Journal: Cell Stem Cell

Article Title: Human naive epiblast cells possess unrestricted lineage potential

doi: 10.1016/j.stem.2021.02.025

Figure Lengend Snippet:

Article Snippet: SUSD2 Antibody, anti-human, VioBright FITC , Miltenyi Biotech , Cat#130-106-401; RRID: AB_2653618.

Techniques: Recombinant, Cell Attachment Assay, Transfection, Plasmid Preparation, Software

Journal: eLife

Article Title: Mutationally-activated PI3’-kinase-α promotes de-differentiation of lung tumors initiated by the BRAF V600E oncoprotein kinase

doi: 10.7554/eLife.43668

Figure Lengend Snippet: ( A ) Images were only compared when they had been immunostained in parallel, imaged on the same microscope with the same exposure settings, and not been processed or scaled post image acquisition. ( B ) Individual tumors were traced in NIH imageJ. ( C ) Color images were split in to component channels in NIH imageJ. ( D ) CellProfiler was used to identify tumor nuclei based on NKX2-1 staining when done, or based on DAPI. ( E ) Cells were identified based on propagation from nuclear objects. ( F ) Cytoplasm was defined as Cell objects minus nuclei. ( G ) Measurements taken always used median fluorescent intensity with no outlier removal. Data were imported and graphed using a custom R script in RStudio. ( H ) Statistical detail of comparisons done with a custom R script in RStudio.

Article Snippet: Recombinant DNA reagent , MSCV-NKX2.1 , Addgene , Plasmid #31271 RRID: Addgene_31271 , NKX2.1 expression vector.

Techniques: Microscopy, Staining

Journal: eLife

Article Title: Mutationally-activated PI3’-kinase-α promotes de-differentiation of lung tumors initiated by the BRAF V600E oncoprotein kinase

doi: 10.7554/eLife.43668

Figure Lengend Snippet: ( A ) BRAF V600E driven hyperplasia and tumors display widespread expression of both SFTPA and nuclear localization of the lung lineage transcription factor, NKX2-1, at 2 and 12 weeks post initiation. Scale bar = 10 um. ( B ) BRAF V600E /PI3Kα H1047R driven hyperplasia and tumors show decreased SFTPA expression at 2 and 12 weeks post initiation. These tumors maintain broad nuclear expression of NKX2-1, including those cells with decreased SFTPA expression (yellow arrowheads). ( C ) Quantitation showing no significant difference in NKX2-1 immunoreactivity at 2 weeks post initiation, but a slight increase in nuclear NKX2-1 at 12 weeks post initiation. Wilcoxon rank sum p values = 0.2,. 02 respectively. ( D ) Significant reduction of SFTPA immunoreactivity seen in BRAF V600E /PI3Kα H1047R driven hyperplasia and tumors at both 2 and 12 weeks post initiation. Wilcoxon rank sum p values = 5e-5, 4e-5 respectively. ( E ) Cytoplasmic SFTPA immunoreactivity plotted versus nuclear NKX2-1 immunoreactivity in BRAF V600E driven hyperplasia and tumors at 2 and 12 weeks post initiation. Similar association seen at both time points (Rho = 0.23,. 27 respectively). ( F ) Cytoplasmic SFTPA immunoreactivity plotted versus nuclear NKX2-1 immunoreactivity in BRAF V600E /PI3Kα H1047R driven hyperplasia and tumors at 2 and 12 weeks post initiation. Relatively lower association seen at 2 weeks compared to 12 weeks (Rho = 0.07,. 40 respectively). ( G ) Overlay of BRAF V600E /PI3Kα H1047R and BRAF V600E driven hyperplasia 2 weeks post initiation. Dashed line for each marker drawn at mean - one standard deviation of BRAF V600E driven tumors. BRAF V600E /PI3Kα H1047R driven tumors show fewer SFTPA+, NKX2−1 + cells, most strongly accounted for by an increase in SFTPA-, NKX2−1 + cells. Chi square test associates genotype with distribution, p val <1e-5. ( H ) Overlay of BRAF V600E /PI3Kα H1047R and BRAF V600E driven tumors 12 weeks post initiation. Dashed line for each marker drawn at mean - one standard deviation of BRAF V600E driven tumors. BRAF V600E /PI3Kα H1047R driven tumors show fewer SFTPA+, NKX2−1 + cells, most strongly accounted for by an increase in SFTPA-, NKX2−1 + cells. Chi square test associates genotype with distribution, p val <1e-5. 10.7554/eLife.43668.020 Figure 4—source code 1. R script to perform statistics on – , as well as plot these results. 10.7554/eLife.43668.021 Figure 4—source code 2. Cellprofiler pipeline to quantify raw images from BRAF V600E /PI3Kα H1047R and BRAF V600E driven tumors, producing . 10.7554/eLife.43668.022 Figure 4—source code 3. Cellprofiler pipeline to quantify raw images from KRAS G12D /PIK3CA H1047R and KRAS G12D driven tumors, producing . 10.7554/eLife.43668.023 Figure 4—source data 1. Cellprofiler output quantifying immunofluorescence of SFTPA and NKX2-1 in BRAF V600E /PI3Kα H1047R and BRAF V600E driven tumors. 10.7554/eLife.43668.024 Figure 4—source data 2. Cellprofiler output quantifying immunofluorescence of SFTPA and NKX2-1 in KRAS G12D /PIK3CA H1047R and KRAS G12D driven tumors.

Article Snippet: Recombinant DNA reagent , MSCV-NKX2.1 , Addgene , Plasmid #31271 RRID: Addgene_31271 , NKX2.1 expression vector.

Techniques: Expressing, Quantitation Assay, Marker, Standard Deviation, Immunofluorescence

Journal: eLife

Article Title: Mutationally-activated PI3’-kinase-α promotes de-differentiation of lung tumors initiated by the BRAF V600E oncoprotein kinase

doi: 10.7554/eLife.43668

Figure Lengend Snippet: ( A ) Tumor cells with decreased SFTPA maintain expression and nuclear localization of the lung lineage transcription factor, FOXA1. Scale bars = 10 um. ( B ) Tumor cells with decreased SFTPA maintain expression and nuclear localization of the lung lineage transcription factor, FOXA2. ( C ) Tumor cells with decreased SFTPA maintain phosphorylation of the lung lineage transcription factor, NKX2-1. ( D ) Expression of SFTPA and NKX2.1 are maintained in KRAS G12D driven tumors, 16 weeks post initiation. ( E ) Activation of PI3K in KRAS G12D driven tumors causes a decrease of SFTPA immunoreactivity not apparently associated with a decrease in NKX2-1 immunostaining. ( F ) No significant difference in NKX2-1 immunoreactivity upon PI3K activation in KRAS G12D driven tumors. ( G ) Significant reduction in cytoplasmic staining of SFTPA upon PI3K activation in KRAS G12D driven tumors. Wilcoxon rank sum p=0.0354. ( H ) Modest association between immunoreactivity of SFTPA and NKX2-1 in KRAS G12D driven tumors (Spearman Rho = 0.256). ( I ) Modest association between immunoreactivity of SFTPA and NKX2-1 in in KRAS G12D /PI3Kα H1047R driven tumors (Spearman Rho = 0.203).

Article Snippet: Recombinant DNA reagent , MSCV-NKX2.1 , Addgene , Plasmid #31271 RRID: Addgene_31271 , NKX2.1 expression vector.

Techniques: Expressing, Activation Assay, Immunostaining, Staining

Journal: eLife

Article Title: Mutationally-activated PI3’-kinase-α promotes de-differentiation of lung tumors initiated by the BRAF V600E oncoprotein kinase

doi: 10.7554/eLife.43668

Figure Lengend Snippet: ( A ) Tumors were induced in cohorts of Braf CAT/+ and Braf CAT/+ ;Ppargc1a f/f mice via intranasal instillation of 10 6 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 BRAF V600E /PGC1α NULL tumors. ( C ) Quantitation demonstrating a significant decrease of LYZ immunoreactivity in BRAF V600E /PGC1α NULL 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. 10.7554/eLife.43668.038 Figure 7—source code 1. R script to perform gene set enrichment analysis on , as well as plot these results. 10.7554/eLife.43668.039 Figure 7—source code 2. R script to perform statistics on , as well as plot these results eLife’s transparent reporting form. 10.7554/eLife.43668.040 Figure 7—source data 1. DEseq2 output of differentially expressed genes comparing BRAF V600E /PGC1α NULL and BRAF V600E /PGC1α HET driven tumors. 10.7554/eLife.43668.041 Figure 7—source data 2. Cellprofiler output quantifying immunofluorescence of LYZ in BRAF V600E /PGC1α NULL and BRAF V600E /PGC1α WT driven tumors. 10.7554/eLife.43668.042 Figure 7—source data 3. Data from luciferase assays looking for transactivation of Sftpa , Sftpb , and Sftpc promoters.

Article Snippet: Recombinant DNA reagent , MSCV-NKX2.1 , Addgene , Plasmid #31271 RRID: Addgene_31271 , NKX2.1 expression vector.

Techniques: Immunostaining, Expressing, Quantitation Assay, Luciferase, Transfection, Immunoprecipitation, Immunofluorescence

Journal: eLife

Article Title: Mutationally-activated PI3’-kinase-α promotes de-differentiation of lung tumors initiated by the BRAF V600E oncoprotein kinase

doi: 10.7554/eLife.43668

Figure Lengend Snippet: ( A ) GSEA mountain plot comparing BRAF V600E /PGC1α NULL driven tumors to BRAF V600E /PGC1α HET 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.

Article Snippet: Recombinant DNA reagent , MSCV-NKX2.1 , Addgene , Plasmid #31271 RRID: Addgene_31271 , NKX2.1 expression vector.

Techniques: Expressing, Western Blot

Journal: eLife

Article Title: Mutationally-activated PI3’-kinase-α promotes de-differentiation of lung tumors initiated by the BRAF V600E oncoprotein kinase

doi: 10.7554/eLife.43668

Figure Lengend Snippet: 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.

Article Snippet: Recombinant DNA reagent , MSCV-NKX2.1 , Addgene , Plasmid #31271 RRID: Addgene_31271 , NKX2.1 expression vector.

Techniques: Activation Assay, Expressing

Journal: eLife

Article Title: Mutationally-activated PI3’-kinase-α promotes de-differentiation of lung tumors initiated by the BRAF V600E oncoprotein kinase

doi: 10.7554/eLife.43668

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , MSCV-NKX2.1 , Addgene , Plasmid #31271 RRID: Addgene_31271 , NKX2.1 expression vector.

Techniques: Recombinant, Plasmid Preparation, Luciferase, Expressing, Transfection, Software, Immunostaining

Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: (A) Western blot analysis of PRDM16, PPARγ, and α-Actin protein levels in whole iWAT (left) or in isolated adipocytes and stromal-vascular cells (SVC) from iWAT of young and aged mice acclimated to thermoneutrality.

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository {"type":"entrez-geo","attrs":{"text":"GSE129083","term_id":"129083"}} GSE129083 {"type":"entrez-geo","attrs":{"text":"GSE129084","term_id":"129084"}} GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE Beiging stimuli decrease the fibrotic phenotype of adipose precursor cells.

Techniques: Western Blot, Isolation

Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: (A-D) iWAT precursor cells were transduced with control (CTL) or PRDM16-expressing retrovirus and treated with either vehicle control (CTL) or DMOG. After 2 days, cells were immunostained for ACTA2 or induced to differentiate into adipocytes for 5 days.

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository {"type":"entrez-geo","attrs":{"text":"GSE129083","term_id":"129083"}} GSE129083 {"type":"entrez-geo","attrs":{"text":"GSE129084","term_id":"129084"}} GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE Beiging stimuli decrease the fibrotic phenotype of adipose precursor cells.

Techniques: Transduction, Expressing

Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: (A) Gene Set Enrichment Analysis (GSEA) of genes upregulated in PRDM16-expressing vs. control cells. Enriched pathways were defined by a normalized enrichment score > 1.3 and FDR < 0.05.

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository {"type":"entrez-geo","attrs":{"text":"GSE129083","term_id":"129083"}} GSE129083 {"type":"entrez-geo","attrs":{"text":"GSE129084","term_id":"129084"}} GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE Beiging stimuli decrease the fibrotic phenotype of adipose precursor cells.

Techniques: Expressing

Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository {"type":"entrez-geo","attrs":{"text":"GSE129083","term_id":"129083"}} GSE129083 {"type":"entrez-geo","attrs":{"text":"GSE129084","term_id":"129084"}} GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE Beiging stimuli decrease the fibrotic phenotype of adipose precursor cells.

Techniques: Generated, Recombinant, Sample Prep, SYBR Green Assay, RNA Sequencing Assay, Mouse Assay, Software