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: Genes

Article Title: Screening Methods to Discover the FDA-Approved Cancer Drug Encorafenib as Optimally Selective for Metallothionein Gene Loss Ovarian Cancer.

doi: 10.3390/genes16010042

Figure Lengend Snippet: Figure 2. Zinc and cadmium metal toxicity in MT2A knockdown. (A) Amino acid alignment of the 11 expressed human metallothioneins. Note that cysteine residues, which chelate metals, are highly conserved among the proteins and represent a third of all the amino acids in metallothioneins. (B) Nuclear magnetic resonance structure of metallothionein MT2A alpha (PDB ID: 2MRB) and beta (PDB ID:1MRB) domains, with bound Cd2+ ions shown. Zn2+ ions are predicted to be similarly located within the protein in the absence of Cd2+. (C) CAOV3 cells, which only express MT2A (higher) and MT1X (lower), were knocked down by lentiviral shMT2A and validated by RT-qPCR as reduced in expression relative to scrambled shRNA control (shScr). (D) ZnCl2 toxicity was assessed by Hoechst-33342-stained nuclei counts relative to control treated cells, with 2-day (2 d) or 3 d exposure. (E) CdCl2 toxicity was assessed as in (D). Error bars are standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 by t-test of technical replicates relative to shScr control from a representative experiment.

Article Snippet: Fluorescent protein nls-GFP (Addgene, #126688) or nls-BFP (Addgene, #36085) was stably added via lentivirus using third generation lentiviral packaging vectors (Addgene, #12251, 12253, 12259) packaged in HEK293T cells (ATCC, #CRL-3216).

Techniques: Knockdown, Nuclear Magnetic Resonance, Quantitative RT-PCR, Expressing, shRNA, Control, Staining