bassik human crispr deletion library-drug targets, kinases, and phosphatases  (Addgene inc)

Journal: Cancers

Article Title: CRISPR/Cas9 Screening Highlights PFKFB3 Gene as a Major Contributor to 5-Fluorouracil Resistance in Esophageal Cancer

doi: 10.3390/cancers17101637

Figure Lengend Snippet: Human protein kinase library screen identified the PFKFB3 gene involved in esophageal cancer chemoresistance ( A ) Schematic outline of 5-FU drug resistance screening. ( B ) Identification of good sgRNA using MAGeCK-VISPR analysis. ( C – E ) PFKFB3 knockout KYSE-70, KYSE-270, and KYSE-150 cell lines were generated using CRISPR/Cas9 technology, and Western blot analysis was employed to confirm the efficiency of the PFKFB3 gene knockout.

Article Snippet: The human protein kinase library, which was a gift from John Doench and David Root (RRID: Addgene_75312), and additional sgRNAs designed to target genes involved in protein kinase [ ] contained 6934 sgRNAs targeting 1053 human genes and 107 NTCs (non-targeting-controls).

Techniques: Knock-Out, Generated, CRISPR, Western Blot, Gene Knockout

Journal: Cell Reports

Article Title: AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection

doi: 10.1016/j.celrep.2024.115001

Figure Lengend Snippet: AMPKα2 is the kinase responsible for Bcl2-L-13 phosphorylation at Ser272 (A) Schematic of the responsible kinase screening workflow. (B) In vitro kinase assay in the third screening. Bacterially synthesized HA-Bcl2-L-13 was mixed with purified candidate proteins and ATP. After incubation at 37°C for 30 min, the reaction mix was subjected to western blotting using an anti-phospho-Bcl2-L-13 (Ser272) antibody. (C) The effect of AMPKα2 knockdown in CCCP-induced Bcl2-L-13 phosphorylation. HEK293A cells stably expressing HA-Bcl2-L-13 were transfected with control siRNA or siAMPKα2 for 72 h. Then, the cells were treated with DMSO or 15 μM CCCP for the indicated times, and cell lysates were subjected to western blot analysis. Densitometric analysis of phospho-Bcl2-L-13 (Ser272) is shown in the bar graph. The value for the group with control siRNA (siCtrl) transfection and 15 min of DMSO treatment in each experiment was set to 1 ( n = 3). (D) Upregulation of AMPKα2 activity by CCCP treatment. To analyze AMPKα2-specific activity, HEK293A cells stably expressing HA-Bcl2-L-13 were transfected with siAMPKα1 for 72 h and then treated with DMSO or 15 μM CCCP. The value for the group with 15-min DMSO treatment in each experiment was set to 1 ( n = 4). (E and F) HEK293A cells were transfected with control siRNA (siCtrl) or siAMPKα2 for 72 h, followed by transfection with an empty vector or HA-Bcl2-L-13. Forty-four hours after transfection, cells were treated with 100 nM bafilomycin A1 for 4 h and immunostained with anti-LC3B and anti-ATP synthase antibodies. Images in the box at higher magnification are shown on the right. White arrows indicate the puncta recognized as colocalized by the software. The number of LC3B dots colocalized with ATP synthase dots per cell is shown in (F). At least 20 cells were counted for each group ( n = 3). Scale bar: 10 μm. (G) Upregulation of AMPKα2 phosphorylation 5 days after TAC operation. To analyze AMPKα2-specific phosphorylation, lysates from the left ventricle were subjected to immunoprecipitation with an anti-AMPKα2 antibody followed by immunoblotting with an anti-phospho-AMPKα (Thr172) antibody. Densitometric analysis of phospho-AMPKα (Thr172) is shown in the right bar graph. The value for the WT sham group in each experiment was set to 1 ( n = 3). (H) Interaction between Bcl2-L-13 and AMPKα2 5 days after TAC. Lysates from the left ventricle were subjected to immunoprecipitation with an anti-AMPKα2 antibody. Co-precipitated Bcl2-L-13 was detected by immunoblotting. Densitometric analysis of Bcl2-L-13 is shown in the graph below. The value for the WT sham group in each experiment was set to 1 ( n = 3). Results are shown as mean with 95% CI. Statistical analysis by unpaired, two-tailed t tests in (C), (D), (F), and (H) and one-way ANOVA followed by Tukey-Kramer’s post hoc test in (G). All pairwise comparisons were performed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant. See also Figures S6 and .

Article Snippet: SilencerTM Human Kinase siRNA Library , ThermoFisher Scientific , Cat# A30079.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Synthesized, Purification, Incubation, Western Blot, Knockdown, Stable Transfection, Expressing, Transfection, Control, Activity Assay, Plasmid Preparation, Software, Immunoprecipitation, Two Tailed Test

Journal: Cell Reports

Article Title: AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection

doi: 10.1016/j.celrep.2024.115001

Figure Lengend Snippet:

Article Snippet: SilencerTM Human Kinase siRNA Library , ThermoFisher Scientific , Cat# A30079.

Techniques: Transduction, Virus, Recombinant, Western Blot, Protease Inhibitor, Immunoprecipitation, Purification, ATP Assay, In Situ, Kinase Assay, Real-time Polymerase Chain Reaction, Knock-Out, Knock-In, Negative Control, Software

Journal: Cell Reports

Article Title: AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection

doi: 10.1016/j.celrep.2024.115001

Figure Lengend Snippet:

Article Snippet: For the primary screen, the Silencer Human Kinase siRNA Library (three siRNAs per gene) targeting 708 genes (ThermoFisher Scientific, A30079) was used.

Techniques: Transduction, Virus, Recombinant, Western Blot, Protease Inhibitor, Immunoprecipitation, Purification, ATP Assay, In Situ, Kinase Assay, Real-time Polymerase Chain Reaction, Knock-Out, Knock-In, Negative Control, Software