cas9  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cas9
    (A) OMM 1.5 cells expressing <t>Cas9</t> were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.
    Cas9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas9/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas9 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Synthetic Lethal Screens Reveal Co-Targeting FAK and MEK as a Multimodal Precision Therapy for GNAQ -Driven Uveal Melanoma"

    Article Title: Synthetic Lethal Screens Reveal Co-Targeting FAK and MEK as a Multimodal Precision Therapy for GNAQ -Driven Uveal Melanoma

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-20-3363

    (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.
    Figure Legend Snippet: (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.

    Techniques Used: Expressing, Infection, CRISPR, Selection, Western Blot

    cas9  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc cas9
    (A) OMM 1.5 cells expressing <t>Cas9</t> were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.
    Cas9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    1) Product Images from "Synthetic Lethal Screens Reveal Co-Targeting FAK and MEK as a Multimodal Precision Therapy for GNAQ -Driven Uveal Melanoma"

    Article Title: Synthetic Lethal Screens Reveal Co-Targeting FAK and MEK as a Multimodal Precision Therapy for GNAQ -Driven Uveal Melanoma

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-20-3363

    (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.
    Figure Legend Snippet: (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.

    Techniques Used: Expressing, Infection, CRISPR, Selection, Western Blot

    cas9  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cas9
    (A) OMM 1.5 cells expressing <t>Cas9</t> were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.
    Cas9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas9/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    cas9 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Synthetic Lethal Screens Reveal Co-Targeting FAK and MEK as a Multimodal Precision Therapy for GNAQ -Driven Uveal Melanoma"

    Article Title: Synthetic Lethal Screens Reveal Co-Targeting FAK and MEK as a Multimodal Precision Therapy for GNAQ -Driven Uveal Melanoma

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-20-3363

    (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.
    Figure Legend Snippet: (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.

    Techniques Used: Expressing, Infection, CRISPR, Selection, Western Blot

    cas 9  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cas 9
    Knockdown of TUG1 hindered OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a) The transfection efficiency of si-TUG1 (si-TUG1 #1 , si-TUG1 #2 ) was assessed by RT-qPCR in N2a cells. (b) The effect of si-TUG1 #2 on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (c) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells treated by OGD/R. (d and e) The proteins levels of apoptosis-related markers (cleaved-cas-3 and <t>cleaved-cas-9)</t> were performed by western blot assay in N2a cells treated by OGD/R. * P < 0.05.
    Cas 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas 9/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas 9 - by Bioz Stars, 2023-02
    94/100 stars

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    1) Product Images from "Long non-coding RNA TUG1 aggravates cerebral ischemia and reperfusion injury by sponging miR-493-3p/miR-410-3p"

    Article Title: Long non-coding RNA TUG1 aggravates cerebral ischemia and reperfusion injury by sponging miR-493-3p/miR-410-3p

    Journal: Open Medicine

    doi: 10.1515/med-2021-0253

    Knockdown of TUG1 hindered OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a) The transfection efficiency of si-TUG1 (si-TUG1 #1 , si-TUG1 #2 ) was assessed by RT-qPCR in N2a cells. (b) The effect of si-TUG1 #2 on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (c) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells treated by OGD/R. (d and e) The proteins levels of apoptosis-related markers (cleaved-cas-3 and cleaved-cas-9) were performed by western blot assay in N2a cells treated by OGD/R. * P < 0.05.
    Figure Legend Snippet: Knockdown of TUG1 hindered OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a) The transfection efficiency of si-TUG1 (si-TUG1 #1 , si-TUG1 #2 ) was assessed by RT-qPCR in N2a cells. (b) The effect of si-TUG1 #2 on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (c) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells treated by OGD/R. (d and e) The proteins levels of apoptosis-related markers (cleaved-cas-3 and cleaved-cas-9) were performed by western blot assay in N2a cells treated by OGD/R. * P < 0.05.

    Techniques Used: In Vitro, Transfection, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Western Blot

    The inhibition of miR-493-3p or miR-410-3p could restore the reverse effects of TUG1 knockdown on OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a and b) The effect of si-TUG1 #2 and anti-miR-493-3p or anti-miR-410-3p on the expression of miR-493-3p or miR-410-3p was detected by RT-qPCR. (c) The effect of si-TUG1 #2 and anti-miR-493-3p or anti-miR-410-3p on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (d) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells, which transfected with si-TUG1 #2 + anti-miR-493-3p or si-TUG1 #2 + anti-miR-410-3p or negative controls, with or without being treated by OGD/R. (e and f) The proteins levels of apoptosis-related markers (cleaved-cas-3 and cleaved-cas-9) were performed by western blot assay in N2a cells, which transfected with si-TUG1 #2 + anti-miR-493-3p or si-TUG1 #2 + anti-miR-410-3p or negative controls, with or without being treated by OGD/R. * P < 0.05.
    Figure Legend Snippet: The inhibition of miR-493-3p or miR-410-3p could restore the reverse effects of TUG1 knockdown on OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a and b) The effect of si-TUG1 #2 and anti-miR-493-3p or anti-miR-410-3p on the expression of miR-493-3p or miR-410-3p was detected by RT-qPCR. (c) The effect of si-TUG1 #2 and anti-miR-493-3p or anti-miR-410-3p on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (d) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells, which transfected with si-TUG1 #2 + anti-miR-493-3p or si-TUG1 #2 + anti-miR-410-3p or negative controls, with or without being treated by OGD/R. (e and f) The proteins levels of apoptosis-related markers (cleaved-cas-3 and cleaved-cas-9) were performed by western blot assay in N2a cells, which transfected with si-TUG1 #2 + anti-miR-493-3p or si-TUG1 #2 + anti-miR-410-3p or negative controls, with or without being treated by OGD/R. * P < 0.05.

    Techniques Used: Inhibition, In Vitro, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Transfection, Western Blot

    egfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egfp
    Egfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    spcas9  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc spcas9
    Spcas9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    anti cas9  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cas9
    (A–C) Mouse splenocytes were either freshly harvested (naive) or cultured for 16 h with recombinant mouse (rm) IL-15 (activated NK and T cells), FLT3-L (activated cDC1, cDC2), or M-CSF (activated macrophage) in complete media and subsequently electroporated in the presence of 40 pmol <t>Cas9</t> protein alone. (A) Frequency of intracellular Cas9+ naive and activated NK (TCRβ−CD3ε−NK1.1+) and T cells (TCRβ+CD3ε+NK1.1−) (B) Representative histogram of intracellular Cas9 levels for rmIL-15 stimulated un-electroporated NK cells and NK cells electroporated in the presence of <t>Cas9</t> <t>protein</t> alone. (C) Frequency of intracellular Cas9+ macrophages (TCRβ−CD3ε−NK1.1−CD64+CD11b+), cDC1s (TCRβ−CD3ε−NK1.1−CD64−CD11β−XCR1+), and cDC2s (TCRβ−CD3ε−NK1.1−CD64−CD11b+XCR1−) following electroporation at the indicated voltages.
    Anti Cas9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells"

    Article Title: CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.107651

    (A–C) Mouse splenocytes were either freshly harvested (naive) or cultured for 16 h with recombinant mouse (rm) IL-15 (activated NK and T cells), FLT3-L (activated cDC1, cDC2), or M-CSF (activated macrophage) in complete media and subsequently electroporated in the presence of 40 pmol Cas9 protein alone. (A) Frequency of intracellular Cas9+ naive and activated NK (TCRβ−CD3ε−NK1.1+) and T cells (TCRβ+CD3ε+NK1.1−) (B) Representative histogram of intracellular Cas9 levels for rmIL-15 stimulated un-electroporated NK cells and NK cells electroporated in the presence of Cas9 protein alone. (C) Frequency of intracellular Cas9+ macrophages (TCRβ−CD3ε−NK1.1−CD64+CD11b+), cDC1s (TCRβ−CD3ε−NK1.1−CD64−CD11β−XCR1+), and cDC2s (TCRβ−CD3ε−NK1.1−CD64−CD11b+XCR1−) following electroporation at the indicated voltages.
    Figure Legend Snippet: (A–C) Mouse splenocytes were either freshly harvested (naive) or cultured for 16 h with recombinant mouse (rm) IL-15 (activated NK and T cells), FLT3-L (activated cDC1, cDC2), or M-CSF (activated macrophage) in complete media and subsequently electroporated in the presence of 40 pmol Cas9 protein alone. (A) Frequency of intracellular Cas9+ naive and activated NK (TCRβ−CD3ε−NK1.1+) and T cells (TCRβ+CD3ε+NK1.1−) (B) Representative histogram of intracellular Cas9 levels for rmIL-15 stimulated un-electroporated NK cells and NK cells electroporated in the presence of Cas9 protein alone. (C) Frequency of intracellular Cas9+ macrophages (TCRβ−CD3ε−NK1.1−CD64+CD11b+), cDC1s (TCRβ−CD3ε−NK1.1−CD64−CD11β−XCR1+), and cDC2s (TCRβ−CD3ε−NK1.1−CD64−CD11b+XCR1−) following electroporation at the indicated voltages.

    Techniques Used: Cell Culture, Recombinant, Electroporation

    (A–E) Purified splenic NK cells were cultured for 16 h with 50 ng/mL rmIL-15 in complete media or freshly isolated before electroporation. (A) Frequency of intracellular Cas9+ NK cells (left) and percentage of viable NK cells (right) 2 h following electroporation with 40 pmol Cas9 of 5 × 105 rmIL-15-preactivated NK cells at the indicated voltages compared to un-electroporated controls. (B and C) Percentage of NK1.1− rmIL-15-preactivated NK cells 3 days after electroporation with cRNP complex consisting of Cas9 and Klrb1c gRNA compared to un-electroporated controls with varying (B) voltages or (C) concentrations of Cas9 in the Klrb1c cRNP complex electroporated at 1,900 V. Representative histogram of NK1.1 expression (D) and percentage of NK1.1− NK cells (E) 3 days after electroporation at 1,900 V of 5 × 105 rmIL-15-preactivated activated or freshly isolated naive cells compared to controls electroporated in the presence of Cas9 protein alone.
    Figure Legend Snippet: (A–E) Purified splenic NK cells were cultured for 16 h with 50 ng/mL rmIL-15 in complete media or freshly isolated before electroporation. (A) Frequency of intracellular Cas9+ NK cells (left) and percentage of viable NK cells (right) 2 h following electroporation with 40 pmol Cas9 of 5 × 105 rmIL-15-preactivated NK cells at the indicated voltages compared to un-electroporated controls. (B and C) Percentage of NK1.1− rmIL-15-preactivated NK cells 3 days after electroporation with cRNP complex consisting of Cas9 and Klrb1c gRNA compared to un-electroporated controls with varying (B) voltages or (C) concentrations of Cas9 in the Klrb1c cRNP complex electroporated at 1,900 V. Representative histogram of NK1.1 expression (D) and percentage of NK1.1− NK cells (E) 3 days after electroporation at 1,900 V of 5 × 105 rmIL-15-preactivated activated or freshly isolated naive cells compared to controls electroporated in the presence of Cas9 protein alone.

    Techniques Used: Purification, Cell Culture, Isolation, Electroporation, Expressing

    (A) Frequency of intracellular Cas9+ bone-marrow-derived macrophage (BMDM) or bone marrow-derived cDC1s (BM-cDC1s) 2 h post-electroporation with 40 pmol Cas9 compared to un-electroporated controls.
    Figure Legend Snippet: (A) Frequency of intracellular Cas9+ bone-marrow-derived macrophage (BMDM) or bone marrow-derived cDC1s (BM-cDC1s) 2 h post-electroporation with 40 pmol Cas9 compared to un-electroporated controls.

    Techniques Used: Derivative Assay, Electroporation

    (A–C) NK cells (preactivated 50 ng rmIL-15), BM-cDC1s, and BMDMs were electroporated at 1,900 V in the presence of indicated single cRNP complexes or pooled cRNP1 and cRNP2 complexes targeting Klrb1c and Thy1.2 for NK cells, Itgax and B2m for BM-cDC1s, and Itgam and B2m for BMDMs. (A) Representative flow plots of NK cell NK1.1 and CD90 expression 3 days after electroporation of 5 × 105 cells with (Right panel) pooled cRNP complexes or (Left panel) control Cas9 protein electroporated cells (B) Percent of NK1.1−, CD90− or NK1.1− and CD90− (“Both”) NK cells and (C) percentage of viable NK cells after electroporation in the presence of indicated cRNP complexes relative to un-electroporated controls (unedited).
    Figure Legend Snippet: (A–C) NK cells (preactivated 50 ng rmIL-15), BM-cDC1s, and BMDMs were electroporated at 1,900 V in the presence of indicated single cRNP complexes or pooled cRNP1 and cRNP2 complexes targeting Klrb1c and Thy1.2 for NK cells, Itgax and B2m for BM-cDC1s, and Itgam and B2m for BMDMs. (A) Representative flow plots of NK cell NK1.1 and CD90 expression 3 days after electroporation of 5 × 105 cells with (Right panel) pooled cRNP complexes or (Left panel) control Cas9 protein electroporated cells (B) Percent of NK1.1−, CD90− or NK1.1− and CD90− (“Both”) NK cells and (C) percentage of viable NK cells after electroporation in the presence of indicated cRNP complexes relative to un-electroporated controls (unedited).

    Techniques Used: Expressing, Electroporation

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Electroporation, Cell Isolation, Software, Transfection

    anti cas9 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cas9 antibody
    Blanks is not required for cytoplasmic RNAi. ( A ) Cartoon drawing of the modified Flag-blanks locus (top); transcription of the gene is now controlled by the copper-inducible mtn promoter contained in our marker/tag cassette. If all alleles of the targeted gene are modified, then gene activity can be fully controlled by the addition or omission of copper in the culture medium (bottom, normalized to blanks levels in non-edited cells). ( B ) We used the inducible Flag-blanks cell line to test the efficiency of cytoplasmic dsRNA-triggered RNA interference as a function of the presence or absence of blanks . We chose the <t>cas9</t> gene as a target because it is constitutively expressed in these cells as well as their progenitors and there should be no secondary effects resulting from the loss of this heterologous gene. A non-specific dsRNA targeting Renilla luciferase was used as a control (RLuc). ( C ) Quantification of the Western Blots from B; the <t>Cas9</t> <t>protein</t> signal was normalized to the tubulin loading control, then the ratio between specific and control-knockdown was calculated. The graph shows the average of three independent biological replicates ± Standard Deviation (SD), n.s.: not significantly different (Student's t -test).
    Anti Cas9 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila"

    Article Title: Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa072

    Blanks is not required for cytoplasmic RNAi. ( A ) Cartoon drawing of the modified Flag-blanks locus (top); transcription of the gene is now controlled by the copper-inducible mtn promoter contained in our marker/tag cassette. If all alleles of the targeted gene are modified, then gene activity can be fully controlled by the addition or omission of copper in the culture medium (bottom, normalized to blanks levels in non-edited cells). ( B ) We used the inducible Flag-blanks cell line to test the efficiency of cytoplasmic dsRNA-triggered RNA interference as a function of the presence or absence of blanks . We chose the cas9 gene as a target because it is constitutively expressed in these cells as well as their progenitors and there should be no secondary effects resulting from the loss of this heterologous gene. A non-specific dsRNA targeting Renilla luciferase was used as a control (RLuc). ( C ) Quantification of the Western Blots from B; the Cas9 protein signal was normalized to the tubulin loading control, then the ratio between specific and control-knockdown was calculated. The graph shows the average of three independent biological replicates ± Standard Deviation (SD), n.s.: not significantly different (Student's t -test).
    Figure Legend Snippet: Blanks is not required for cytoplasmic RNAi. ( A ) Cartoon drawing of the modified Flag-blanks locus (top); transcription of the gene is now controlled by the copper-inducible mtn promoter contained in our marker/tag cassette. If all alleles of the targeted gene are modified, then gene activity can be fully controlled by the addition or omission of copper in the culture medium (bottom, normalized to blanks levels in non-edited cells). ( B ) We used the inducible Flag-blanks cell line to test the efficiency of cytoplasmic dsRNA-triggered RNA interference as a function of the presence or absence of blanks . We chose the cas9 gene as a target because it is constitutively expressed in these cells as well as their progenitors and there should be no secondary effects resulting from the loss of this heterologous gene. A non-specific dsRNA targeting Renilla luciferase was used as a control (RLuc). ( C ) Quantification of the Western Blots from B; the Cas9 protein signal was normalized to the tubulin loading control, then the ratio between specific and control-knockdown was calculated. The graph shows the average of three independent biological replicates ± Standard Deviation (SD), n.s.: not significantly different (Student's t -test).

    Techniques Used: Modification, Marker, Activity Assay, Luciferase, Western Blot, Standard Deviation

    anti cas9  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cas9
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    spcas9  (Cell Signaling Technology Inc)


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    egfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cas9
    (A) OMM 1.5 cells expressing <t>Cas9</t> were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.
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    Knockdown of TUG1 hindered OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a) The transfection efficiency of si-TUG1 (si-TUG1 #1 , si-TUG1 #2 ) was assessed by RT-qPCR in N2a cells. (b) The effect of si-TUG1 #2 on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (c) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells treated by OGD/R. (d and e) The proteins levels of apoptosis-related markers (cleaved-cas-3 and <t>cleaved-cas-9)</t> were performed by western blot assay in N2a cells treated by OGD/R. * P < 0.05.
    Cas 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc egfp
    Knockdown of TUG1 hindered OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a) The transfection efficiency of si-TUG1 (si-TUG1 #1 , si-TUG1 #2 ) was assessed by RT-qPCR in N2a cells. (b) The effect of si-TUG1 #2 on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (c) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells treated by OGD/R. (d and e) The proteins levels of apoptosis-related markers (cleaved-cas-3 and <t>cleaved-cas-9)</t> were performed by western blot assay in N2a cells treated by OGD/R. * P < 0.05.
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    Knockdown of TUG1 hindered OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a) The transfection efficiency of si-TUG1 (si-TUG1 #1 , si-TUG1 #2 ) was assessed by RT-qPCR in N2a cells. (b) The effect of si-TUG1 #2 on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (c) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells treated by OGD/R. (d and e) The proteins levels of apoptosis-related markers (cleaved-cas-3 and <t>cleaved-cas-9)</t> were performed by western blot assay in N2a cells treated by OGD/R. * P < 0.05.
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    (A–C) Mouse splenocytes were either freshly harvested (naive) or cultured for 16 h with recombinant mouse (rm) IL-15 (activated NK and T cells), FLT3-L (activated cDC1, cDC2), or M-CSF (activated macrophage) in complete media and subsequently electroporated in the presence of 40 pmol <t>Cas9</t> protein alone. (A) Frequency of intracellular Cas9+ naive and activated NK (TCRβ−CD3ε−NK1.1+) and T cells (TCRβ+CD3ε+NK1.1−) (B) Representative histogram of intracellular Cas9 levels for rmIL-15 stimulated un-electroporated NK cells and NK cells electroporated in the presence of <t>Cas9</t> <t>protein</t> alone. (C) Frequency of intracellular Cas9+ macrophages (TCRβ−CD3ε−NK1.1−CD64+CD11b+), cDC1s (TCRβ−CD3ε−NK1.1−CD64−CD11β−XCR1+), and cDC2s (TCRβ−CD3ε−NK1.1−CD64−CD11b+XCR1−) following electroporation at the indicated voltages.
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    Cell Signaling Technology Inc anti cas9 antibody
    Blanks is not required for cytoplasmic RNAi. ( A ) Cartoon drawing of the modified Flag-blanks locus (top); transcription of the gene is now controlled by the copper-inducible mtn promoter contained in our marker/tag cassette. If all alleles of the targeted gene are modified, then gene activity can be fully controlled by the addition or omission of copper in the culture medium (bottom, normalized to blanks levels in non-edited cells). ( B ) We used the inducible Flag-blanks cell line to test the efficiency of cytoplasmic dsRNA-triggered RNA interference as a function of the presence or absence of blanks . We chose the <t>cas9</t> gene as a target because it is constitutively expressed in these cells as well as their progenitors and there should be no secondary effects resulting from the loss of this heterologous gene. A non-specific dsRNA targeting Renilla luciferase was used as a control (RLuc). ( C ) Quantification of the Western Blots from B; the <t>Cas9</t> <t>protein</t> signal was normalized to the tubulin loading control, then the ratio between specific and control-knockdown was calculated. The graph shows the average of three independent biological replicates ± Standard Deviation (SD), n.s.: not significantly different (Student's t -test).
    Anti Cas9 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Synthetic Lethal Screens Reveal Co-Targeting FAK and MEK as a Multimodal Precision Therapy for GNAQ -Driven Uveal Melanoma

    doi: 10.1158/1078-0432.CCR-20-3363

    Figure Lengend Snippet: (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.

    Article Snippet: FAK, phospho-FAK, ERK, phospho-ERK, cleaved PARP1, cleaved Caspase 3, YAP, Cas9, and GAPDH antibodies were purchased from Cell Signaling.

    Techniques: Expressing, Infection, CRISPR, Selection, Western Blot

    Knockdown of TUG1 hindered OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a) The transfection efficiency of si-TUG1 (si-TUG1 #1 , si-TUG1 #2 ) was assessed by RT-qPCR in N2a cells. (b) The effect of si-TUG1 #2 on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (c) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells treated by OGD/R. (d and e) The proteins levels of apoptosis-related markers (cleaved-cas-3 and cleaved-cas-9) were performed by western blot assay in N2a cells treated by OGD/R. * P < 0.05.

    Journal: Open Medicine

    Article Title: Long non-coding RNA TUG1 aggravates cerebral ischemia and reperfusion injury by sponging miR-493-3p/miR-410-3p

    doi: 10.1515/med-2021-0253

    Figure Lengend Snippet: Knockdown of TUG1 hindered OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a) The transfection efficiency of si-TUG1 (si-TUG1 #1 , si-TUG1 #2 ) was assessed by RT-qPCR in N2a cells. (b) The effect of si-TUG1 #2 on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (c) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells treated by OGD/R. (d and e) The proteins levels of apoptosis-related markers (cleaved-cas-3 and cleaved-cas-9) were performed by western blot assay in N2a cells treated by OGD/R. * P < 0.05.

    Article Snippet: The antibodies were cleaved-cas-3, cleaved-cas-9, p-JNK and JNK, p-p38, p38 (1:1,000; Cell Signaling, Danvers, MA, USA), GAPDH (1:500; Santa Cruz, Dallas, TX, USA) and HRP-conjugated secondary antibodies (1:5,000; Southern-Biotech, Birmingham, AL, USA).

    Techniques: In Vitro, Transfection, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Western Blot

    The inhibition of miR-493-3p or miR-410-3p could restore the reverse effects of TUG1 knockdown on OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a and b) The effect of si-TUG1 #2 and anti-miR-493-3p or anti-miR-410-3p on the expression of miR-493-3p or miR-410-3p was detected by RT-qPCR. (c) The effect of si-TUG1 #2 and anti-miR-493-3p or anti-miR-410-3p on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (d) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells, which transfected with si-TUG1 #2 + anti-miR-493-3p or si-TUG1 #2 + anti-miR-410-3p or negative controls, with or without being treated by OGD/R. (e and f) The proteins levels of apoptosis-related markers (cleaved-cas-3 and cleaved-cas-9) were performed by western blot assay in N2a cells, which transfected with si-TUG1 #2 + anti-miR-493-3p or si-TUG1 #2 + anti-miR-410-3p or negative controls, with or without being treated by OGD/R. * P < 0.05.

    Journal: Open Medicine

    Article Title: Long non-coding RNA TUG1 aggravates cerebral ischemia and reperfusion injury by sponging miR-493-3p/miR-410-3p

    doi: 10.1515/med-2021-0253

    Figure Lengend Snippet: The inhibition of miR-493-3p or miR-410-3p could restore the reverse effects of TUG1 knockdown on OGD/R-mediated decrease in viability and increase in apoptosis in N2a cells in vitro . (a and b) The effect of si-TUG1 #2 and anti-miR-493-3p or anti-miR-410-3p on the expression of miR-493-3p or miR-410-3p was detected by RT-qPCR. (c) The effect of si-TUG1 #2 and anti-miR-493-3p or anti-miR-410-3p on cell viability was evaluated by CCK-8 assay in N2a cells treated by OGD/R. (d) Flow cytometry assay was applied to detect the apoptosis rate in N2a cells, which transfected with si-TUG1 #2 + anti-miR-493-3p or si-TUG1 #2 + anti-miR-410-3p or negative controls, with or without being treated by OGD/R. (e and f) The proteins levels of apoptosis-related markers (cleaved-cas-3 and cleaved-cas-9) were performed by western blot assay in N2a cells, which transfected with si-TUG1 #2 + anti-miR-493-3p or si-TUG1 #2 + anti-miR-410-3p or negative controls, with or without being treated by OGD/R. * P < 0.05.

    Article Snippet: The antibodies were cleaved-cas-3, cleaved-cas-9, p-JNK and JNK, p-p38, p38 (1:1,000; Cell Signaling, Danvers, MA, USA), GAPDH (1:500; Santa Cruz, Dallas, TX, USA) and HRP-conjugated secondary antibodies (1:5,000; Southern-Biotech, Birmingham, AL, USA).

    Techniques: Inhibition, In Vitro, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Transfection, Western Blot

    (A–C) Mouse splenocytes were either freshly harvested (naive) or cultured for 16 h with recombinant mouse (rm) IL-15 (activated NK and T cells), FLT3-L (activated cDC1, cDC2), or M-CSF (activated macrophage) in complete media and subsequently electroporated in the presence of 40 pmol Cas9 protein alone. (A) Frequency of intracellular Cas9+ naive and activated NK (TCRβ−CD3ε−NK1.1+) and T cells (TCRβ+CD3ε+NK1.1−) (B) Representative histogram of intracellular Cas9 levels for rmIL-15 stimulated un-electroporated NK cells and NK cells electroporated in the presence of Cas9 protein alone. (C) Frequency of intracellular Cas9+ macrophages (TCRβ−CD3ε−NK1.1−CD64+CD11b+), cDC1s (TCRβ−CD3ε−NK1.1−CD64−CD11β−XCR1+), and cDC2s (TCRβ−CD3ε−NK1.1−CD64−CD11b+XCR1−) following electroporation at the indicated voltages.

    Journal: Cell reports

    Article Title: CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells

    doi: 10.1016/j.celrep.2020.107651

    Figure Lengend Snippet: (A–C) Mouse splenocytes were either freshly harvested (naive) or cultured for 16 h with recombinant mouse (rm) IL-15 (activated NK and T cells), FLT3-L (activated cDC1, cDC2), or M-CSF (activated macrophage) in complete media and subsequently electroporated in the presence of 40 pmol Cas9 protein alone. (A) Frequency of intracellular Cas9+ naive and activated NK (TCRβ−CD3ε−NK1.1+) and T cells (TCRβ+CD3ε+NK1.1−) (B) Representative histogram of intracellular Cas9 levels for rmIL-15 stimulated un-electroporated NK cells and NK cells electroporated in the presence of Cas9 protein alone. (C) Frequency of intracellular Cas9+ macrophages (TCRβ−CD3ε−NK1.1−CD64+CD11b+), cDC1s (TCRβ−CD3ε−NK1.1−CD64−CD11β−XCR1+), and cDC2s (TCRβ−CD3ε−NK1.1−CD64−CD11b+XCR1−) following electroporation at the indicated voltages.

    Article Snippet: Anti-Cas9 , Cell Signaling Technology , 7A9-3A3.

    Techniques: Cell Culture, Recombinant, Electroporation

    (A–E) Purified splenic NK cells were cultured for 16 h with 50 ng/mL rmIL-15 in complete media or freshly isolated before electroporation. (A) Frequency of intracellular Cas9+ NK cells (left) and percentage of viable NK cells (right) 2 h following electroporation with 40 pmol Cas9 of 5 × 105 rmIL-15-preactivated NK cells at the indicated voltages compared to un-electroporated controls. (B and C) Percentage of NK1.1− rmIL-15-preactivated NK cells 3 days after electroporation with cRNP complex consisting of Cas9 and Klrb1c gRNA compared to un-electroporated controls with varying (B) voltages or (C) concentrations of Cas9 in the Klrb1c cRNP complex electroporated at 1,900 V. Representative histogram of NK1.1 expression (D) and percentage of NK1.1− NK cells (E) 3 days after electroporation at 1,900 V of 5 × 105 rmIL-15-preactivated activated or freshly isolated naive cells compared to controls electroporated in the presence of Cas9 protein alone.

    Journal: Cell reports

    Article Title: CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells

    doi: 10.1016/j.celrep.2020.107651

    Figure Lengend Snippet: (A–E) Purified splenic NK cells were cultured for 16 h with 50 ng/mL rmIL-15 in complete media or freshly isolated before electroporation. (A) Frequency of intracellular Cas9+ NK cells (left) and percentage of viable NK cells (right) 2 h following electroporation with 40 pmol Cas9 of 5 × 105 rmIL-15-preactivated NK cells at the indicated voltages compared to un-electroporated controls. (B and C) Percentage of NK1.1− rmIL-15-preactivated NK cells 3 days after electroporation with cRNP complex consisting of Cas9 and Klrb1c gRNA compared to un-electroporated controls with varying (B) voltages or (C) concentrations of Cas9 in the Klrb1c cRNP complex electroporated at 1,900 V. Representative histogram of NK1.1 expression (D) and percentage of NK1.1− NK cells (E) 3 days after electroporation at 1,900 V of 5 × 105 rmIL-15-preactivated activated or freshly isolated naive cells compared to controls electroporated in the presence of Cas9 protein alone.

    Article Snippet: Anti-Cas9 , Cell Signaling Technology , 7A9-3A3.

    Techniques: Purification, Cell Culture, Isolation, Electroporation, Expressing

    (A) Frequency of intracellular Cas9+ bone-marrow-derived macrophage (BMDM) or bone marrow-derived cDC1s (BM-cDC1s) 2 h post-electroporation with 40 pmol Cas9 compared to un-electroporated controls.

    Journal: Cell reports

    Article Title: CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells

    doi: 10.1016/j.celrep.2020.107651

    Figure Lengend Snippet: (A) Frequency of intracellular Cas9+ bone-marrow-derived macrophage (BMDM) or bone marrow-derived cDC1s (BM-cDC1s) 2 h post-electroporation with 40 pmol Cas9 compared to un-electroporated controls.

    Article Snippet: Anti-Cas9 , Cell Signaling Technology , 7A9-3A3.

    Techniques: Derivative Assay, Electroporation

    (A–C) NK cells (preactivated 50 ng rmIL-15), BM-cDC1s, and BMDMs were electroporated at 1,900 V in the presence of indicated single cRNP complexes or pooled cRNP1 and cRNP2 complexes targeting Klrb1c and Thy1.2 for NK cells, Itgax and B2m for BM-cDC1s, and Itgam and B2m for BMDMs. (A) Representative flow plots of NK cell NK1.1 and CD90 expression 3 days after electroporation of 5 × 105 cells with (Right panel) pooled cRNP complexes or (Left panel) control Cas9 protein electroporated cells (B) Percent of NK1.1−, CD90− or NK1.1− and CD90− (“Both”) NK cells and (C) percentage of viable NK cells after electroporation in the presence of indicated cRNP complexes relative to un-electroporated controls (unedited).

    Journal: Cell reports

    Article Title: CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells

    doi: 10.1016/j.celrep.2020.107651

    Figure Lengend Snippet: (A–C) NK cells (preactivated 50 ng rmIL-15), BM-cDC1s, and BMDMs were electroporated at 1,900 V in the presence of indicated single cRNP complexes or pooled cRNP1 and cRNP2 complexes targeting Klrb1c and Thy1.2 for NK cells, Itgax and B2m for BM-cDC1s, and Itgam and B2m for BMDMs. (A) Representative flow plots of NK cell NK1.1 and CD90 expression 3 days after electroporation of 5 × 105 cells with (Right panel) pooled cRNP complexes or (Left panel) control Cas9 protein electroporated cells (B) Percent of NK1.1−, CD90− or NK1.1− and CD90− (“Both”) NK cells and (C) percentage of viable NK cells after electroporation in the presence of indicated cRNP complexes relative to un-electroporated controls (unedited).

    Article Snippet: Anti-Cas9 , Cell Signaling Technology , 7A9-3A3.

    Techniques: Expressing, Electroporation

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells

    doi: 10.1016/j.celrep.2020.107651

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Anti-Cas9 , Cell Signaling Technology , 7A9-3A3.

    Techniques: Recombinant, Electroporation, Cell Isolation, Software, Transfection

    Blanks is not required for cytoplasmic RNAi. ( A ) Cartoon drawing of the modified Flag-blanks locus (top); transcription of the gene is now controlled by the copper-inducible mtn promoter contained in our marker/tag cassette. If all alleles of the targeted gene are modified, then gene activity can be fully controlled by the addition or omission of copper in the culture medium (bottom, normalized to blanks levels in non-edited cells). ( B ) We used the inducible Flag-blanks cell line to test the efficiency of cytoplasmic dsRNA-triggered RNA interference as a function of the presence or absence of blanks . We chose the cas9 gene as a target because it is constitutively expressed in these cells as well as their progenitors and there should be no secondary effects resulting from the loss of this heterologous gene. A non-specific dsRNA targeting Renilla luciferase was used as a control (RLuc). ( C ) Quantification of the Western Blots from B; the Cas9 protein signal was normalized to the tubulin loading control, then the ratio between specific and control-knockdown was calculated. The graph shows the average of three independent biological replicates ± Standard Deviation (SD), n.s.: not significantly different (Student's t -test).

    Journal: Nucleic Acids Research

    Article Title: Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

    doi: 10.1093/nar/gkaa072

    Figure Lengend Snippet: Blanks is not required for cytoplasmic RNAi. ( A ) Cartoon drawing of the modified Flag-blanks locus (top); transcription of the gene is now controlled by the copper-inducible mtn promoter contained in our marker/tag cassette. If all alleles of the targeted gene are modified, then gene activity can be fully controlled by the addition or omission of copper in the culture medium (bottom, normalized to blanks levels in non-edited cells). ( B ) We used the inducible Flag-blanks cell line to test the efficiency of cytoplasmic dsRNA-triggered RNA interference as a function of the presence or absence of blanks . We chose the cas9 gene as a target because it is constitutively expressed in these cells as well as their progenitors and there should be no secondary effects resulting from the loss of this heterologous gene. A non-specific dsRNA targeting Renilla luciferase was used as a control (RLuc). ( C ) Quantification of the Western Blots from B; the Cas9 protein signal was normalized to the tubulin loading control, then the ratio between specific and control-knockdown was calculated. The graph shows the average of three independent biological replicates ± Standard Deviation (SD), n.s.: not significantly different (Student's t -test).

    Article Snippet: The anti-cas9 antibody was obtained from Cell Signaling Technology (clone 7A9-3A3).

    Techniques: Modification, Marker, Activity Assay, Luciferase, Western Blot, Standard Deviation