Journal: bioRxiv
Article Title: FORMATION OF MALIGNANT, METASTATIC SMALL CELL LUNG CANCERS THROUGH OVERPRODUCTION OF cMYC PROTEIN IN TP53 AND RB1 DEPLETED PULMONARY NEUROENDOCRINE CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS
doi: 10.1101/2023.10.06.561244
Figure Lengend Snippet: A) Schematic of the protocol used to generate PNECs by stepwise differentiation of hPSCs to form: definitive endoderm (DE), day 3; anterior foregut endoderm (AFE), day 6; and lung progenitor cells (LPs) days 15 -25. LPs were further differentiated into the types of lung cells (LCs) found in mature human lung parenchyma and airway epithelium, days 25 -55. DAPT (10 μM) encourages the formation of PNECs, and addition of doxycycline (1 μM; DOX) induces expression of shRNAs against RB1 and TP53 mRNAs, as well as expression of cMYC or cMYC (T58A), as described in the text. B) Western blot of extracts of RUES2 LCs at day 25 of differentiation protocol treated with DOX (1 μM for 72hrs); cells unexposed to DOX served as negative expression controls. Apparent differences in cMYC protein levels may be attributable to the HA-tagged version of cMYC (T58A), which migrates slightly slower that wildtype cMYC protein. C) Schematic representation of tumorigenesis experiments comparing injection sites (renal capsule or subcutaneous), DOX treatment (+/-DOX diet), and genotypes (see Methods for additional details). Total numbers of animals are 6-7 per experimental arm with 2 injection sites per mouse (right and left flank). Renal capsule injections were performed on a single kidney. Transgenic lines of RUES2 hESCs were differentiated and grown in DAPT (10 μM) from days 25 -55. At day 55, PNECs were separated from other LCs by sorting for PE+ CGRP-expressing cells ( see Methods ). PNECs were then injected either subcutaneously or into the renal capsular space in NOG mice, half of which then received DOX in their feed as described in Materials and Methods. D) Table summary of experiments with xenografted mice, indicating the number of animals that developed visible tumors (≥250 mm 3 in volume) at the site of injection or the number of visible metastases in the liver or lung. *, P < 0.05; **, P < 0.01 by Fisher’s test to denote significant differences between mice that did and did not receive DOX diet. As before, abbreviations for cell lines are: RP = shRB1 + shTP53; RPM = shRB1 + shTP53 + WT cMYC; RPM (T58A) = shRB1 + shTP53 + cMYC (T58A).
Article Snippet: The lentiviral vectors expressing TET-inducible shRNAs against human RB1 construct (“pSLIK sh human Rb 1534 hyg” was a gift from Julien Sage; plasmid # 31500) , TET-inducible wild type cMYC (FUW-tetO-hMYC was a gift from Rudolf Jaenisch; plasmid # 20723) or mutant cMYC (T58A) tagged at the N-terminus with three copies of a hemagglutinin tag (3X-HA) (pLV-tetO-myc T58A was a gift from Konrad Hochedlinger; plasmid # 19763) were obtained from Addgene and sequence verified prior to use.
Techniques: Expressing, Western Blot, Injection, Transgenic Assay
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