Journal: bioRxiv

Article Title: Studying a human genetic model of lung squamous cell carcinoma with organotypic cultures and xenografts uncovers distinct advantages of each system and implicates NOTCH1 loss in tumour development

doi: 10.1101/2025.08.28.672595

Figure Lengend Snippet: Protocol for genetic manipulation of hBECs and correlation between xenograft volume and ALI thickness. A. Schematics depicting the protocol of hBEC genetic manipulation using CRISPR/Cas9 and lentiviral transduction. Wild type hBECs were electroporated to transfect multiplex CRISPR constructs and treated with Nutlin-3a to select for TP53 truncations as all constructs include a TP53 -targeting gRNA. Nutlin-3a resistant hBECs were transduced with polycistronic lentiviral constructs that harbour mCherry+ SOX2 or mCherry only. B. Correlation between mean xenograft volume and mean ALI thickness for each genotype. Line shows a linear regression of the data. Correlation coefficient, and the p-value was calculated using Pearson analysis. C. Correlation between mean xenografts volume and mean ALI thickness for each genotype except TC+PK. Line shows a linear regression of the data. Correlation coefficient and p-value were calculated using Pearson analysis.

Article Snippet: To induce SOX2 overexpression, we employed the third-generation lentiviral vector pUltra-hot (Addgene #24130), generously shared by Malcolm Moore.

Techniques: CRISPR, Transduction, Multiplex Assay, Construct

Journal: bioRxiv

Article Title: Studying a human genetic model of lung squamous cell carcinoma with organotypic cultures and xenografts uncovers distinct advantages of each system and implicates NOTCH1 loss in tumour development

doi: 10.1101/2025.08.28.672595

Figure Lengend Snippet: Histological analysis of xenografts generated with mutant hBECs (continued). A and B: Representative images of acetylated-tubulin and MUC5AC immunostaining of xenografts to identify the presence of ciliated ( A ) and goblet cells ( B ) respectively. For TC+P and TC+PS mutants, representative images of areas with squamous and cystic morphologies are shown separately. L=Lumen. C. Schematic depicting the expression cassette of the lentiviral construct pUltrahot for SOX2 overexpression. 5’LTR and 3’LTR= 5’ and 3’ long terminal repeats; UbC=Ubiquinone promoter; WPRE= Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element; P2A and T2A=ribosome skipping sequences. D. Quantification of mCherry staining in areas juxtaposed mucociliary and squamous morphology in TC+PS xenografts (see ). mCherry staining was quantified as brown DAB mean pixel intensity for each region analysed. Data is show as mean±SEM (n=9). P-values were calculated using a paired t-test. E and F. Correlation analysis between the frequency of p63-positive cells in xenografts and ALI cultures for each genotype. Panel E includes all genotypes that generated xenografts, whereas panel F excludes the outlier TC+P and TC+PS genotypes. Line shows a linear regression of the data. Correlation coefficient and the p-value were calculated using Pearson analysis. ns=not significant

Article Snippet: To induce SOX2 overexpression, we employed the third-generation lentiviral vector pUltra-hot (Addgene #24130), generously shared by Malcolm Moore.

Techniques: Generated, Mutagenesis, Immunostaining, Expressing, Construct, Over Expression, Virus, Staining

Journal: Scientific Reports

Article Title: Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing

doi: 10.1038/s41598-020-68257-y

Figure Lengend Snippet: The dCas9-KRAB system represses PTEN in the HEK 293T cell line and human mesenchymal precursor cells (hMPCs). dCas9-KRAB was stably expressed with no gRNA, with individual gRNAs targeting the PTEN proximal promoter and 5′ untranslated region (UTR), with a mix of gRNAs C and D, or with a mix of all four gRNAs. Empty vector and the combination of four shRNAs targeting the PTEN transcript were also stably expressed by lentiviral transduction. ( A , C ) Fold change in PTEN mRNA expression in qRT-PCR relative to empty vector in the HEK 293T cell line ( A ) and hMPCs ( C ). *p < 0.05, **p < 0.01 (Kruskall–Wallis test with Dunn’s multiple comparisons test to compare each condition to empty vector control), n = 3, error bars show standard error of the mean (SEM). ( B , D ) Western blot of PTEN and GAPDH in HEK 293T ( B ) and hMPCs ( D ). Conditions correspond to qRT-PCR data labeled above.

Article Snippet: The third-generation lentiviral packaging plasmids pMDLg/pRRE (Addgene plasmid #12251) and pRSV-Rev (Addgene plasmid #12253), and envelope plasmid pMD2.G (Addgene plasmid #12259; all gifts from Didier Trono), were used for lentiviral production.

Techniques: Stable Transfection, Plasmid Preparation, Transduction, Expressing, Quantitative RT-PCR, Control, Western Blot, Labeling