Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet:

Article Snippet: For inducible overexpression (OE), a pLVX-IRES-ZsGreen1 vector (Clontech) containing the gene of interest and pLVX-TRE3G vector (Clontech) were transfected separately with packaging plasmids.

Techniques: Transfection, Recombinant, Knock-Out, LDH Cytotoxicity Assay, SYBR Green Assay, Membrane, Luciferase, Binding Assay, Software, Microscopy

Journal: Viruses

Article Title: Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors

doi: 10.3390/v11030275

Figure Lengend Snippet: SH-SY5Y cells do not express a dominant entry restriction factor. Huh-7.5 or SH-SY5Y cells stably expressing the tetracycline-inducible Tet On 3G transactivator protein were cocultured with HEK293T-H6 cells stably expressing transactivator-inducible ZsGreen1 green fluorescent protein for 24 h. Cells were chemically fused with PEG. One hour after fusion, cells were transduced with NoEnv, EBOV, MARV, or VSV-G pseudoparticles encoding mCherry for 6 h at 37 °C. Seventy-two hours post transduction, cells were fixed with 3% PFA and analyzed for heterokaryon formation (ZsGreen1 protein expression) and susceptibility to pseudoparticle infection (mCherry protein expression). ( A ) Confocal microscopy images of one representative experiment with SH-SY5Y and HEK293T-H6 cells. Scale bars = 50 µm ( B ) Quantification of transduced cells by flow cytometry. Left-hand side of graph represents transduction percentage of single cell line controls and right-hand side from heterokaryons (discriminated previously by gating on ZsGreen1 positive cells). Mean ± SD. of three independent cell fusions and subsequent transduction experiments ( n = 3).

Article Snippet: Lenti-X TM Tet-On ® inducible expression system plasmids pLVX-Tet3G and pLVX-TRE3G-ZsGreen1 were purchased from Takara Bio. (Mountain View, CA, USA).

Techniques: Stable Transfection, Expressing, Transduction, Infection, Confocal Microscopy, Flow Cytometry

Journal: Nature genetics

Article Title: Silencing of LINE-1 Retrotransposons is a Selective Dependency of Myeloid Leukemia

doi: 10.1038/s41588-021-00829-8

Figure Lengend Snippet: a, Schematic of the CRISPR screen system containing a TET-on-3G trans-activator, a tetracycline (TRE3G)-inducible Cas9 with an mCherry reporter, and the sgRNA. b, Schematic of the experiments to validate the inducible CRISPR screen system by targeting the stably expressed GFP reporter gene in MOLM-13 cells. c, Dox-induced expression of Cas9 and GFP-targeting sgRNAs in mCherry+ cells significantly decreased GFP expression compared to Cas9-negative (mCherry−) cells after 4 days of doxycycline treatment. d, Schematic of the customized sgRNA library containing 3,248 sgRNAs targeting 498 conserved chromatin-interacting domains in 266 annotated human epigenetic regulators and 1,800 sgRNAs targeting the 5’ exon of the other epigenetic regulators, together with 239 positive and 390 negative control sgRNAs. e, Comparisons of replicate experiments of CRISPR screens in MOLM-13 cells. The x- and y-axis of each graph represent the normalized sgRNA counts at day0 and day14. Spearman correlation value is shown for each comparison. f, MPP8 expression was determined by Western blot in MOLM-13 cells before xenograft (control), one week after Dox-induced MPP8 KO in xenografted NSG mice, and in the moribund mice (6-week post-xenograft) by independent MPP8-targeting sgRNAs (sg1 and sg2). g, Depletion of MPP8 by RNAi-mediated knockdown using shRNAs against the coding or 3’UTR sequences. shRNA against the luciferase gene (shLuc) was used as a negative control. Schematic of the MPP8 gene and the positions of shRNAs (sh1 to sh4) are shown. Results are mean ± SD (N = 3 independent experiments) and analyzed by a one-way ANOVA with Dunnett’s test. h, Growth curves of MOLM-13 AML cells after doxycycline-induced expression of MPP8-targeting shRNAs. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Tukey’s test.

Article Snippet: Then the eSpCas9 1.1 sequence (Addgene, #71814) was cloned to the BamHI and NotI sites of the pLVX-TRE3G-IRES-mCherry vector using the In-Fusion HD cloning kit (Takara).

Techniques: CRISPR, Stable Transfection, Expressing, Negative Control, Western Blot, shRNA, Luciferase