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: PLoS ONE

Article Title: EGFR in enterocytes & endothelium and HIF1α in enterocytes are dispensable for massive small bowel resection induced angiogenesis

doi: 10.1371/journal.pone.0236964

Figure Lengend Snippet: (A) Transwell co-culture of ECs and ISEMFs was performed as depicted. (B) After 24h in culture, RNA was isolated and quantitative RT-PCR performed for EGFR expression (n = 3 per condition). (C) Western blot for EGFR protein expression in ECs and ISEMFs cultured either alone or together. (D) Expression of EGFR mRNA (upper panel) and protein (lower panel) in ISEMFs transduced with EGFR shRNA lentiviral particles, with copGFP and shRNA lentiviral controls as indicated. (E) Schematic of microfluidic device system. (F) Representative images of vessel networks formed in microfluidic devices with fluorescent ECs and ISEMFs transduced with EGFR shRNA silencing (left), as opposed to copGFP (middle) and shRNA (right) lentiviral controls. (G) Quantitative analysis of vessel networks with number of junctions per high power field and average vessel length in microfluidic devices with fluorescent ECs and ISEMFs transduced with EGFR shRNA silencing (n = 4,3), copGFP (n = 6,3), and shRNA (n = 1,1) lentiviral controls, respectively.

Article Snippet: In accordance with Washington University protocol (#2947), HEK293T was transduced using a three helper plasmid vectors (Addgene pMDLg-pRRE (Plasmid #12251), Addgene pRSV-Rev (Plasmid #12253), Addgene pMD2.G (Plasmid #12259)) and a third-generation lentiviral vector (Addgene pLV-Azurite, Plasmid #36086) to generate an azurite fluorescent protein lentiviral plasmid.

Techniques: Co-Culture Assay, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Cell Culture, Transduction, shRNA

Journal: The Biochemical journal

Article Title: FoxA2 and RNA Pol II Mediate Human Islet Amyloid Polypeptide Turnover in ER-stressed Pancreatic β-cells

doi: 10.1042/BCJ20200984

Figure Lengend Snippet: RIN-m5f cells were transduced with empty vector (EV), hIAPP or rIAPP encoding lentivirus particles for 48h. (A) Immuno-confocal microscopy optical section (1μm-Z plane) of RIN-m5f cells transduced with empty vector (EV) and hIAPP encoding lentivirus. Cells were co-stained with hIAPP-specific IAPP monoclonal antibody (red) and DAPI (blue). (B) Representative single (1μm-Z plane) fluorescence confocal sections of hIAPP intracellular accumulation sites in hIAPP lentivirus-transduced cells. Arrows denote hIAPP’s nuclear locations in micrographs. Bars, 5μm.

Article Snippet: Flag-tagged ORF sequences for pre-pro-hIAPP and rIAPP, flanked with restriction enzyme sites (5’ SgfI and 3’ Mlu1) were synthesized using gBlock gene synthesis (Integrated DNA Technology) and cloned into a third-generation lentivirus destination vector (pLenti-C-Myc-DDK-IRES-Puro, Origene, cat# PS100069).

Techniques: Transduction, Plasmid Preparation, Confocal Microscopy, Staining, Fluorescence

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology

doi: 10.1016/j.omtm.2022.07.017

Figure Lengend Snippet: Primary human myoblasts are efficiently immortalized after transduction with lentiviruses encoding for CDK4 and hTERT (A) Primary myoblast cells were transduced with lentiviral vectors encoding the CDK4 and hTERT proteins. Primary and immortalized myoblasts were maintained in culture and proliferation was quantified by determination of population doubling. Each dot represents the mean of two cell counts. (B) Human immortalized myoblasts were expanded and cell differentiation was promoted by removing growth factors from the culture medium. The expression levels of MEF2 and MyHC proteins were quantified by in vitro immunofluorescence to assess the myogenic differentiation process. The image is representative of at least seven images from one biologically independent experiment. Scale bar, 50 μm.

Article Snippet: The cDNAs for the catalytic subunit of the hTERT, CDK4, and GM-CSF were provided by Geneva University’s Vectorlab platform and subcloned into a third-generation pCLX lentiviral vector backbone (Addgene, 45956) under the expression of the human PGK promoter., , Lentiviral particles encoding hTERT, CDK4, and GM-CSF were produced by co-transfection of the pCLX, pMD2.G (Addgene, 12259), and psPAX2 plasmids (Addgene, 12260), as described elsewhere.

Techniques: Transduction, Cell Differentiation, Expressing, In Vitro, Immunofluorescence

Journal: Nature

Article Title: Progenitor identification and SARS-CoV-2 infection in human distal lung organoids

doi: 10.1038/s41586-020-3014-1

Figure Lengend Snippet: a, Left, confocal images of a live AT2 organoid at 67 days of culture labelled with Hoescht nuclear stain and LysoTracker Red DND-99. Top right, isolation of purified AT2 organoids. Representative FACS plots showing LysoTracker Red AT2 purification from unfractionated organoid cultures. Bottom right, immunostaining of cytospin of LysoTracker-sorted AT2 cells show high purity (100/100 cells SPC+ SCGB1A1− KRT5); scale bar = 50 μm). b, Schema of FACS isolation of AT2 cells from human mixed distal lung organoids as EPCAM+LysoTracker+ AT2 cells followed by long-term clonogenic organoid culture. c, Representative image of clonal mixing studies from stroma-depleted, EPCAM+LysoTracker+, lentivirally-marked AT2 cells demonstrating presence of completely mCherry+ or GFP+ but not chimaeric organoids carried out as in Extended Data Fig. 1e–g, passage 1 after lentiviral infection, scale bar = 200 μm. d, Quantitation of red, green, or chimaeric AT2 organoid cultures as in (c) from two individuals (1, 2) after initial and serial passaging (P1 = passage 1). e, AT2 organoid proliferation with differing combinations of recombinant niche factors and PORCUPINE inhibitor C59 (1 mM), NOGGIN (N), EGF (E), WNT3A (W), R-SPONDIN1 (R). n = 3 per condition, data are mean ± s.e.m., * = P < 0.05, *** = P < 0.001, two-tailed student’s t-test. f, Brightfield microscopy comparing pure AT2 organoid growth enhancement in chemically defined lung organoid media (EN) versus serum-containing L-cell conditioned media containing WNT3A, NOGGIN, and R-SPONDIN3 (L-WRN CM) supplemented with recombinant EGF, one experiment. Scale bar = 200 μm. g, Transmission electron microscopy image of representative AT2 organoid at 28 days of culture. Note apical microvilli (black arrows) and lamellar bodies (red arrows); scale bar = 10 μm.

Article Snippet: FACS EPCAM + stroma-depleted organoids at D14 were infected with lentivirus at an estimated MOI of 0.9 as described previously 43 with third-generation lentiviral vectors (PGK-GFP T2A Puro, SBI cat. no. CD550A-1; mCherry modified from pLentiCRISPRv1 (Addgene no. 49545) to incorporate an EF-1a-mCherry P2A Puro cassette, a gift from Paul Rack).

Techniques: Staining, Isolation, Purification, Immunostaining, Infection, Quantitation Assay, Passaging, Recombinant, Two Tailed Test, Microscopy, Transmission Assay, Electron Microscopy

Journal: Nature

Article Title: Progenitor identification and SARS-CoV-2 infection in human distal lung organoids

doi: 10.1038/s41586-020-3014-1

Figure Lengend Snippet: a–c, Basal organoids in mixed culture progressively form internal lumens, which is not associated with apoptosis. a, KRT5 IF, day 26 culture, scale bar = 200 μm. b. Lumen quantitation, d12 versus d26 culture, single determination. c, Absence of apoptosis in d26 basal cell organoid internal lumen, cleaved caspase IF, from Fig. 2b, scale bar = 20 μm. d, e, Isolation of purified basal cell organoids via differential sedimentation in Ficoll. d, Schema and enrichment to >90% KRT5+ cells as measured by intracellular KRT5 FACS of sedimented basal organoid cells; scale bar = 100 μm. e, Serial time lapse microscopy of sedimented basal organoids reveals spontaneous cavitation within two weeks post passage or within four weeks of culture initiation; scale bar = 25 μm. f, g, Clonal mixing studies from stroma-depleted, Ficoll-purified and lentivirally-marked basal organoid cells demonstrating fully mCherry+ or GFP+ but not chimaeric organoids as in Extended Data Fig. 1f, passage 1 after lentiviral infection, scale bar = 200 μm. f, Representative clonal mixing image study. g, Quantitation. h, Growth factor evaluation for basal organoids after d14 sedimentation, enzymatic dissociation and clonogenic culture. Growth was not affected by the PORCUPINE inhibitor C59 (1 μM). n = 4 per condition, data are mean ± s.e.m., *** = P < 0.001, two-tailed student’s t-test.

Article Snippet: FACS EPCAM + stroma-depleted organoids at D14 were infected with lentivirus at an estimated MOI of 0.9 as described previously 43 with third-generation lentiviral vectors (PGK-GFP T2A Puro, SBI cat. no. CD550A-1; mCherry modified from pLentiCRISPRv1 (Addgene no. 49545) to incorporate an EF-1a-mCherry P2A Puro cassette, a gift from Paul Rack).

Techniques: Quantitation Assay, Isolation, Purification, Sedimentation, Time-lapse Microscopy, Infection, Two Tailed Test