one sgrna crispr cas9 lentivectors  (Applied Biological Materials Inc)

Journal: bioRxiv

Article Title: Orai1 is required for Ca 2+ -dependent plasma membrane repair and mechanoadaptation

doi: 10.64898/2026.05.13.724989

Figure Lengend Snippet: A. Western blot confirmation of loss of Orai1 expression upon CRISPR/Cas9-mediated KO in MDA-MB-231 and Hs578T cells; shown are pools of three clones maintained individually. B-C. Impairment of SOCE upon Orai1 KO in TNBC cells. SOCE was measured using thapsigargin-induced ER Ca 2+ depletion. Typical profiles are shown on left and quantification of fold-change in peak fluorescence intensity from 3 independent experiments is shown on right. Welch’s t test, ***p<0.001. D-E. Orai1-KO impairs the repair of mechanically induced plasma membrane injury. WT vs. Orai1-KO cells were subjected to mechanical injury to plasma membrane and repair assay was performed as in . Representative confocal images are shown in D and quantified data are shown in E. Scale bar, 200 µm. Data represents mean +/- SEM of three experiments, two-way ANOVA, **,p<0.01. F-G. Orai1-KO impairs the repair of SLO-induced membrane injury. Plasma membrane damage using SLO and the repair assay on MDA-MB-231 ( F ) and Hs578T ( G ) cell lines were as in . Representative confocal images are shown on left. Quantification of PI-stained cells from 3 independent experiments is shown on right. Welch’s t test, ***p<0.001. H-J. Orai1 inhibitors impair plasma membrane repair. Panel H shows Thapsigargin (TG)-induced SOCE measurements (initial peak, Ca 2+ store release in the absence of extracellular Ca 2+ ; second peak, SOCE in the presence of extracellular Ca 2+ ) performed on the indicated cell lines cultured with or without CM4620 (10 μM, 4 hours pretreatment). Note significant SOCE inhibition by CM4620 in WT cells but not in Orai1-KO cells, supporting Orai1-selelctive effect of CM4620. Data represents mean +/- SEM of three experiments, Welch’s t test, ***p<0.001. The I panel shows the impairment of mechanically induced plasma membrane repair by CM4620 in MDA-MB-231. Cells were cultured without or with pretreatment with CM4620 (10 μM) and analyzed for repair as in . Scale bar, 200 µm. Data represents mean +/- SEM of three experiments, two-way ANOVA, **,p<0.01. The J panel shows the impairment of SLO-induced membrane damage in MDA-MB-231. Left panel shows representative FACS analysis of membrane repair in cells without or with CM4620 treatment. Right panel shows quantification of cells that failed to repair (PI staining) from 3 independent experiments. Data represents mean +/-SEM of three experiments. Welch’s t test, ***, p<0.001.

Article Snippet: All-in-One sgRNA CRISPR/Cas9 Lentivectors from Applied Biological Materials (Richmond, BC, Canada) were used to derive Orai1 (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro, #35720125) KO cell lines.

Techniques: Western Blot, Expressing, CRISPR, Clone Assay, Fluorescence, Clinical Proteomics, Membrane, Staining, Cell Culture, Inhibition

Journal: Cell Reports

Article Title: YAP1 is a key regulator of EWS::FLI1-dependent malignant transformation upon IGF-1-mediated reprogramming of bone mesenchymal stem cells

doi: 10.1016/j.celrep.2025.115381

Figure Lengend Snippet: Comparative analysis of differentially accessible chromatin modules in MSCLCs (A) Heatmap showing row-scaled normalized read counts for all peaks that were considered differentially accessible in at least one comparison (EF Prx1 MSCLCs/WT MSCLCs, hr-EF Prx1 MSCLCs/EF Prx1 MSCLCs, or MSCLC + IGF-1/WT MSCLCs; n total = 2,038). Peaks are grouped into five “modules” (M1–M5). GGAA motif density in differentially open regions is indicated to the right of the heatmap. See Table S4 . (B) Scatterplot comparing changes in the chromatin accessibility of ATAC-seq peaks (x axis) with the corresponding changes in gene expression of the nearest genes (y axis). One point is indicated for each combination of differentially accessible peak (from A) and differentially expressed gene. Color indicates the mean of both fold changes. (C) The protein levels of YAP1, LAMA5, and β-actin were detected by western blot for WT MSCLCs, EF Prx1 MSCLCs, and hr-EF Prx1 MSCLCs (top) and those of Yap1 by immunofluorescence staining (bottom). Scale bars: 50 μm. (D) Bar plots showing DNA sequence motifs (mouse and human TF motifs from JASPAR 2022 ) overrepresented in peaks belonging to each of the five modules from (A). Each plot chart lists the top three motifs per module and each bar indicates the percentage of peaks with at least one match to the given motif. Enrichment was calculated using Fisher’s exact test (one-tailed). ∗ p adj < 0.05, ∗∗ p adj ≤ 0.01, and ∗∗∗ p adj ≤ 0.005. See Table S9 . (E) Left: Western blot analysis of YAP1 levels in hr-EF Prx1 MSCLCs #2 upon knockout of Yap1 using three CRISPR single-guide (sg)RNAs (sg-Yap1). Middle: Representative soft-agar assay for hr-EF Prx1 cells transduced with sg-Ctrl versus sg-Yap1. Right: The number of cell colonies was counted on days 21 after plating. Data are presented as the mean ± SE ( n = 3), ∗∗∗ p < 0.001. Statistics were calculated by one-tailed, paired Student’s t test. (F) Quantitative analysis by RT-qPCR of relative mRNA expression levels of Yap1, Igf2bp1, Hpf1, Cenpq, and Ccnd1 after knockout of Yap1. Data are presented as the mean ± SE ( n = 3), ∗∗∗ p < 0.001 and ∗∗ p < 0.001. Statistics were calculated by one-tailed, paired Student’s t test.

Article Snippet: LentiCRISPR Yap1 sgRNA Crispr/Cas9 all in-one lentivector set mouse , Applied Biological Materials (abm) , Cat# 505841140595.

Techniques: Comparison, Gene Expression, Western Blot, Immunofluorescence, Staining, Sequencing, One-tailed Test, Knock-Out, CRISPR, Soft Agar Assay, Transduction, Quantitative RT-PCR, Expressing

Journal: Cell Reports

Article Title: YAP1 is a key regulator of EWS::FLI1-dependent malignant transformation upon IGF-1-mediated reprogramming of bone mesenchymal stem cells

doi: 10.1016/j.celrep.2025.115381

Figure Lengend Snippet:

Article Snippet: LentiCRISPR Yap1 sgRNA Crispr/Cas9 all in-one lentivector set mouse , Applied Biological Materials (abm) , Cat# 505841140595.

Techniques: Recombinant, Cell Viability Assay, CRISPR, Isolation, DNA Purification, Generated, Software