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

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

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

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

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

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

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

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

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