Journal: Oncogene
Article Title: Human cell transformation by combined lineage conversion and oncogene expression.
doi: 10.1038/s41388-021-01940-0
Figure Lengend Snippet: Fig. 2 Oncogene exposure transforms induced hepatocytes but not control fibroblasts. A Phase contrast microscope images showing the phenotype and morphology of the cells in the course of conversion of fibroblasts to iHeps at different times points after transduction with a cocktail of three TFs HNF1A, HNF4A and FOXA3 [36]. B Generation of highly proliferative iHep cells by transducing iHeps with two pools of liver cancer-specific oncogenic drivers, a list of xenograft experiments in nude mice that were used to test the tumorigenicity of different conditions, and mutation rates of the oncogenic drivers as reported in the COSMIC database for HCC and MYC amplification as reported in [43]. CMT pool contains three oncogenes CTNNB1T41A, MYC, and TERT, and CMT + sgTP53 pool contains the same oncogenes along with constructs for TP53 inactivation by CRISPR-Cas9. Phase contrast microscope images showing the phenotype and morphology of the cells. Oncogenes are co-transduced with fluorescent reporter mCherry for the detection of transduced cells. Oncogene transduction to fibroblasts fails to transform the cells, passaging of oncogene-expressing fibroblasts results in cellular senescence as demonstrated by β-galactosidase staining and loss of mCherry-positive oncogene-expressing cells from the fibroblast population. iHeps maintained in defined culture medium become senescent around week four of transdifferentiation although they can survive in culture for several weeks after that if not passaged. Passaging of iHeps without oncogenes results in apoptosis after few passages. Scale bar 1000 μm unless otherwise specified.
Article Snippet: Expression construct for mCherry (#36084), lentiviral Cas9 expression construct LentiCas9-Blast (#52962), a cloning backbone lentiGuide-Puro (#52963), and the constructs for neuronal conversion (Tet-O-FUW-Ascl1, #27150; TetO-FUW-Brn2, #27151; Tet-O-FUW-Myt1l, #27152; Tet-O-FUW-NeuroD1, # 30129; pTetO-Ngn2-Puro, #52047; Tet-O-FUW-EGFP, # 30130; FUW-M2rtTA, #20342) were obtained from Addgene (Watertown, MA).
Techniques: Control, Microscopy, Transduction, Mutagenesis, Construct, CRISPR, Passaging, Expressing, Staining
Addgene inc is a verified supplier 
![( A ) Plot of the candidate genes from <t>CRISPR-Cas9</t> knockout (KO) screen in LNCaP cells treated with 450 nM ipatasertib on day 18. ( B ) Top genes identified from the CRISPR-Cas9 KO screen on day 18 (ipatasertib-treated LNCaP cells compared to DMSO-treated cells). ( C ) TSC2-KO LNCaP cells were subjected to a 4-hour treatment with either DMSO or 500 nM ipatasertib. Expression levels of downstream targets in the PI3K pathway were evaluated using Western Blot analysis. ( D and E ) Outcomes of a competition assay involving four candidate genes in LNCaP cells. Cells were transduced with lentivirus carrying sgNT-GFP or sgRNA-GFP targeting the candidate genes, each with two specific sgRNAs. Following transduction, cells were treated with either DMSO or 500 nM ipatasertib. The ratio of GFP + cells was normalized to the DMSO treatment group. The data represent the results of three independent experiments ( n = 3). ( F ) LNCaP, CWR22Pc-EP-sgPTEN, and PCa12, each stably expressing sgNT or sgNPRL3, were subjected to treatment with either ipatasertib (500 nM) or DMSO. Cell viability on day 7 was assessed. ( G ) WT, TSC2-KO, or NPRL3-KO LNCaP cells were exposed to 6 hours of starvation and a 4-hour treatment with either DMSO or 500 nM ipatasertib. Following this, cells were stimulated with or without amino acids for 10 min. The expression levels of downstream targets in the PI3K pathway were evaluated using Western Blot analysis {* P < 0.05, ** P < 0.01, and **** P < 0.0001; [(D) to (F)] Welch’s t test; error bar represents ±SEM}.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4928/pmc11804928/pmc11804928__sciadv.adq3802-f3.jpg)
![Fig. 2 Oncogene exposure transforms induced hepatocytes but not control fibroblasts. A Phase contrast microscope images showing the phenotype and morphology of the cells in the course of conversion of fibroblasts to iHeps at different times points after transduction with a cocktail of three TFs HNF1A, HNF4A and FOXA3 [36]. B Generation of highly proliferative iHep cells by transducing iHeps with two pools of liver cancer-specific oncogenic drivers, a list of xenograft experiments in nude mice that were used to test the tumorigenicity of different conditions, and mutation rates of the oncogenic drivers as reported in the COSMIC database for HCC and MYC amplification as reported in [43]. CMT pool contains three oncogenes CTNNB1T41A, MYC, and TERT, and CMT + sgTP53 pool contains the same oncogenes along with constructs for TP53 inactivation by <t>CRISPR-Cas9.</t> Phase contrast microscope images showing the phenotype and morphology of the cells. Oncogenes are co-transduced with fluorescent reporter mCherry for the detection of transduced cells. Oncogene transduction to fibroblasts fails to transform the cells, passaging of oncogene-expressing fibroblasts results in cellular senescence as demonstrated by β-galactosidase staining and loss of mCherry-positive oncogene-expressing cells from the fibroblast population. iHeps maintained in defined culture medium become senescent around week four of transdifferentiation although they can survive in culture for several weeks after that if not passaged. Passaging of iHeps without oncogenes results in apoptosis after few passages. Scale bar 1000 μm unless otherwise specified.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_2118/pm34302118/pm34302118__page3_image1.jpg)
