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. 2019 Aug 27;8:e43668. doi: 10.7554/eLife.43668

Figure 7. PGC1α is required for maintenance of lung identity in BRAFV600E driven tumors.

(A) Tumors were induced in cohorts of BrafCAT/+ and BrafCAT/+;Ppargc1af/f mice via intranasal instillation of 106 PFU Ad5-SpC-CRE and harvested from each genotype 12 weeks post tumor induction via tissue dissociation and FACS. GSEA analyses of hallmark pathways and lung identity gene sets. Black bars indicate adjP <.05, gray bars indicate Benjamini-Hochberg corrected enrichment statistic adjP ≥. 05. (B) Immunostaining confirms decreased expression of the AT2 markers SFTPA and LYZ in BRAFV600E/PGC1αNULL tumors. (C) Quantitation demonstrating a significant decrease of LYZ immunoreactivity in BRAFV600E/PGC1αNULL tumors. Wilcoxon rank sum p val. = 0.0288. (D) Luciferase assays in HEK293T cells demonstrating the cooperation of NKX2-1, FOXA1, PGC1α, and NR5A2 in transactivation of surfactant promoters. All three promoters showed significant induction by ordinary one-way ANOVA (p<0.0001). Comparison of individual groups to mock transfected controls by Dunnett’s test for multiple comparisons: (*) p=0.0189, (****) p<0.0001. (E) Co-Immunoprecipitation of NKX2-1 by immunoprecipitation with a mouse monoclonal antibody recognizing PGC1α but not with IgG. (F) Co-Immunoprecipitation of PGC1α by immunoprecipitation with a mouse monoclonal antibody recognizing NKX2-1 but not with mouse IgG.

Figure 7—source code 1. R script to perform gene set enrichment analysis on Figure 7—source data 1, as well as plot these results.
elife-43668-fig7-code1.r (1.2KB, r)
DOI: 10.7554/eLife.43668.038
Figure 7—source code 2. R script to perform statistics on Figure 7—source data 2, as well as plot these results eLife’s transparent reporting form.
elife-43668-fig7-code2.r (2KB, r)
DOI: 10.7554/eLife.43668.039
Figure 7—source data 1. DEseq2 output of differentially expressed genes comparing BRAFV600E/PGC1αNULL and BRAFV600E/PGC1αHET driven tumors.
elife-43668-fig7-data1.zip (1.1MB, zip)
DOI: 10.7554/eLife.43668.040
Figure 7—source data 2. Cellprofiler output quantifying immunofluorescence of LYZ in BRAFV600E/PGC1αNULL and BRAFV600E/PGC1αWT driven tumors.
elife-43668-fig7-data2.zip (1.1MB, zip)
DOI: 10.7554/eLife.43668.041
Figure 7—source data 3. Data from luciferase assays looking for transactivation of Sftpa, Sftpb, and Sftpc promoters.
elife-43668-fig7-data3.xlsx (36.2KB, xlsx)
DOI: 10.7554/eLife.43668.042

Figure 7.

Figure 7—figure supplement 1. PGC1α is required for maintenance of lung identity in BRAFV600E driven tumors.

Figure 7—figure supplement 1.

(A) GSEA mountain plot comparing BRAFV600E/PGC1αNULL driven tumors to BRAFV600E/PGC1αHET driven tumors indicates that reduced PGC1α expression results in a widespread decrease in markers of AT2 pneumocyte identity, P is enrichment statistic. (B) Western blot showing expression of NKX2-1, FOXA1, NR5A2, PGC1α, SFTPA, and SFTPC in the immortalized AT2 cell line, MLE-12.
Figure 7—figure supplement 2. Proposed mechanism of PI3’-kinase-α promoting de-differentiation of lung tumors initiated by the BRAFV600E oncoprotein kinase.

Figure 7—figure supplement 2.

Model in which dual arm mutational activation of growth factor signaling results in the loss of AT2 identity by repressing expression of PGC1α but without affecting expression of the lineage survival transcription factors NKX2-1 or FOXA1/FOXA2.