Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: A) Principal component analysis of ATAC-seq data from cells treated as indicated for two weeks. B) ATAC-seq signal intensities centered on up-regulated (UP) or down-regulated (DOWN) peaks in dormant, OT-treated cells vs. control cells. C) Analysis for enriched transcription factor motifs D) GSEA of YAP/TEAD signature (Zhang et al., 2009) E) Left: ATAC-seq signal intensities centered on upregulated (UP) or down-regulated (DOWN) peaks in OT-treated vs. O-treated cells. Right: Analysis for transcription factor motifs enriched in upregulated peaks. F) QPCR analysis of YAP target gene expression. G) Regrowth of EGFR-mutant NSCLC cells after washout following a 3-week treatment with the indicated drug combinations. H) Western blot analysis of YAP protein levels in YAP1 knock-out (KO) and control (CTRL) cells. I) Proliferation of cells in (H) treated as indicated for 21 days, followed by drug washout. J) Mice bearing CTRL or YAP1 KO cell xenograft tumors were treated with vehicle or OT followed by treatment cessation and follow-up. Data from 6/8 live mice per group are plotted. Right: tumor volumes at time of regrowth, indicated by an arrow. Mean ± SEM are shown in all plots except (F), where mean ± SD are shown. ANOVA was used for statistical analyses in all but (J), where t-test was used. ***, P<0.001; *, P<0.05. See also Figure S4.

Article Snippet: The YAP1-TAdel cDNA was amplified from pLX304-YAP1_TA (gift from William Hahn, Addgene plasmid #59143) using primers 5’-ATCAACGGGACTTTCCAAAATGTCG-3’ and 5’-TTTTTTTCTAGACTATAACCATGTAAGAAAGCTTTCTGGGCT-3’ and subcloned into pDNR-dual- YAP1 using BamHI and XbaI restriction sites.

Techniques: Expressing, Mutagenesis, Western Blot, Knock-Out

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: A) YAP activity following indicated treatments in PC-9 cells transduced with a fluorescent YAP/Hippo pathway reporter (PC-9 YAP reporter cells). B) IF staining for YAP nuclear localization following the indicated treatments. C) YAP activity and apoptosis in PC-9 YAP reporter cells treated with OT. D) Analysis of overlap between YAPhigh cells (red) and apoptotic cells (green) after 80h of treatment in PC-9 YAP reporter cells. E) Apoptosis in PC-9 cells treated with the indicated drugs or drug combinations. F) Apoptosis in EGFR-mutant NSCLC cells treated as indicated. Peak apoptosis values over 72h are shown. G) Apoptosis in YAP1 knock-out (KO) or control (CTRL) cells treated as indicated. H) Left: Western blot analysis of YAP protein levels in YAP1 KO cells transduced with wild-type YAP1. Right: cells were treated with OT and analyzed as in (G). Only data from drug-treated cells is shown. I) Proportions of YAPhigh cells in PC-9 YAP reporter cell populations treated as indicated. J) Different means for EGFR-mutant NSCLC cells to avoid apoptosis following EGFR inhibition. Mean ± SEM are shown in all plots except (I), where SD is shown. ANOVA was used for statistical analyses in all but (D), where Fisher’s exact test was used. ***, P<0.001. See also Figure S5.

Article Snippet: The YAP1-TAdel cDNA was amplified from pLX304-YAP1_TA (gift from William Hahn, Addgene plasmid #59143) using primers 5’-ATCAACGGGACTTTCCAAAATGTCG-3’ and 5’-TTTTTTTCTAGACTATAACCATGTAAGAAAGCTTTCTGGGCT-3’ and subcloned into pDNR-dual- YAP1 using BamHI and XbaI restriction sites.

Techniques: Activity Assay, Transduction, Staining, Mutagenesis, Knock-Out, Western Blot, Inhibition

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: A) Western blot analysis of EGFR downstream signaling following 24h treatment as indicated. B) RNA-seq samples used in (C). C) Expression of genes regulating apoptosis in OT-treated YAP1 KO cells vs OT-treated CTRL cells. Colors indicate log2 fold change values with p<0.001. D) QPCR analysis of BMF expression in CTRL or YAP1 KO cells treated as indicated for 24h in vitro or 3 days in vivo. E) Schematic representation of the endogenous BMF locus in PC-9 HA-BMF cells. F) Western blot analysis of BMF, BIM, and YAP expression in PC-9 HA-BMF cells transfected with non-targeting (NT) or YAP siRNA and treated as indicated for 24h. G) QPCR analysis of BMF expression in CTRL or YAP1 KO cells transduced as indicated, and following treatment with either DMSO or OT for 24h. H) Peak apoptosis over 72h treatment in PC-9 and HCC4006 cells transfected with NT or BMF siRNA. I) The mechanism of YAP/TEAD-mediated suppression of apoptosis in EGFR-mutant NSCLC cells following EGFR/MEK inhibition. Mean ± SD are shown in all plots except (H), where mean ± SEM is shown. ANOVA was used for statistical analyses. ***, P<0.001; **, P<0.01; n.s., not significant (P>0.05). See also Figure S7.

Article Snippet: The YAP1-TAdel cDNA was amplified from pLX304-YAP1_TA (gift from William Hahn, Addgene plasmid #59143) using primers 5’-ATCAACGGGACTTTCCAAAATGTCG-3’ and 5’-TTTTTTTCTAGACTATAACCATGTAAGAAAGCTTTCTGGGCT-3’ and subcloned into pDNR-dual- YAP1 using BamHI and XbaI restriction sites.

Techniques: Western Blot, RNA Sequencing Assay, Expressing, In Vitro, In Vivo, Transfection, Mutagenesis, Inhibition

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: A) GSEA of EMT signature in YAP1 knock-out (KO) vs control cells treated with OT for 24 hours. B) QPCR analysis of EMT transcription factor expression in untreated EGFR-mutant NSCLC cells. C) Co-immunoprecipitation analysis of the interaction between YAP, TEAD, and SLUG in PC-9 cells following treatment with DMSO or OT for 48h. D) Western blot analysis of YAP and SLUG protein levels in PC-9 or HCC4006 cells transfected with non-targeting (NT), YAP or SLUG siRNA. E) QPCR analysis of BMF expression in cells in (D) following 24h treatment with DMSO or OT. F) Apoptosis in cells in (D) following treatment with DMSO or OT. G) Number of peaks called by MACS2 (FDR<0.01). H) ChIP-seq signal traces in BMF locus. H3K27Ac was used to identify enhancer regions. I) The mechanism by which YAP/TEAD/SLUG complex represses BMF expression upon combined EGFR/MEK inhibition. Mean ± SD (E), or mean ± SEM (F) are shown. ANOVA was used for statistical analyses. ***, P<0.001; **, P<0.01.

Article Snippet: The YAP1-TAdel cDNA was amplified from pLX304-YAP1_TA (gift from William Hahn, Addgene plasmid #59143) using primers 5’-ATCAACGGGACTTTCCAAAATGTCG-3’ and 5’-TTTTTTTCTAGACTATAACCATGTAAGAAAGCTTTCTGGGCT-3’ and subcloned into pDNR-dual- YAP1 using BamHI and XbaI restriction sites.

Techniques: Knock-Out, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Transfection, ChIP-sequencing, Inhibition

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: A) YAP1 mutants used in the rescue experiment in (B). B) Viability (Cell Titer Glo) of CTRL cells or PC-9 YAP1 KO cells transduced with YAP1 mutants (A) following 72h treatment with OT. C) Top: the structure of MYF-01–37. Bottom: MYF-01–37 binding to the palmitoylation pocket in TEAD1 based on molecular docking. The cysteine 359 targeted by MYF-01–37 is indicated. D) Effect of MYF-01–37 or the corresponding reversible control on YAP/TEAD interaction measured using Split Gaussia Luciferase Assay. E) Left: Western bot analysis of the expression of myc-tagged TEAD1 in PC-9 cells transduced as indicated. Right: QPCR analysis of CTGF expression after 24h treatment with XAV939 or MYF-01–37 in the transduced PC-9 cells. F) YAP activity in PC-9 YAP reporter cells after 72h treatment with OT or OT in combination with XAV939 (XAV) or MYF-01–37 (MYF). G) QPCR analysis of BMF expression in cells in (E) following 24h treatment as indicated. H) Apoptosis in PC-9 and HCC4006 cells treated as indicated. G) Regrowth of PC-9 and HCC4006 cells after drug washout following a two-week treatment as indicated. Mean ± SEM are shown in all plots except (E), where mean ± SD is shown. ANOVA was used for statistical analyses. ***, P<0.001; **, P<0.01. See also Figure S8.

Article Snippet: The YAP1-TAdel cDNA was amplified from pLX304-YAP1_TA (gift from William Hahn, Addgene plasmid #59143) using primers 5’-ATCAACGGGACTTTCCAAAATGTCG-3’ and 5’-TTTTTTTCTAGACTATAACCATGTAAGAAAGCTTTCTGGGCT-3’ and subcloned into pDNR-dual- YAP1 using BamHI and XbaI restriction sites.

Techniques: Transduction, Binding Assay, Luciferase, Western Blot, Expressing, Activity Assay

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: A–B) Apoptosis in NSCLC cell lines treated as indicated. C) Left: Western blot analysis of YAP expression in control (CTRL) and YAP1 KO H3122 and EBC-1 cells. Right: apoptosis in CTRL and YAP1 KO H3122 and EBC-1 cells treated as indicated. D) PC-9 cells were treated as indicated in the scheme on the left, followed by drug washout. Regrowth of cells was monitored and quantified as in Figure 2G. Mean ± SEM are shown. ANOVA was used for statistical analyses. ***, P<0.001.

Article Snippet: The YAP1-TAdel cDNA was amplified from pLX304-YAP1_TA (gift from William Hahn, Addgene plasmid #59143) using primers 5’-ATCAACGGGACTTTCCAAAATGTCG-3’ and 5’-TTTTTTTCTAGACTATAACCATGTAAGAAAGCTTTCTGGGCT-3’ and subcloned into pDNR-dual- YAP1 using BamHI and XbaI restriction sites.

Techniques: Western Blot, Expressing

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The YAP1-TAdel cDNA was amplified from pLX304-YAP1_TA (gift from William Hahn, Addgene plasmid #59143) using primers 5’-ATCAACGGGACTTTCCAAAATGTCG-3’ and 5’-TTTTTTTCTAGACTATAACCATGTAAGAAAGCTTTCTGGGCT-3’ and subcloned into pDNR-dual- YAP1 using BamHI and XbaI restriction sites.

Techniques: Recombinant, In Vivo, In Vitro, Synthesized, Expressing, Staining, Cell Viability Assay, TaqMan Assay, Plasmid Preparation, Software

Journal: Microvascular research

Article Title: YAP1-TEAD1 signaling controls angiogenesis and mitochondrial biogenesis through PGC1α

doi: 10.1016/j.mvr.2018.04.003

Figure Lengend Snippet: A) Graphs showing TEAD1 and PGC1α mRNA levels in HUVE cells treated with TEAD1 siRNA#1, #2, or control siRNA with irrelevant sequences (*, p<0.05). Error bars represent s.e.m. of three independent experiments. B) Immunoblots showing TEAD1, PGC1α, and β-actin protein levels in HUVE cells treated with TEAD1 siRNA#1 or control siRNA with irrelevant sequences (top). Graph showing TEAD1 and PGC1α protein levels normalized by β-actin protein levels in HUVE cells treated with TEAD1 siRNA #1 or control siRNA with irrelevant sequences (bottom, *, p<0.05). Error bars represent s.e.m. of three independent experiments. C) Diagram showing the structure of full-length YAP1 and the YAP1S127A and S94A mutant constructs (top). Immunoblots showing YAP1 and TEAD1 immunoprecipitated with TEAD1 antibody in HUVE cells overexpressing full-length YAP1, YAP1S127A, YAP1S94A, or control vector (middle). Immunoblots showing YAP1 and TEAD1 immunoprecipitated with YAP1 antibody in HUVE cells overexpressing full-length YAP1, YAP1S127A, YAP1S94A, or control vector (bottom). D) Graph showing YAP1 and PGC1α mRNA expression levels in HUVE cells overexpressing full-length YAP1, YAP1S127A, YAP1S94A, or control vector alone (*, P<0.05). Error bars represent s.e.m. of three independent experiments.

Article Snippet: The retroviral full-length pQCXIH-myc-YAP1 (human) and pQCXIH-flag-YAP1-S127A (human) were gifts from Kunliang Guan (Addgene plasmid # 33091 and # 33092) ( Zhao et al., 2007 ) and pLX304-YAP1 (S94A) (human) construct was a gift from William Hahn (Addgene plasmid # 59145) ( Shao et al., 2014 ). pLenti-PGC1α (mouse) construct was kindly provided by Dimitri Krainc (Northwestern University) ( Cui et al., 2006 ).

Techniques: Control, Western Blot, Mutagenesis, Construct, Immunoprecipitation, Plasmid Preparation, Expressing

Journal: Microvascular research

Article Title: YAP1-TEAD1 signaling controls angiogenesis and mitochondrial biogenesis through PGC1α

doi: 10.1016/j.mvr.2018.04.003

Figure Lengend Snippet: A) Oxygen consumption rate (OCR) of HUVE cells treated with virus overexpressing full-length YAP1, YAP1S127A or S94A mutant construct, TEAD1 siRNA or control vector alone. Error bars represent s.e.m. of three independent experiments. B) Graphs showing maximal respiration following carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) treatment (left) and ATP-dependent OCR (right, *, p<0.05). Error bars represent s.e.m. of three independent experiments. C) Extracellular acidification rate (ECAR) of HUVE cells treated with virus overexpressing full-length YAP1, YAP1S127A or S94A mutant construct, TEAD1 siRNA or control vector alone. Error bars represent s.e.m. of three independent experiments. D) Graphs showing glycolysis (left) and glycolytic reserve (right, *, p<0.05). Error bars represent s.e.m. of three independent experiments.

Article Snippet: The retroviral full-length pQCXIH-myc-YAP1 (human) and pQCXIH-flag-YAP1-S127A (human) were gifts from Kunliang Guan (Addgene plasmid # 33091 and # 33092) ( Zhao et al., 2007 ) and pLX304-YAP1 (S94A) (human) construct was a gift from William Hahn (Addgene plasmid # 59145) ( Shao et al., 2014 ). pLenti-PGC1α (mouse) construct was kindly provided by Dimitri Krainc (Northwestern University) ( Cui et al., 2006 ).

Techniques: Virus, Mutagenesis, Construct, Control, Plasmid Preparation