Journal: Cell reports

Article Title: Modeling Patient-Derived Glioblastoma with Cerebral Organoids

doi: 10.1016/j.celrep.2019.02.063

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To establish cells that stably express GFP, GSCs were transfected with pLenti PGK GFP Blast (w510–5) ( Campeau et al., 2009 ) (Addgene) and were cultured under blasticidin selection in NBE medium.

Techniques: Recombinant, Plasmid Preparation, Imaging, SYBR Green Assay, Luciferase, Construct, Software

Journal: bioRxiv

Article Title: A versatile ES cell-based melanoma mouse modeling platform

doi: 10.1101/658260

Figure Lengend Snippet: Murine melanoma cell lines as a resource to complement chimera experiments in vitro and in vivo. (A) Melanoma cells from B TRE-shPten chimeras are maintained in Dox-containing media and restore Pten expression upon Dox removal. Western blot shows increased expression of Pten 3 and 6 days after Dox removal, while expression of GFP and AKT pS473 decrease. Total Akt and Hsp90 were used as loading controls. (B) Proliferation curve of B TRE-shPten melanoma cells under on-Dox and off-Dox conditions. (C) Focus formation of B TRE-shPten melanoma cells under on- Dox and off-Dox conditions. (D) Tumor growth of B TRE-shPten melanoma cells in Nu/Nu recipient mice. 5 mice each were kept on a regular diet (Off Dox, n=10 tumors), placed on a Dox diet two days prior to transplantation of tumor cells (On Dox, n=10 tumors), or switched from the On Dox condition to a regular diet 18 days after transplantation of tumor cells (On-Off Dox, n=10 tumors). (E) Survival of Nu/Nu mice shown in (E) bearing B TRE-shPten melanomas (n=5 for each cohort). (F) Western blot of BPP melanoma cells infected with pLenti-TRE-PtenWT, pLenti-TRE-PtenC124S, or pLenti-TRE-GFP lentiviruses. The effect of Dox-mediated induction of Pten expression on Akt phosphorylation (pS473 and pT308) is shown. BCC cells are used as a control for Pten expression, and total Akt and actin were used as loading controls. (G) Proliferation curve of BPP melanoma cells expressing TRE-PtenWT, TRE-PtenC124S, or TRE-GFP (H) Focus formation of BPP melanoma cells expressing TRE-PtenWT, TRE-PtenC124S, or TRE-GFP. (I) Tumor growth of BPP melanoma cells expressing TRE-PtenWT (n=10 tumors) or TRE-PtenC124S (n=10 tumors) transplanted in C57BL/6 mice. Recipient mice were placed on a Dox diet 2 days prior to melanoma cell transplantation. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The Cre reporter was a gift from N. Geijsen (Addgene plasmid #62732). col1a1-EF1 -GFP was generated by removing TRE from col1a1-TRE and GFP from pLenti-GFP (Addgene plasmid #17448) and the EF1α core promoter from lentiCRISPRv2_puro (from T. Jacks) were inserted using standard cloning techniques. pLenti-TRE-GFP-Blast was generated by replacing the CMV promoter in pLenti-GFP-Blast with the TRE3G promoter from pRRL-TRE3G-GFP-PGK-Puro-IRES-rtTA3 (from J. Zuber). pLenti-TRE-PtenWT-Blast and pLenti-TRE-PtenC124S-Blast were created by replacing GFP in pLenti-TRE-GFP-Blast with mouse Pten wildtype or mutant cDNA.

Techniques: In Vitro, In Vivo, Expressing, Western Blot, Transplantation Assay, Infection

Journal: bioRxiv

Article Title: A versatile ES cell-based melanoma mouse modeling platform

doi: 10.1101/658260

Figure Lengend Snippet: Restoration of endogenous Pten expression in B TRE-shPten chimeras halts melanoma growth. (A) Fluorescent imaging of gross melanomas in B TRE-shPten chimeras where the inducible expression of shPten is linked to GFP. Taking chimeras off Dox for 14 days results in cessation of GFP expression. (B) Western blot showing expression of GFP, Pten, and phosphorylated (pS473 and pT308) and total Akt in melanomas from chimeras on Dox, or taken off Dox for 7 days. Actin was used as a loading control, and a tumor from a BCC chimera is shown for comparison. (C) Immunohistochemistry staining of Pten, GFP and Ki67 on tumors from B TRE-shPten chimeras on Dox or off Dox for 7 or 14 days. (D) Graph displaying the fold change in tumor volume over time in chimeras on Dox compared to chimeras that were taken off Dox on day 0. (E) Quantification of Ki67-positive nuclei per field in tumors from B TRE-shPten chimeras on Dox or off Dox for 7 or 14 days. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The Cre reporter was a gift from N. Geijsen (Addgene plasmid #62732). col1a1-EF1 -GFP was generated by removing TRE from col1a1-TRE and GFP from pLenti-GFP (Addgene plasmid #17448) and the EF1α core promoter from lentiCRISPRv2_puro (from T. Jacks) were inserted using standard cloning techniques. pLenti-TRE-GFP-Blast was generated by replacing the CMV promoter in pLenti-GFP-Blast with the TRE3G promoter from pRRL-TRE3G-GFP-PGK-Puro-IRES-rtTA3 (from J. Zuber). pLenti-TRE-PtenWT-Blast and pLenti-TRE-PtenC124S-Blast were created by replacing GFP in pLenti-TRE-GFP-Blast with mouse Pten wildtype or mutant cDNA.

Techniques: Expressing, Imaging, Western Blot, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: A versatile ES cell-based melanoma mouse modeling platform

doi: 10.1101/658260

Figure Lengend Snippet: Ectopic Pten re-expression impairs melanoma formation. (A) Kaplan-Meier analysis of tumor-free survival depicting the onset of melanoma formation in BPP TRE-Pten and BPP TRE-GFP chimeras. (B) Kaplan-Meier analysis showing the overall survival of BPP TRE-Pten and BPP TRE-GFP chimeras. (C) Quantification of tumor numbers in BPP TRE-Pten and BPP TRE-GFP chimeras. (D) Immunohistochemistry staining of Pten and GFP in BPP TRE-Pten and BPP TRE-GFP chimeras. (E) Quantification of melanomas with strong or weak/absent Pten staining intensity in melanomas from BPP TRE-Pten and BPP TRE-GFP chimeras. ns, not significant; * p < 0.05.

Article Snippet: The Cre reporter was a gift from N. Geijsen (Addgene plasmid #62732). col1a1-EF1 -GFP was generated by removing TRE from col1a1-TRE and GFP from pLenti-GFP (Addgene plasmid #17448) and the EF1α core promoter from lentiCRISPRv2_puro (from T. Jacks) were inserted using standard cloning techniques. pLenti-TRE-GFP-Blast was generated by replacing the CMV promoter in pLenti-GFP-Blast with the TRE3G promoter from pRRL-TRE3G-GFP-PGK-Puro-IRES-rtTA3 (from J. Zuber). pLenti-TRE-PtenWT-Blast and pLenti-TRE-PtenC124S-Blast were created by replacing GFP in pLenti-TRE-GFP-Blast with mouse Pten wildtype or mutant cDNA.

Techniques: Expressing, Immunohistochemistry, Staining