Journal: eLife
Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs
doi: 10.7554/eLife.93170
Figure Lengend Snippet: ( A ) Schematic of the protocol used to generate pulmonary neuroendocrine cells (PNECs) by stepwise differentiation of human pluripotent stem cells (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 MYC or MYC (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 72 hr); cells unexposed to DOX served as negative expression controls. Apparent differences in MYC protein levels may be attributable to the HA-tagged version of MYC (T58A), which migrates slightly slower than wild-type MYC protein. ( C ) Schematic representation of tumorigenesis experiments comparing injection sites (renal capsule or subcutaneous), DOX treatment (+/-DOX diet), and genotypes (see Materials and methods for additional details). Total numbers of animals are six to seven per experimental arm with two 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 to 55. At day 55, PNECs were separated from other LCs by sorting for PE+ CGRP-expressing cells (see Materials and 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 not receive DOX diet. As before, abbreviations for cell lines are: RP = shRB1+shTP53; RPM = shRB1+shTP53+WT MYC; RPM (T58A)=shRB1+shTP53+MYC (T58A). Figure 1—source data 1. Raw data of western blot bands in . Figure 1—source data 2. Raw data of .
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 WT MYC (FUW-tetO-hMYC was a gift from Rudolf Jaenisch; plasmid # 20723) or mutant MYC (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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