Journal: Nature protocols

Article Title: Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells

doi: 10.1038/s41596-019-0220-0

Figure Lengend Snippet: a, Schematic showing method to sort SFTPCtdTomato cells and passage them sequentially, after which the majority of cells within alveolospheres are SFTPCtdTomato+. b, Electron micrograph of passage 10 alveolospheres shows expression of lamellar body–like inclusions. c, Proliferation kinetic of cell yield per sorted and replated SFTPCtdTomato+ cell in BU3 NGST and RUES2 ST lines over three passages. d, Representative phase-contrast images of alveolospheres at an optimal density for passaging (left) versus too dense for reliable passaging (right). Scale bars, 200 μm. e, Representative flow cytometry of BU3 NGST alveolospheres at passages 3 and 9 after SFTPCtdTomato+ sorting. The percentage of SFTPCtdTomato+ is more variable in early passages (top), although the majority of cells maintain expression of NKX2–1GFP. Representative image of passage 9 alveolospheres expressing SFTPCtdTomato. f, Mean colony-forming efficiency ± s.d., n = 4 biological replicates for RUES2 SFTPCtdTomato+-sorted alveolospheres (~95% SFTPCtdTomato+) and primary adult human AEC2 cells (Adult AEC2) in either SAGM or CK+DCI medium ± MRC5 fibroblast feeder cells. *P ≤ 0.05, ANOVA. g, Assessment of clonality of alveolosphere outgrowth by fluorescent tagging of iAEC2s in separate wells (~day 100) using lentiviral vectors constitutively expressing either TagBFP (blue) or GFP (green). Subsequent mixing of flow cytometry–sorted GFP+ and TagBFP+ iAEC2s at a 1:1 ratio at various densities in 3D Matrigel produces sphere outgrowths that are predominantly monocolored. Photomicrograph represents merged BFP (blue) and GFP (green) channels. Bars represent average sphere numbers and color percentages across five random fields, n = 689 spheres scored, and downward facing error bars represent s.d. for each color; upward error bars represent mixed color scores. Results are representative of two repeated independent experiments on spheres that were >90% SFTPCtdTomato+ at the time of mixing. NS, nonsignificant; Pass, passage; Phase, phase-contrast. Scale bars, 500 nm (b); 100 μm (d); 200 μm (e); 300 μm (g). c, f adapted with permission from ref. 3, Elsevier.

Article Snippet: Growth factor–reduced Matrigel (Corning, cat. no. 356231) Human embryonic stem cell (hESC)-qualified Matrigel (5-ml vial; Corning, cat. no. 354277) ▲CRITICAL Use the dilution factor recommended by the manufacturer on the certificate of analysis, because the concentration varies from lot to lot.

Techniques: Expressing, Passaging, Flow Cytometry

Journal: Nature protocols

Article Title: Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells

doi: 10.1038/s41596-019-0220-0

Figure Lengend Snippet: a, Schematic of experimental plan and bright-field images of spheres analyzed on day 32 after day 14 NKX2–1+ lung progenitor plating at densities ranging from 8 to 1,000 cells/μl of Matrigel. Scale bars, 200 μm. b, Representative flow cytometry of day 32 alveolospheres originally plated at 1,000 versus 65 cells/μl of Matrigel. c, Graphs show day 32 percentage of SFTPCtdTomato+ and NKX2–1GFP+ cells for each plating density, with error bars showing mean ± s.d.

Article Snippet: Growth factor–reduced Matrigel (Corning, cat. no. 356231) Human embryonic stem cell (hESC)-qualified Matrigel (5-ml vial; Corning, cat. no. 354277) ▲CRITICAL Use the dilution factor recommended by the manufacturer on the certificate of analysis, because the concentration varies from lot to lot.

Techniques: Flow Cytometry

Journal: Nature protocols

Article Title: Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells

doi: 10.1038/s41596-019-0220-0

Figure Lengend Snippet: a, Protocol schematic and tSNE plot of 675 cells captured on day 41 of distal differentiation by 10x Genomics platform for single-cell RNA sequencing. Cells were derived from BU3 iPSCs sorted on day 15 of differentiation on the basis of NKX2–1GFP expression and were further differentiated to iAEC2s in ‘distal medium (CK+DCI)’ in 3D Matrigel with a single sphere passage before harvest for computational analysis, as described in McCauley et al.14. Cells in ‘distal media (CK+DCI)’ from McCauley et al. have been reanalyzed to generate the tSNE plots and normalized gene expression overlays shown in a and b, whereas cell cluster identities have all been maintained from McCauley et al.14, where further extensive discussion is available. b, tSNE plots with overlaid normalized expression of indicated marker genes. Datasets are available for download under GEO accession no. GSE103918 or through the bioinformatics portal at www.kottonlab.com. BMPi, inhibition of BMP signaling; RA, retinoic acid; TGFbi, inhibition of TGF signaling.

Article Snippet: Growth factor–reduced Matrigel (Corning, cat. no. 356231) Human embryonic stem cell (hESC)-qualified Matrigel (5-ml vial; Corning, cat. no. 354277) ▲CRITICAL Use the dilution factor recommended by the manufacturer on the certificate of analysis, because the concentration varies from lot to lot.

Techniques: RNA Sequencing, Derivative Assay, Expressing, Gene Expression, Marker, Inhibition

Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: CD36 expression is positively correlated with cell proliferation and migration in vitro . A–F A549 cells were transfected with pCMV or pCMV-CD36 plasmid, and NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h in serum-free medium and then cultured in complete medium for another 24 h. Cells were collected for determination of cell viability ( A ), cell cycle ( B , C ), migration ( D ), and apoptosis ( E ) by MTT assay, FACS, wound healing test, and Annexin V-FITC/PI staining, respectively. Protein expression of CD36, CDH1, PCNA, vimentin, Bcl-2, and BAX was determined by Western blot with density quantitative analysis ( F ). G , H NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by FFAs (150 µM) treatment for 24 h. Cells were collected for determination of cell viability ( G ) and apoptosis ( H ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A , G : n = 6; B – F , H : n = 3

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Expressing, Migration, In Vitro, Transfection, Plasmid Preparation, Cell Culture, MTT Assay, Staining, Western Blot

Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: The effects of pitavastatin on cell proliferation, migration, and apoptosis are related to CD36 expression. A549 cells were transfected with pCMV or pCMV-CD36 plasmid, and NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by received pitavastatin (5 µM) treatment for 24 h. Cells were collected for determination of cell viability ( A ), cell cycle ( B , C ), migration ( D ), and apoptosis ( E ). Protein expression of CD36, PCNA, Bcl-2, and BAX was determined by Western blot with density quantitative analysis ( F , G ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A : n = 6; B – F : n = 3; Pita, pitavastatin

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Migration, Expressing, Transfection, Plasmid Preparation, Cell Culture, Western Blot

Journal: Cell Biology and Toxicology

Article Title: CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

doi: 10.1007/s10565-024-09848-7

Figure Lengend Snippet: The reduction effects of pitavastatin on tumor progression are regulated by CD36/AKT/mTOR pathway. A–E A549 ( A ) and NCI-H520 ( B ) cells were treated with 150 µM FFAs or 5 µM pitavastatin plus FFAs for 24 h. A549 cells were transfected with pCMV or pCMV-CD36 plasmid for 12 h ( C ); NCI-H520 cells were transfected with CasRX or CasRX-CD36 plasmid ( D , E ) for 12 h and then cultured in complete medium for 24 h, followed by treatment with 5 µM pitavastatin ( C , D ) or 150 µM FFAs ( E ) for 24 h. Protein expression of p-AKT, AKT, p-mTOR, and mTOR was detected by Western blot. F , G Tumor paraffin sections collected from Fig. A ( F ) or Fig. A ( G ) were performed IHC staining to detect the expression of p-AKT and p-mTOR with MD quantified by ImageJ software. H–L A549 cells were transfected with pCMV or pCMV-CD36 plasmid for 12 h and then cultured in complete medium for 24 h, followed by received LY294002 (10 µM) treatment for 24 h. Cells were collected for determination of cell viability ( H ) and apoptosis ( I , J ). Protein expression of CD36, vimentin, PCNA, BAX, p-AKT, AKT, p-mTOR, and mTOR was determined by Western blot ( K , L ). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; A – E , I – L : n = 3; F , G : n = 5; H : n = 6; Pita, pitavastatin; LY, LY294002

Article Snippet: CasRx gRNA cloning backbone (pXR003, #109053, Addgene) was digested with BbsI and then ligated with annealed oligo duplex using T4 ligase.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Expressing, Western Blot, Immunohistochemistry, Software