Journal: International Journal of Molecular Sciences

Article Title: Genome-Wide CRISPR Screening Identifies Genetic Modulators of Amyloid Precursor Protein Processing

doi: 10.3390/ijms27093926

Figure Lengend Snippet: Design of the dual-fluorescence reporter system and genome-wide CRISPR screening workflow. ( A ) Schematic representation of the lentiviral dual-fluorescence reporter constructs. In the experimental vector (bottom), a constitutive EF-1α promoter drives the expression of the APP-GAL4 fusion protein, while a separate SV40 promoter drives the constitutive expression of the mCherry internal reference, with a 5×UAS element governing enhanced green fluorescent protein (EGFP) expression. The control vector (top) lacks the APP-GAL4 expression cassette. ( B ) Mechanism of reporter transactivation. Endogenous secretase-mediated cleavage releases the AICD-GAL4 domain, which translocates to the nucleus to drive EGFP transcription. ( C ) Representative fluorescence microscopy images of the monoclonal HEK293FT reporter cell line, demonstrating stable mCherry expression and dynamic basal EGFP signal. Scale bars = 500 μm. ( D ) Schematic outline of the genome-wide CRISPR-Cas9 screening pipeline, including low-MOI lentiviral library transduction, phenotypic cell sorting, and next-generation sequencing (NGS). ( E ) Representative flow cytometry histogram illustrating the EGFP fluorescence distribution of the transduced mutant cell pool. The defined sorting windows are shown for the isolation of the EGFP-low (enriched for sgRNAs targeting positive regulators of cleavage) and EGFP-high (enriched for sgRNAs targeting negative regulators of cleavage) populations.

Article Snippet: The human embryonic kidney cell line HEK293FT (CRL-1573) was obtained from the American Type Culture Collection (ATCC).

Techniques: Fluorescence, Genome Wide, CRISPR, Construct, Plasmid Preparation, Expressing, Control, Microscopy, Transduction, FACS, Next-Generation Sequencing, Flow Cytometry, Mutagenesis, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Genome-Wide CRISPR Screening Identifies Genetic Modulators of Amyloid Precursor Protein Processing

doi: 10.3390/ijms27093926

Figure Lengend Snippet: Biochemical, cellular, and clinical validation of core APP processing regulators. ( A , B ) Flow cytometry-based quantification showing changes in the EGFP/mCherry reporter profile following CRISPR-Cas9-mediated knockout, using two independent sgRNAs per gene for PIAS2 , LDHB , CCDC53 , and TRIM61 . BACE1 overexpression (OE) serves as a positive control for amyloidogenic APP processing, and a non-targeting sgRNA serves as the negative control ( A ). Quantification of the percentage of cells in the EGFP-low and EGFP-high populations across independent biological replicates ( B ). ( C , D ) Representative immunoblots assessing the steady-state protein levels of full-length APP, key secretases (BACE1, ADAM10, and γ-secretase components Presenilin 1 (PS1) and Presenilin 2 (PS2)), and the GAPDH loading control in HEK293FT cells, following CRISPR-Cas9-mediated knockout with two independent sgRNAs per gene ( C ): CCDC53, TRIM61; ( D ): PIAS2, LDHB). ( E , F ) ELISA quantification of the secreted neuroprotective sAPPα fragment ( E ) and amyloidogenic Aβ 42 peptide ( F ) in conditioned media from each knockout cell line. Data are presented as mean ± SD ( n = 5). Statistical significance was calculated using a two-tailed Student’s t -test (* p < 0.05, ** p < 0.01, *** p < 0.001). ( G ) Cross-regional clinical transcriptomic analysis showing the expression dysregulation (Log2(AD/Control)) of validated candidate regulators across multiple brain regions in human AD cohorts. Data are derived from the AMP-AD Agora portal. Significant transcriptional dysregulation is observed in pathologically vulnerable brain regions (e.g., parahippocampal gyrus, temporal cortex) compared to the pathologically resilient cerebellum. Statistical significance was calculated using a two-tailed Student’s t -test (* p < 0.05, ** p < 0.01, *** p < 0.001, ns , not significant).

Article Snippet: The human embryonic kidney cell line HEK293FT (CRL-1573) was obtained from the American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Flow Cytometry, CRISPR, Knock-Out, Over Expression, Positive Control, Negative Control, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Expressing, Derivative Assay

Journal: Biology Direct

Article Title: The MST4–14-3-3ζ complex promotes pancreatic cancer by activating YAP

doi: 10.1186/s13062-026-00784-6

Figure Lengend Snippet: MST4 Directly Binds to 14-3-3ζ via a Phosphorylation-dependent manner. ( A ). Left: Confocal microscope analysis of endogenous 14-3-3ζ and overexpressed Flag-MST4 in PANC-1 cells. DAPI represents the nuclear region. Scale bar: 10 μm. Right: Quantitative colocalization of MST4 and 14-3-3ζ in PANC-1 cells. Red pixel intensity presents Flag-MST4; Green pixel intensity presents 14-3-3ζ; Pearson r = 0.7441 ( n = 40). Colocalization quantification was performed using Pearson’s correlation coefficient analysis, based on methods reported in the literature . ( B ). Co-IP and immunoblot analysis of the interaction between exogenous Flag-tagged MST4 and 14-3-3ζ in HEK293FT cells. ( C ). MBP pulldown analysis between MST4/pMST4 and 14-3-3ζ. Purified protein of MST4 was first dephosphorylated by λPPase or further phosphorylated by ATP. The input and output samples were loaded on SDS-PAGE followed by CBB and immunoblot staining. ( D ). Microscale thermophoresis (MST) assay showing the binding affinity between 14-3-3ζ and the pMST4 (pT320) peptide. ( E ). Alignment of 14-3-3ζ binding motif sequence and the putative phosphorylation sites of MST4. The phosphorylation sites were labelled red. ( F ). Co-IP and immunoblot analysis of the interaction between exogenous Flag-tagged wildtype or T320A mutant of MST4 and endogenous 14-3-3ζ in HEK293FT cells

Article Snippet: HEK293FT cells were obtained from Shanghai Life Academy of Sciences cell library (Shanghai, China), and PANC-1 and KPC cells were purchased from Shanghai Model Organisms Center, Inc and Shanghai YuanChuang biotechnology Co.,Ltd (Shanghai, China), respectively.

Techniques: Phospho-proteomics, Microscopy, Co-Immunoprecipitation Assay, Western Blot, Purification, SDS Page, Staining, Microscale Thermophoresis, Binding Assay, Sequencing, Mutagenesis