for an additional 4 d. Data represent 3 experiments. (H–J) CD4 Tn cells isolated from Tet1 - 3 fl/fl mice were cultured in T reg -induction medium. Retroviral Cre was transduced at d 1 and PBS or ASC was added from d 3 to d 5. Cells were then grown in media containing DMSO (with IL-2), aIL-2, or JQ1 (with IL-2) with TCR agonists for an additional 3 d before analyzing Foxp3 + cells (H, I). Total live cells were also used to quantify Foxp3 and Gapdh mRNA with RT-qPCR (J). Data show means + SEMs of triplicates and represent 2 experiments. Unpaired, two-tailed t test. See also .
Figure Legend Snippet: (A) Schematic processes of T reg cell lineage commitment. mCpG, methylated CpG. Off rate indicates unstable Foxp3 expression. (B) Experimental procedures for assaying the stability of Foxp3 expression in iT reg cells. (C) The stability and levels of Foxp3 expression in mock- and ASC-pretreated iT reg cells. Technical replicates are shown. Data represent 2 experiments. (D and E) Quantification of Foxp3 transcription by EU pulse labeling and RT-qPCR. Triplicates are shown. Data represent 2 experiments. (F and G) Foxp3 expression in mock- and ASC-pretreated iT reg cells grown in media containing titrated JQ1 for an additional 4 d. Data represent 3 experiments. (H–J) CD4 Tn cells isolated from Tet1 - 3 fl/fl mice were cultured in T reg -induction medium. Retroviral Cre was transduced at d 1 and PBS or ASC was added from d 3 to d 5. Cells were then grown in media containing DMSO (with IL-2), aIL-2, or JQ1 (with IL-2) with TCR agonists for an additional 3 d before analyzing Foxp3 + cells (H, I). Total live cells were also used to quantify Foxp3 and Gapdh mRNA with RT-qPCR (J). Data show means + SEMs of triplicates and represent 2 experiments. Unpaired, two-tailed t test. See also .

Techniques Used: Methylation, Expressing, Labeling, Quantitative RT-PCR, Isolation, Cell Culture, Two Tailed Test

(A) Epigenetic modifications and RNA Pol II and Stat5 binding around the Foxp3 locus in mock- and ASC-treated iT reg cells. Data represent 2 biological replicates. (B-E) Effects of ASC on H3K27ac (B), chromatin accessibility (C), and Stat5 (D) and RNA Pol II binding (E) in iT reg cells after 4 d treatment. Data were pooled from 2 biological replicates. Differentially represented peaks are highlighted and counted (p < 0.01). (F and G) Cross comparison of the levels of mCpG and H3K27ac in regions showing differential (p < 0.05) Stat5 (F) or RNA Pol II (G) binding in ASC- versus mock-treated iT reg cells. Unpaired two-samples Wilcoxon test. (H) Stat5 ChIP-qPCR with mock- and ASC-treated iT reg and Th0 cells after 30 min of IL-2 stimulation. Data represent 2 experiments. (I and J) Stat5 (I) and Foxp3 (J) ChIP qPCR at CNS2 in iT reg cells upon acute deletion of Tet1 - 3 followed by ASC treatment. Tet1 - 3 fl/fl CD4 Tn cells were transduced with retroviral Cre 1 d after activation. ASC or PBS was added to media at d 3. Transduced cells were sorted 3–4 d later for ChIP qPCR. Data show means + SEMs of triplicates and represent 2 experiments. See also .
Figure Legend Snippet: (A) Epigenetic modifications and RNA Pol II and Stat5 binding around the Foxp3 locus in mock- and ASC-treated iT reg cells. Data represent 2 biological replicates. (B-E) Effects of ASC on H3K27ac (B), chromatin accessibility (C), and Stat5 (D) and RNA Pol II binding (E) in iT reg cells after 4 d treatment. Data were pooled from 2 biological replicates. Differentially represented peaks are highlighted and counted (p < 0.01). (F and G) Cross comparison of the levels of mCpG and H3K27ac in regions showing differential (p < 0.05) Stat5 (F) or RNA Pol II (G) binding in ASC- versus mock-treated iT reg cells. Unpaired two-samples Wilcoxon test. (H) Stat5 ChIP-qPCR with mock- and ASC-treated iT reg and Th0 cells after 30 min of IL-2 stimulation. Data represent 2 experiments. (I and J) Stat5 (I) and Foxp3 (J) ChIP qPCR at CNS2 in iT reg cells upon acute deletion of Tet1 - 3 followed by ASC treatment. Tet1 - 3 fl/fl CD4 Tn cells were transduced with retroviral Cre 1 d after activation. ASC or PBS was added to media at d 3. Transduced cells were sorted 3–4 d later for ChIP qPCR. Data show means + SEMs of triplicates and represent 2 experiments. See also .

Techniques Used: Binding Assay, Transduction, Activation Assay

(A–C) Foxp3 expression in iT reg cells upon CRISPR knockdown of known regulators. CD4 Tn cells isolated from Foxp3 gfp Rosa Cas9 mice were cultured in T reg -induction media with or without supplemented ASC. Cells were transduced by retroviral sgRNA at d 3 and GFP-Foxp3 + cells were sorted at d 4 to assay the stability of Foxp3 expression (A). Live cells were gated to quantify Foxp3 + cells (B) and Foxp3 MFI (C). NC, non-targeting control sgRNA. Data show triplicates and represent 2 experiments. Unpaired, two-tailed t test. (D) A model of transcriptional drivers of Foxp3 expression during iT reg cell development. At the initiation stage, T reg -induction cues deposit histone acetylation at Foxp3 promoter to promote Foxp3 transcription. During the transition state before Tet-induced DNA demethylation, Foxp3 transcription is maintained by histone acetylation signal in trans via multiple positive regulators. After DNA demethylation, both cis and trans mechanisms of histone acetylation signal are dispensable for maintaining Foxp3 expression. Ac, histone acetylation.
Figure Legend Snippet: (A–C) Foxp3 expression in iT reg cells upon CRISPR knockdown of known regulators. CD4 Tn cells isolated from Foxp3 gfp Rosa Cas9 mice were cultured in T reg -induction media with or without supplemented ASC. Cells were transduced by retroviral sgRNA at d 3 and GFP-Foxp3 + cells were sorted at d 4 to assay the stability of Foxp3 expression (A). Live cells were gated to quantify Foxp3 + cells (B) and Foxp3 MFI (C). NC, non-targeting control sgRNA. Data show triplicates and represent 2 experiments. Unpaired, two-tailed t test. (D) A model of transcriptional drivers of Foxp3 expression during iT reg cell development. At the initiation stage, T reg -induction cues deposit histone acetylation at Foxp3 promoter to promote Foxp3 transcription. During the transition state before Tet-induced DNA demethylation, Foxp3 transcription is maintained by histone acetylation signal in trans via multiple positive regulators. After DNA demethylation, both cis and trans mechanisms of histone acetylation signal are dispensable for maintaining Foxp3 expression. Ac, histone acetylation.

Techniques Used: Expressing, CRISPR, Isolation, Cell Culture, Two Tailed Test

KEY RESOURCES TABLE
Figure Legend Snippet: KEY RESOURCES TABLE

Techniques Used: Purification, Recombinant, Cell Isolation, Staining, SYBR Green Assay, Transfection, Clone Assay, Amplification, Methylation Sequencing


Structured Review

Addgene inc retroviral backbone sfg
A GL261 were transduced with a <t>retroviral</t> vector to express the murine version of EGFRvIII. The mutated portion of EGFRvIII was fused with the transmembrane domain of the mouse EGFR to obtain cells which express the epitope on the surface. Left panel: flow cytometry staining of wild type GL261 (blue) and GL261 transduced to express EGFRvIII (red). Right panel: immunohistochemistry staining for EGFRvIII on orthotopically implanted tumors (scale bar represents 100 μm). One representative tumor is shown of four mice. B Murine CAR construct. The C-terminal portion of murine CD34 was included as marker gene and separated by a T2A peptide from the CAR construct, which included an ScFv to graft specificity, a CD8 stalk, and CD28-CD3ζ as activation domains. MR1 was used as ScFv specific for EGFRvIII, while 4g7 was used as ScFv specific for human CD19, used as negative control CAR. C “Stress experiment”. Direct comparison of the effect on tumor control of intravenous CAR-T cell administration on day 11 or 17 post tumor implantation. D Representative MRI images (axial orientation) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 11 post tumor implantation. E Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment, p = 0.0288 (*), Log-rank test) and F tumor volume quantification. G Representative MRI images (axial plane) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 17 post tumor implantation. H Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment) and I tumor volume quantification. Source data are provided as a Source Data file.
Retroviral Backbone Sfg, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma"

Article Title: Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma

Journal: Nature Communications

doi: 10.1038/s41467-020-20599-x

A GL261 were transduced with a retroviral vector to express the murine version of EGFRvIII. The mutated portion of EGFRvIII was fused with the transmembrane domain of the mouse EGFR to obtain cells which express the epitope on the surface. Left panel: flow cytometry staining of wild type GL261 (blue) and GL261 transduced to express EGFRvIII (red). Right panel: immunohistochemistry staining for EGFRvIII on orthotopically implanted tumors (scale bar represents 100 μm). One representative tumor is shown of four mice. B Murine CAR construct. The C-terminal portion of murine CD34 was included as marker gene and separated by a T2A peptide from the CAR construct, which included an ScFv to graft specificity, a CD8 stalk, and CD28-CD3ζ as activation domains. MR1 was used as ScFv specific for EGFRvIII, while 4g7 was used as ScFv specific for human CD19, used as negative control CAR. C “Stress experiment”. Direct comparison of the effect on tumor control of intravenous CAR-T cell administration on day 11 or 17 post tumor implantation. D Representative MRI images (axial orientation) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 11 post tumor implantation. E Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment, p = 0.0288 (*), Log-rank test) and F tumor volume quantification. G Representative MRI images (axial plane) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 17 post tumor implantation. H Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment) and I tumor volume quantification. Source data are provided as a Source Data file.
Figure Legend Snippet: A GL261 were transduced with a retroviral vector to express the murine version of EGFRvIII. The mutated portion of EGFRvIII was fused with the transmembrane domain of the mouse EGFR to obtain cells which express the epitope on the surface. Left panel: flow cytometry staining of wild type GL261 (blue) and GL261 transduced to express EGFRvIII (red). Right panel: immunohistochemistry staining for EGFRvIII on orthotopically implanted tumors (scale bar represents 100 μm). One representative tumor is shown of four mice. B Murine CAR construct. The C-terminal portion of murine CD34 was included as marker gene and separated by a T2A peptide from the CAR construct, which included an ScFv to graft specificity, a CD8 stalk, and CD28-CD3ζ as activation domains. MR1 was used as ScFv specific for EGFRvIII, while 4g7 was used as ScFv specific for human CD19, used as negative control CAR. C “Stress experiment”. Direct comparison of the effect on tumor control of intravenous CAR-T cell administration on day 11 or 17 post tumor implantation. D Representative MRI images (axial orientation) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 11 post tumor implantation. E Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment, p = 0.0288 (*), Log-rank test) and F tumor volume quantification. G Representative MRI images (axial plane) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 17 post tumor implantation. H Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment) and I tumor volume quantification. Source data are provided as a Source Data file.

Techniques Used: Transduction, Plasmid Preparation, Flow Cytometry, Staining, Immunohistochemistry, Construct, Marker, Activation Assay, Negative Control, Tumor Implantation


Structured Review

Addgene inc retroviral empty backbone construct

Retroviral Empty Backbone Construct, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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retroviral empty backbone construct - by Bioz Stars, 2024-06
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1) Product Images from "PTEN and DNA-PK determine sensitivity and recovery in response to WEE1 inhibition in human breast cancer"

Article Title: PTEN and DNA-PK determine sensitivity and recovery in response to WEE1 inhibition in human breast cancer

Journal: eLife

doi: 10.7554/eLife.57894


Figure Legend Snippet:

Techniques Used: CRISPR, Knock-Out, Plasmid Preparation, Expressing, Recombinant, Construct, Software


Structured Review

Addgene inc retroviral empty backbone vector pbabe puro
Retroviral Empty Backbone Vector Pbabe Puro, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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retroviral empty backbone vector pbabe puro - by Bioz Stars, 2024-06
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Addgene inc retroviral plasmid backbone
(A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero <t>retroviral</t> infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.
Retroviral Plasmid Backbone, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/retroviral plasmid backbone/product/Addgene inc
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retroviral plasmid backbone - by Bioz Stars, 2024-06
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Images

1) Product Images from "Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling"

Article Title: Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling

Journal: bioRxiv

doi: 10.1101/2020.02.07.938985

(A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero retroviral infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.
Figure Legend Snippet: (A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero retroviral infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.

Techniques Used: In Utero, Infection, Labeling, Marker, Expressing

(A) Representative images of mitochondrial morphology in SOX2+ (RGC) and βIII-tub+ (Newborn neuron) human ESC-derived cortical cell 3 days post mito-GFP retroviral infection. Quantification of mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; Mann Whitney test. (B) Timeline and representative images of Drp1- and Mff-overexpressing human cortical cells (6 days post Cre-expressing retrovirus infection). Arrow head: Neuron (Tbr1+), Arrow: Progenitor (Sox2+). Quantification of TBR1+ cells among GFP-labeled cells from three biological replicate experiments. Data are shown as mean ± SEM. ****P < 0.001; Dunnett’s multiple comparisons test. (C,D) Representative time-lapse images of mitochondrial dynamics in (C) Symmetric non-neurogenic division and (D) Symmetric neurogenic division. Asterisks indicate tracked cells. (E) Timeline of PC experiment and quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. (F,H) Timing of M1 treatment in (F) mouse and (H) human cortical cells. Quantification of βIII-tub+ cells among PC cells from three biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; Dunnett’s multiple comparisons test. (G,I) Quantification of mitochondrial length in mouse (G) and human (I) cortical cells from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. *P < 0.05, **P < 0.01, ****P < 0.0001; Dunn’s multiple comparisons test.
Figure Legend Snippet: (A) Representative images of mitochondrial morphology in SOX2+ (RGC) and βIII-tub+ (Newborn neuron) human ESC-derived cortical cell 3 days post mito-GFP retroviral infection. Quantification of mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; Mann Whitney test. (B) Timeline and representative images of Drp1- and Mff-overexpressing human cortical cells (6 days post Cre-expressing retrovirus infection). Arrow head: Neuron (Tbr1+), Arrow: Progenitor (Sox2+). Quantification of TBR1+ cells among GFP-labeled cells from three biological replicate experiments. Data are shown as mean ± SEM. ****P < 0.001; Dunnett’s multiple comparisons test. (C,D) Representative time-lapse images of mitochondrial dynamics in (C) Symmetric non-neurogenic division and (D) Symmetric neurogenic division. Asterisks indicate tracked cells. (E) Timeline of PC experiment and quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. (F,H) Timing of M1 treatment in (F) mouse and (H) human cortical cells. Quantification of βIII-tub+ cells among PC cells from three biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; Dunnett’s multiple comparisons test. (G,I) Quantification of mitochondrial length in mouse (G) and human (I) cortical cells from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. *P < 0.05, **P < 0.01, ****P < 0.0001; Dunn’s multiple comparisons test.

Techniques Used: Derivative Assay, Infection, MANN-WHITNEY, Expressing, Labeling


Structured Review

Addgene inc retroviral plasmid backbone
(A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero <t>retroviral</t> infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.
Retroviral Plasmid Backbone, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/retroviral plasmid backbone/product/Addgene inc
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Price from $9.99 to $1999.99
retroviral plasmid backbone - by Bioz Stars, 2024-06
92/100 stars

Images

1) Product Images from "Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling"

Article Title: Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling

Journal: bioRxiv

doi: 10.1101/2020.02.07.938985

(A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero retroviral infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.
Figure Legend Snippet: (A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero retroviral infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.

Techniques Used: In Utero, Infection, Labeling, Marker, Expressing

(A) Representative images of mitochondrial morphology in SOX2+ (RGC) and βIII-tub+ (Newborn neuron) human ESC-derived cortical cell 3 days post mito-GFP retroviral infection. Quantification of mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; Mann Whitney test. (B) Timeline and representative images of Drp1- and Mff-overexpressing human cortical cells (6 days post Cre-expressing retrovirus infection). Arrow head: Neuron (Tbr1+), Arrow: Progenitor (Sox2+). Quantification of TBR1+ cells among GFP-labeled cells from three biological replicate experiments. Data are shown as mean ± SEM. ****P < 0.001; Dunnett’s multiple comparisons test. (C,D) Representative time-lapse images of mitochondrial dynamics in (C) Symmetric non-neurogenic division and (D) Symmetric neurogenic division. Asterisks indicate tracked cells. (E) Timeline of PC experiment and quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. (F,H) Timing of M1 treatment in (F) mouse and (H) human cortical cells. Quantification of βIII-tub+ cells among PC cells from three biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; Dunnett’s multiple comparisons test. (G,I) Quantification of mitochondrial length in mouse (G) and human (I) cortical cells from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. *P < 0.05, **P < 0.01, ****P < 0.0001; Dunn’s multiple comparisons test.
Figure Legend Snippet: (A) Representative images of mitochondrial morphology in SOX2+ (RGC) and βIII-tub+ (Newborn neuron) human ESC-derived cortical cell 3 days post mito-GFP retroviral infection. Quantification of mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; Mann Whitney test. (B) Timeline and representative images of Drp1- and Mff-overexpressing human cortical cells (6 days post Cre-expressing retrovirus infection). Arrow head: Neuron (Tbr1+), Arrow: Progenitor (Sox2+). Quantification of TBR1+ cells among GFP-labeled cells from three biological replicate experiments. Data are shown as mean ± SEM. ****P < 0.001; Dunnett’s multiple comparisons test. (C,D) Representative time-lapse images of mitochondrial dynamics in (C) Symmetric non-neurogenic division and (D) Symmetric neurogenic division. Asterisks indicate tracked cells. (E) Timeline of PC experiment and quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. (F,H) Timing of M1 treatment in (F) mouse and (H) human cortical cells. Quantification of βIII-tub+ cells among PC cells from three biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; Dunnett’s multiple comparisons test. (G,I) Quantification of mitochondrial length in mouse (G) and human (I) cortical cells from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. *P < 0.05, **P < 0.01, ****P < 0.0001; Dunn’s multiple comparisons test.

Techniques Used: Derivative Assay, Infection, MANN-WHITNEY, Expressing, Labeling

retroviral backbone pmscv ires bfp mib vector  (Addgene inc)

 
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    Structured Review

    Addgene inc retroviral backbone pmscv ires bfp mib vector
    Ro 08–2750 increases differentiation and apoptosis in myeloid leukemia cells. a Cytotoxicity assay (Cell-Titer Glo) of Ro ( red ), Ro-OH ( cyan ) and Ro-NGF ( orange ) in MLL-AF9+ BM cells. 50% Effective Concentration ( EC 50 ) values, average of at least three independent experiments ± standard deviation are shown. b Flow cytometry representative histograms of DMSO ( gray ) and 5 μM Ro ( red ) treated MLL-AF9+ BM cells with myeloid differentiation markers (Mac1 and Gr1); bar graphs ( below ) show average (fold change increase) ± standard error mean of three independent experiments, performed in triplicate. Paired t -test, * p < 0.05; ** p < 0.01. c Representative immunocytochemistry images of cytospun MLL-AF9 + BM cells control (DMSO) or Ro treated (5 and 10 μM) and stained by Eosin Y and Methylene Blue/ Azure A. Scale, 50 μm. d Apoptosis analysis by Annexin V+ (% population) for MLL-AF9 + BM cells cultured in absence (DMSO, black ) or presence of Ro 5 μM ( light red ) or 10 μM ( red ). Results represent at least three independent experiments ± s.e.m. e Colony Formation Unit (CFU) assay of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM cells transduced with <t>MSCV-IRES-BFP</t> <t>(MIB,</t> control) or MSCV-IRES-MSI2-BFP (MSI2-BFP) <t>retroviral</t> vectors after Ro treatment 1, 5 and 10 μM. Results represent the average ± s.e.m. of colony numbers of at least five experiments performed in duplicate. f Representative immunoblot of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM MIB ( black bars ) and MSI2-BFP ( red bars ) cells (used in panel e ) after DMSO or 10 μM Ro treatment for 4 h. β-ACTIN, loading control. g CFU assay of Lin - Sca + cKit + (LSK) versus MLL-AF9+ BM cells demonstrates Ro 08–2750 therapeutic window. Results represent the average ± s.e.m. of colony numbers of three experiments performed in duplicate. Two tailed Paired t -test ( b , d , e and g ), * p < 0.05, ** p < 0.01, *** p < 0.005. Source data are provided as a Source Data file
    Retroviral Backbone Pmscv Ires Bfp Mib Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia"

    Article Title: Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia

    Journal: Nature Communications

    doi: 10.1038/s41467-019-10523-3

    Ro 08–2750 increases differentiation and apoptosis in myeloid leukemia cells. a Cytotoxicity assay (Cell-Titer Glo) of Ro ( red ), Ro-OH ( cyan ) and Ro-NGF ( orange ) in MLL-AF9+ BM cells. 50% Effective Concentration ( EC 50 ) values, average of at least three independent experiments ± standard deviation are shown. b Flow cytometry representative histograms of DMSO ( gray ) and 5 μM Ro ( red ) treated MLL-AF9+ BM cells with myeloid differentiation markers (Mac1 and Gr1); bar graphs ( below ) show average (fold change increase) ± standard error mean of three independent experiments, performed in triplicate. Paired t -test, * p < 0.05; ** p < 0.01. c Representative immunocytochemistry images of cytospun MLL-AF9 + BM cells control (DMSO) or Ro treated (5 and 10 μM) and stained by Eosin Y and Methylene Blue/ Azure A. Scale, 50 μm. d Apoptosis analysis by Annexin V+ (% population) for MLL-AF9 + BM cells cultured in absence (DMSO, black ) or presence of Ro 5 μM ( light red ) or 10 μM ( red ). Results represent at least three independent experiments ± s.e.m. e Colony Formation Unit (CFU) assay of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM cells transduced with MSCV-IRES-BFP (MIB, control) or MSCV-IRES-MSI2-BFP (MSI2-BFP) retroviral vectors after Ro treatment 1, 5 and 10 μM. Results represent the average ± s.e.m. of colony numbers of at least five experiments performed in duplicate. f Representative immunoblot of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM MIB ( black bars ) and MSI2-BFP ( red bars ) cells (used in panel e ) after DMSO or 10 μM Ro treatment for 4 h. β-ACTIN, loading control. g CFU assay of Lin - Sca + cKit + (LSK) versus MLL-AF9+ BM cells demonstrates Ro 08–2750 therapeutic window. Results represent the average ± s.e.m. of colony numbers of three experiments performed in duplicate. Two tailed Paired t -test ( b , d , e and g ), * p < 0.05, ** p < 0.01, *** p < 0.005. Source data are provided as a Source Data file
    Figure Legend Snippet: Ro 08–2750 increases differentiation and apoptosis in myeloid leukemia cells. a Cytotoxicity assay (Cell-Titer Glo) of Ro ( red ), Ro-OH ( cyan ) and Ro-NGF ( orange ) in MLL-AF9+ BM cells. 50% Effective Concentration ( EC 50 ) values, average of at least three independent experiments ± standard deviation are shown. b Flow cytometry representative histograms of DMSO ( gray ) and 5 μM Ro ( red ) treated MLL-AF9+ BM cells with myeloid differentiation markers (Mac1 and Gr1); bar graphs ( below ) show average (fold change increase) ± standard error mean of three independent experiments, performed in triplicate. Paired t -test, * p < 0.05; ** p < 0.01. c Representative immunocytochemistry images of cytospun MLL-AF9 + BM cells control (DMSO) or Ro treated (5 and 10 μM) and stained by Eosin Y and Methylene Blue/ Azure A. Scale, 50 μm. d Apoptosis analysis by Annexin V+ (% population) for MLL-AF9 + BM cells cultured in absence (DMSO, black ) or presence of Ro 5 μM ( light red ) or 10 μM ( red ). Results represent at least three independent experiments ± s.e.m. e Colony Formation Unit (CFU) assay of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM cells transduced with MSCV-IRES-BFP (MIB, control) or MSCV-IRES-MSI2-BFP (MSI2-BFP) retroviral vectors after Ro treatment 1, 5 and 10 μM. Results represent the average ± s.e.m. of colony numbers of at least five experiments performed in duplicate. f Representative immunoblot of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM MIB ( black bars ) and MSI2-BFP ( red bars ) cells (used in panel e ) after DMSO or 10 μM Ro treatment for 4 h. β-ACTIN, loading control. g CFU assay of Lin - Sca + cKit + (LSK) versus MLL-AF9+ BM cells demonstrates Ro 08–2750 therapeutic window. Results represent the average ± s.e.m. of colony numbers of three experiments performed in duplicate. Two tailed Paired t -test ( b , d , e and g ), * p < 0.05, ** p < 0.01, *** p < 0.005. Source data are provided as a Source Data file

    Techniques Used: Cytotoxicity Assay, Concentration Assay, Standard Deviation, Flow Cytometry, Immunocytochemistry, Staining, Cell Culture, Colony-forming Unit Assay, Transduction, Western Blot, Two Tailed Test

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    (A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero <t>retroviral</t> infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.
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    Ro 08–2750 increases differentiation and apoptosis in myeloid leukemia cells. a Cytotoxicity assay (Cell-Titer Glo) of Ro ( red ), Ro-OH ( cyan ) and Ro-NGF ( orange ) in MLL-AF9+ BM cells. 50% Effective Concentration ( EC 50 ) values, average of at least three independent experiments ± standard deviation are shown. b Flow cytometry representative histograms of DMSO ( gray ) and 5 μM Ro ( red ) treated MLL-AF9+ BM cells with myeloid differentiation markers (Mac1 and Gr1); bar graphs ( below ) show average (fold change increase) ± standard error mean of three independent experiments, performed in triplicate. Paired t -test, * p < 0.05; ** p < 0.01. c Representative immunocytochemistry images of cytospun MLL-AF9 + BM cells control (DMSO) or Ro treated (5 and 10 μM) and stained by Eosin Y and Methylene Blue/ Azure A. Scale, 50 μm. d Apoptosis analysis by Annexin V+ (% population) for MLL-AF9 + BM cells cultured in absence (DMSO, black ) or presence of Ro 5 μM ( light red ) or 10 μM ( red ). Results represent at least three independent experiments ± s.e.m. e Colony Formation Unit (CFU) assay of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM cells transduced with <t>MSCV-IRES-BFP</t> <t>(MIB,</t> control) or MSCV-IRES-MSI2-BFP (MSI2-BFP) <t>retroviral</t> vectors after Ro treatment 1, 5 and 10 μM. Results represent the average ± s.e.m. of colony numbers of at least five experiments performed in duplicate. f Representative immunoblot of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM MIB ( black bars ) and MSI2-BFP ( red bars ) cells (used in panel e ) after DMSO or 10 μM Ro treatment for 4 h. β-ACTIN, loading control. g CFU assay of Lin - Sca + cKit + (LSK) versus MLL-AF9+ BM cells demonstrates Ro 08–2750 therapeutic window. Results represent the average ± s.e.m. of colony numbers of three experiments performed in duplicate. Two tailed Paired t -test ( b , d , e and g ), * p < 0.05, ** p < 0.01, *** p < 0.005. Source data are provided as a Source Data file
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    Image Search Results


    (A) Schematic processes of T reg cell lineage commitment. mCpG, methylated CpG. Off rate indicates unstable Foxp3 expression. (B) Experimental procedures for assaying the stability of Foxp3 expression in iT reg cells. (C) The stability and levels of Foxp3 expression in mock- and ASC-pretreated iT reg cells. Technical replicates are shown. Data represent 2 experiments. (D and E) Quantification of Foxp3 transcription by EU pulse labeling and RT-qPCR. Triplicates are shown. Data represent 2 experiments. (F and G) Foxp3 expression in mock- and ASC-pretreated iT reg cells grown in media containing titrated JQ1 for an additional 4 d. Data represent 3 experiments. (H–J) CD4 Tn cells isolated from Tet1 - 3 fl/fl mice were cultured in T reg -induction medium. Retroviral Cre was transduced at d 1 and PBS or ASC was added from d 3 to d 5. Cells were then grown in media containing DMSO (with IL-2), aIL-2, or JQ1 (with IL-2) with TCR agonists for an additional 3 d before analyzing Foxp3 + cells (H, I). Total live cells were also used to quantify Foxp3 and Gapdh mRNA with RT-qPCR (J). Data show means + SEMs of triplicates and represent 2 experiments. Unpaired, two-tailed t test. See also .

    Journal: Cell reports

    Article Title: Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation

    doi: 10.1016/j.celrep.2021.110124

    Figure Lengend Snippet: (A) Schematic processes of T reg cell lineage commitment. mCpG, methylated CpG. Off rate indicates unstable Foxp3 expression. (B) Experimental procedures for assaying the stability of Foxp3 expression in iT reg cells. (C) The stability and levels of Foxp3 expression in mock- and ASC-pretreated iT reg cells. Technical replicates are shown. Data represent 2 experiments. (D and E) Quantification of Foxp3 transcription by EU pulse labeling and RT-qPCR. Triplicates are shown. Data represent 2 experiments. (F and G) Foxp3 expression in mock- and ASC-pretreated iT reg cells grown in media containing titrated JQ1 for an additional 4 d. Data represent 3 experiments. (H–J) CD4 Tn cells isolated from Tet1 - 3 fl/fl mice were cultured in T reg -induction medium. Retroviral Cre was transduced at d 1 and PBS or ASC was added from d 3 to d 5. Cells were then grown in media containing DMSO (with IL-2), aIL-2, or JQ1 (with IL-2) with TCR agonists for an additional 3 d before analyzing Foxp3 + cells (H, I). Total live cells were also used to quantify Foxp3 and Gapdh mRNA with RT-qPCR (J). Data show means + SEMs of triplicates and represent 2 experiments. Unpaired, two-tailed t test. See also .

    Article Snippet: The retroviral backbone pSIR-DsRed-Express2 were purchased from Addgene ( ).

    Techniques: Methylation, Expressing, Labeling, Quantitative RT-PCR, Isolation, Cell Culture, Two Tailed Test

    (A) Epigenetic modifications and RNA Pol II and Stat5 binding around the Foxp3 locus in mock- and ASC-treated iT reg cells. Data represent 2 biological replicates. (B-E) Effects of ASC on H3K27ac (B), chromatin accessibility (C), and Stat5 (D) and RNA Pol II binding (E) in iT reg cells after 4 d treatment. Data were pooled from 2 biological replicates. Differentially represented peaks are highlighted and counted (p < 0.01). (F and G) Cross comparison of the levels of mCpG and H3K27ac in regions showing differential (p < 0.05) Stat5 (F) or RNA Pol II (G) binding in ASC- versus mock-treated iT reg cells. Unpaired two-samples Wilcoxon test. (H) Stat5 ChIP-qPCR with mock- and ASC-treated iT reg and Th0 cells after 30 min of IL-2 stimulation. Data represent 2 experiments. (I and J) Stat5 (I) and Foxp3 (J) ChIP qPCR at CNS2 in iT reg cells upon acute deletion of Tet1 - 3 followed by ASC treatment. Tet1 - 3 fl/fl CD4 Tn cells were transduced with retroviral Cre 1 d after activation. ASC or PBS was added to media at d 3. Transduced cells were sorted 3–4 d later for ChIP qPCR. Data show means + SEMs of triplicates and represent 2 experiments. See also .

    Journal: Cell reports

    Article Title: Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation

    doi: 10.1016/j.celrep.2021.110124

    Figure Lengend Snippet: (A) Epigenetic modifications and RNA Pol II and Stat5 binding around the Foxp3 locus in mock- and ASC-treated iT reg cells. Data represent 2 biological replicates. (B-E) Effects of ASC on H3K27ac (B), chromatin accessibility (C), and Stat5 (D) and RNA Pol II binding (E) in iT reg cells after 4 d treatment. Data were pooled from 2 biological replicates. Differentially represented peaks are highlighted and counted (p < 0.01). (F and G) Cross comparison of the levels of mCpG and H3K27ac in regions showing differential (p < 0.05) Stat5 (F) or RNA Pol II (G) binding in ASC- versus mock-treated iT reg cells. Unpaired two-samples Wilcoxon test. (H) Stat5 ChIP-qPCR with mock- and ASC-treated iT reg and Th0 cells after 30 min of IL-2 stimulation. Data represent 2 experiments. (I and J) Stat5 (I) and Foxp3 (J) ChIP qPCR at CNS2 in iT reg cells upon acute deletion of Tet1 - 3 followed by ASC treatment. Tet1 - 3 fl/fl CD4 Tn cells were transduced with retroviral Cre 1 d after activation. ASC or PBS was added to media at d 3. Transduced cells were sorted 3–4 d later for ChIP qPCR. Data show means + SEMs of triplicates and represent 2 experiments. See also .

    Article Snippet: The retroviral backbone pSIR-DsRed-Express2 were purchased from Addgene ( ).

    Techniques: Binding Assay, Transduction, Activation Assay

    (A–C) Foxp3 expression in iT reg cells upon CRISPR knockdown of known regulators. CD4 Tn cells isolated from Foxp3 gfp Rosa Cas9 mice were cultured in T reg -induction media with or without supplemented ASC. Cells were transduced by retroviral sgRNA at d 3 and GFP-Foxp3 + cells were sorted at d 4 to assay the stability of Foxp3 expression (A). Live cells were gated to quantify Foxp3 + cells (B) and Foxp3 MFI (C). NC, non-targeting control sgRNA. Data show triplicates and represent 2 experiments. Unpaired, two-tailed t test. (D) A model of transcriptional drivers of Foxp3 expression during iT reg cell development. At the initiation stage, T reg -induction cues deposit histone acetylation at Foxp3 promoter to promote Foxp3 transcription. During the transition state before Tet-induced DNA demethylation, Foxp3 transcription is maintained by histone acetylation signal in trans via multiple positive regulators. After DNA demethylation, both cis and trans mechanisms of histone acetylation signal are dispensable for maintaining Foxp3 expression. Ac, histone acetylation.

    Journal: Cell reports

    Article Title: Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation

    doi: 10.1016/j.celrep.2021.110124

    Figure Lengend Snippet: (A–C) Foxp3 expression in iT reg cells upon CRISPR knockdown of known regulators. CD4 Tn cells isolated from Foxp3 gfp Rosa Cas9 mice were cultured in T reg -induction media with or without supplemented ASC. Cells were transduced by retroviral sgRNA at d 3 and GFP-Foxp3 + cells were sorted at d 4 to assay the stability of Foxp3 expression (A). Live cells were gated to quantify Foxp3 + cells (B) and Foxp3 MFI (C). NC, non-targeting control sgRNA. Data show triplicates and represent 2 experiments. Unpaired, two-tailed t test. (D) A model of transcriptional drivers of Foxp3 expression during iT reg cell development. At the initiation stage, T reg -induction cues deposit histone acetylation at Foxp3 promoter to promote Foxp3 transcription. During the transition state before Tet-induced DNA demethylation, Foxp3 transcription is maintained by histone acetylation signal in trans via multiple positive regulators. After DNA demethylation, both cis and trans mechanisms of histone acetylation signal are dispensable for maintaining Foxp3 expression. Ac, histone acetylation.

    Article Snippet: The retroviral backbone pSIR-DsRed-Express2 were purchased from Addgene ( ).

    Techniques: Expressing, CRISPR, Isolation, Cell Culture, Two Tailed Test

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation

    doi: 10.1016/j.celrep.2021.110124

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: The retroviral backbone pSIR-DsRed-Express2 were purchased from Addgene ( ).

    Techniques: Purification, Recombinant, Cell Isolation, Staining, SYBR Green Assay, Transfection, Clone Assay, Amplification, Methylation Sequencing

    A GL261 were transduced with a retroviral vector to express the murine version of EGFRvIII. The mutated portion of EGFRvIII was fused with the transmembrane domain of the mouse EGFR to obtain cells which express the epitope on the surface. Left panel: flow cytometry staining of wild type GL261 (blue) and GL261 transduced to express EGFRvIII (red). Right panel: immunohistochemistry staining for EGFRvIII on orthotopically implanted tumors (scale bar represents 100 μm). One representative tumor is shown of four mice. B Murine CAR construct. The C-terminal portion of murine CD34 was included as marker gene and separated by a T2A peptide from the CAR construct, which included an ScFv to graft specificity, a CD8 stalk, and CD28-CD3ζ as activation domains. MR1 was used as ScFv specific for EGFRvIII, while 4g7 was used as ScFv specific for human CD19, used as negative control CAR. C “Stress experiment”. Direct comparison of the effect on tumor control of intravenous CAR-T cell administration on day 11 or 17 post tumor implantation. D Representative MRI images (axial orientation) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 11 post tumor implantation. E Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment, p = 0.0288 (*), Log-rank test) and F tumor volume quantification. G Representative MRI images (axial plane) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 17 post tumor implantation. H Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment) and I tumor volume quantification. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma

    doi: 10.1038/s41467-020-20599-x

    Figure Lengend Snippet: A GL261 were transduced with a retroviral vector to express the murine version of EGFRvIII. The mutated portion of EGFRvIII was fused with the transmembrane domain of the mouse EGFR to obtain cells which express the epitope on the surface. Left panel: flow cytometry staining of wild type GL261 (blue) and GL261 transduced to express EGFRvIII (red). Right panel: immunohistochemistry staining for EGFRvIII on orthotopically implanted tumors (scale bar represents 100 μm). One representative tumor is shown of four mice. B Murine CAR construct. The C-terminal portion of murine CD34 was included as marker gene and separated by a T2A peptide from the CAR construct, which included an ScFv to graft specificity, a CD8 stalk, and CD28-CD3ζ as activation domains. MR1 was used as ScFv specific for EGFRvIII, while 4g7 was used as ScFv specific for human CD19, used as negative control CAR. C “Stress experiment”. Direct comparison of the effect on tumor control of intravenous CAR-T cell administration on day 11 or 17 post tumor implantation. D Representative MRI images (axial orientation) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 11 post tumor implantation. E Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment, p = 0.0288 (*), Log-rank test) and F tumor volume quantification. G Representative MRI images (axial plane) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 17 post tumor implantation. H Survival curves ( n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment) and I tumor volume quantification. Source data are provided as a Source Data file.

    Article Snippet: The γ-retroviral vector was produced by transient triple transfection of HEK293T using GeneJuice transfection reagent (Merck Millipore) with 4.69 μg of Peq-Pam plasmid (encoding Moloney GagPol), 3.13 μg of VSV-G envelope, and 4.69 μg of retroviral backbone SFG expressing the gene of interest, EGFRvIII or GD2 and GD3 synthase (AddGene plasmid #75013), respectively.

    Techniques: Transduction, Plasmid Preparation, Flow Cytometry, Staining, Immunohistochemistry, Construct, Marker, Activation Assay, Negative Control, Tumor Implantation

    Journal: eLife

    Article Title: PTEN and DNA-PK determine sensitivity and recovery in response to WEE1 inhibition in human breast cancer

    doi: 10.7554/eLife.57894

    Figure Lengend Snippet:

    Article Snippet: Recombinant DNA reagent , pBABE-puro (plasmid) , Addgene , RRID: Addgene_1764 , Retroviral empty backbone construct.

    Techniques: CRISPR, Knock-Out, Plasmid Preparation, Expressing, Recombinant, Construct, Software

    (A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero retroviral infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.

    Journal: bioRxiv

    Article Title: Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling

    doi: 10.1101/2020.02.07.938985

    Figure Lengend Snippet: (A) Representative images of mitochondrial morphology (mito-GFP) in Pax6+ (Apical progenitor in ventricular zone (VZ)) and βIII-tub+ (Newborn neuron in cortical plate (CP)) cell in E16.5 mouse cortex, following in utero retroviral infection at E12.5. Quantified mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; unpaired t test. (B) Schematic of the labeling strategy using photoconverted (PC) histone H2B-mEos4b. Representative images of PC cell labelled with mito-Nep2. (C) Representative images and timeline of PC experiment to determine kinetics of mitochondria dynamics following mitosis in mouse embryonic cortical cells. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size, together with fate marker expression. Red: βIII-tub+ neuron; Green: Tbr2+ intermediate progenitor; Gray: double negative (DN) RGC. (D) Timeline and representative images of PC experiment using M1 and Mdivi-1. (E,G) Quantified mitochondrial length from three biological replicate experiments. (E) 3 hours post-label, (G) 6 hours post-PC. Each data point represents an individual cell average mitochondrial size. **P < 0.01, ***P < 0.001, ****P ≤ 0.0001; Dunnett’s multiple comparisons test. (F,H) Quantification of each cell fate marker+ cells among PC cells from at least four biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Dunn’s multiple comparisons test.

    Article Snippet: DNA fragment of Cre was prepared by PCR and transferred to retroviral plasmid backbone (a gift from Fred Gage (Addgene plasmid # 49054; http://n2t.net/addgene:49054 ; RRID:Addgene_49054)) by restriction digestion and ligation to obtain a pRetro-CAG-Cre-WPRE.

    Techniques: In Utero, Infection, Labeling, Marker, Expressing

    (A) Representative images of mitochondrial morphology in SOX2+ (RGC) and βIII-tub+ (Newborn neuron) human ESC-derived cortical cell 3 days post mito-GFP retroviral infection. Quantification of mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; Mann Whitney test. (B) Timeline and representative images of Drp1- and Mff-overexpressing human cortical cells (6 days post Cre-expressing retrovirus infection). Arrow head: Neuron (Tbr1+), Arrow: Progenitor (Sox2+). Quantification of TBR1+ cells among GFP-labeled cells from three biological replicate experiments. Data are shown as mean ± SEM. ****P < 0.001; Dunnett’s multiple comparisons test. (C,D) Representative time-lapse images of mitochondrial dynamics in (C) Symmetric non-neurogenic division and (D) Symmetric neurogenic division. Asterisks indicate tracked cells. (E) Timeline of PC experiment and quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. (F,H) Timing of M1 treatment in (F) mouse and (H) human cortical cells. Quantification of βIII-tub+ cells among PC cells from three biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; Dunnett’s multiple comparisons test. (G,I) Quantification of mitochondrial length in mouse (G) and human (I) cortical cells from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. *P < 0.05, **P < 0.01, ****P < 0.0001; Dunn’s multiple comparisons test.

    Journal: bioRxiv

    Article Title: Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling

    doi: 10.1101/2020.02.07.938985

    Figure Lengend Snippet: (A) Representative images of mitochondrial morphology in SOX2+ (RGC) and βIII-tub+ (Newborn neuron) human ESC-derived cortical cell 3 days post mito-GFP retroviral infection. Quantification of mitochondrial length from two biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ****P < 0.0001; Mann Whitney test. (B) Timeline and representative images of Drp1- and Mff-overexpressing human cortical cells (6 days post Cre-expressing retrovirus infection). Arrow head: Neuron (Tbr1+), Arrow: Progenitor (Sox2+). Quantification of TBR1+ cells among GFP-labeled cells from three biological replicate experiments. Data are shown as mean ± SEM. ****P < 0.001; Dunnett’s multiple comparisons test. (C,D) Representative time-lapse images of mitochondrial dynamics in (C) Symmetric non-neurogenic division and (D) Symmetric neurogenic division. Asterisks indicate tracked cells. (E) Timeline of PC experiment and quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. (F,H) Timing of M1 treatment in (F) mouse and (H) human cortical cells. Quantification of βIII-tub+ cells among PC cells from three biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; Dunnett’s multiple comparisons test. (G,I) Quantification of mitochondrial length in mouse (G) and human (I) cortical cells from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. *P < 0.05, **P < 0.01, ****P < 0.0001; Dunn’s multiple comparisons test.

    Article Snippet: DNA fragment of Cre was prepared by PCR and transferred to retroviral plasmid backbone (a gift from Fred Gage (Addgene plasmid # 49054; http://n2t.net/addgene:49054 ; RRID:Addgene_49054)) by restriction digestion and ligation to obtain a pRetro-CAG-Cre-WPRE.

    Techniques: Derivative Assay, Infection, MANN-WHITNEY, Expressing, Labeling

    Ro 08–2750 increases differentiation and apoptosis in myeloid leukemia cells. a Cytotoxicity assay (Cell-Titer Glo) of Ro ( red ), Ro-OH ( cyan ) and Ro-NGF ( orange ) in MLL-AF9+ BM cells. 50% Effective Concentration ( EC 50 ) values, average of at least three independent experiments ± standard deviation are shown. b Flow cytometry representative histograms of DMSO ( gray ) and 5 μM Ro ( red ) treated MLL-AF9+ BM cells with myeloid differentiation markers (Mac1 and Gr1); bar graphs ( below ) show average (fold change increase) ± standard error mean of three independent experiments, performed in triplicate. Paired t -test, * p < 0.05; ** p < 0.01. c Representative immunocytochemistry images of cytospun MLL-AF9 + BM cells control (DMSO) or Ro treated (5 and 10 μM) and stained by Eosin Y and Methylene Blue/ Azure A. Scale, 50 μm. d Apoptosis analysis by Annexin V+ (% population) for MLL-AF9 + BM cells cultured in absence (DMSO, black ) or presence of Ro 5 μM ( light red ) or 10 μM ( red ). Results represent at least three independent experiments ± s.e.m. e Colony Formation Unit (CFU) assay of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM cells transduced with MSCV-IRES-BFP (MIB, control) or MSCV-IRES-MSI2-BFP (MSI2-BFP) retroviral vectors after Ro treatment 1, 5 and 10 μM. Results represent the average ± s.e.m. of colony numbers of at least five experiments performed in duplicate. f Representative immunoblot of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM MIB ( black bars ) and MSI2-BFP ( red bars ) cells (used in panel e ) after DMSO or 10 μM Ro treatment for 4 h. β-ACTIN, loading control. g CFU assay of Lin - Sca + cKit + (LSK) versus MLL-AF9+ BM cells demonstrates Ro 08–2750 therapeutic window. Results represent the average ± s.e.m. of colony numbers of three experiments performed in duplicate. Two tailed Paired t -test ( b , d , e and g ), * p < 0.05, ** p < 0.01, *** p < 0.005. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia

    doi: 10.1038/s41467-019-10523-3

    Figure Lengend Snippet: Ro 08–2750 increases differentiation and apoptosis in myeloid leukemia cells. a Cytotoxicity assay (Cell-Titer Glo) of Ro ( red ), Ro-OH ( cyan ) and Ro-NGF ( orange ) in MLL-AF9+ BM cells. 50% Effective Concentration ( EC 50 ) values, average of at least three independent experiments ± standard deviation are shown. b Flow cytometry representative histograms of DMSO ( gray ) and 5 μM Ro ( red ) treated MLL-AF9+ BM cells with myeloid differentiation markers (Mac1 and Gr1); bar graphs ( below ) show average (fold change increase) ± standard error mean of three independent experiments, performed in triplicate. Paired t -test, * p < 0.05; ** p < 0.01. c Representative immunocytochemistry images of cytospun MLL-AF9 + BM cells control (DMSO) or Ro treated (5 and 10 μM) and stained by Eosin Y and Methylene Blue/ Azure A. Scale, 50 μm. d Apoptosis analysis by Annexin V+ (% population) for MLL-AF9 + BM cells cultured in absence (DMSO, black ) or presence of Ro 5 μM ( light red ) or 10 μM ( red ). Results represent at least three independent experiments ± s.e.m. e Colony Formation Unit (CFU) assay of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM cells transduced with MSCV-IRES-BFP (MIB, control) or MSCV-IRES-MSI2-BFP (MSI2-BFP) retroviral vectors after Ro treatment 1, 5 and 10 μM. Results represent the average ± s.e.m. of colony numbers of at least five experiments performed in duplicate. f Representative immunoblot of MLL-AF9 + Puro-CreER + Msi1 fl/fl Msi2 fl/fl BM MIB ( black bars ) and MSI2-BFP ( red bars ) cells (used in panel e ) after DMSO or 10 μM Ro treatment for 4 h. β-ACTIN, loading control. g CFU assay of Lin - Sca + cKit + (LSK) versus MLL-AF9+ BM cells demonstrates Ro 08–2750 therapeutic window. Results represent the average ± s.e.m. of colony numbers of three experiments performed in duplicate. Two tailed Paired t -test ( b , d , e and g ), * p < 0.05, ** p < 0.01, *** p < 0.005. Source data are provided as a Source Data file

    Article Snippet: Human full-length MSI2 was cloned into the retroviral backbone pMSCV-IRES-BFP (MIB) vector (a gift from Dario Vignali; Addgene plasmid #52115) by Custom DNA Constructs (University Heights, Ohio) introducing a 5′Flag tag (5′-ATGGATTACAAGGATGACGACGATAAG-3′) and using BamHI and EcoRI restriction sites.

    Techniques: Cytotoxicity Assay, Concentration Assay, Standard Deviation, Flow Cytometry, Immunocytochemistry, Staining, Cell Culture, Colony-forming Unit Assay, Transduction, Western Blot, Two Tailed Test