Supplementary Figures Supplementary Figure 1 Correlation between LKB1 and YAP expression in human lung cancer samples. (a) Representative photos showing LKB1 and YAP immunohistochemical staining in human lung cancer. Scale bar, 150 m. (b) Correlation analysis between LKB1 and YAP expression (P=0.511) in human lung cancer. 2-test was used to determine the correlation between LKB1 and YAP expression.
Supplementary Figure 2 TAZ expression in human lung ADC and SCC. (a) Representative photos showing TAZ immunohistochemical staining in human lung ADC and SCC. Scale bar, 150 m. (b) Statistical analysis of TAZ expression in human lung ADC and SCC. 2-test was used to determine the expression difference.
Supplementary Figure 3 YAP over-expression validation in KL and YKL mice. (a-b) Realtime PCR quantification (a) and western blot (b) of YAP in lung tumors from KL mice delivered with Lenti-CMV-Cre or Lenti-YAPS127A-Cre (each sample was assayed in triplicates; Representative data of western blot were shown from three independent experiments.). (c) Realtime PCR quantification of YAP in lung tissue from WT and SP-C-YAP transgenic mice (each sample was assayed in triplicates). (d) Western blot of YAP in lung tumors from KL or YKL mice from three SP-C-YAP transgenic lines. Representative data were shown from three independent experiments.
Supplementary Figure 4 Yap knockdown decreases the cell proliferation in lung ADC from KL mouse model. (a) Western blot of Yap in lung tumors from KL mice delivered with Lenti-U6-Cre or Lenti-shYap-Cre. Representative data were shown from three independent experiments. (b-c) Ki-67 and Cleaved Caspase-3 immunohistochemical staining in lung tumors from KL mice delivered with Lenti-U6-Cre or Lenti-shYap-Cre. Scale bar, 150 μm. Data are shown as mean±sem. t-test, ***, P<0.005.
Supplementary Figure 5 YAP knockdown impairs cell proliferation and anchorage independent cell growth in A549 cells. (a) Western blot validation of efficient Yap knockdown in A549 cells. Representative data were shown from three independent experiments. (b) MTT assay showing that YAP knockdown in A549 cells significantly reduced cell proliferation (each sample was assayed in triplicates). (c) Colony formation assay showing that YAP knockdown in A549 cells significantly impaired anchorage independent cell growth in soft agar (each sample was assayed in triplicates). Representative data of MTT assay and colony formation assay were shown from three independent experiments. Data are shown as mean±sem. t-test, ***, P<0.005.
Supplementary Figure 6 YAP negatively regulates DNp63 transcription in human lung cancer cell lines. (a) Ectopic YAP S127A expression down-regulated DNp63 mrna and protein levels in SH-416 cells. Representative data of western blot were shown from three independent experiments. (b)yap S127A over-expression down-regulated DNp63 mrna levels in A549, CRL-5908, CRL-5807 and CRL-5810 cells. (c) Knockdown of YAP up-regulated DNp63 mrna in A549 cells. (d) Ectopic expression of YAP 5SA but not YAP 5SA with C-terminal trans-activation domain deletion dramatically decreased DNp63 mrna levels in A549 cells. The asterisk represents the YAP 5SA-ΔC mutant form with its C-terminal trans-activation domain deletion. Representative data of western blot were shown from three independent experiments. (e) Knockdown of TEADs reversed the decrease of DNp63 mrna and protein levels caused by YAP S127A ectopic expression in CRL-1848 cells. Each sample was assayed in triplicates by quantitative real-time PCR. Representative data of real-time PCR and western blot were shown from three independent experiments. Data are shown as mean±sem. t-test, *, P<0.05; **, P<0.01; ***, P<0.005.
Supplementary Figure 7 Extracellular matrix (ECM) deprivation leads to YAP phosphorylation and inactivation. (a) Realtime PCR quantification of Yap target gene transcripts in ADC and SCC from KL mice (each sample was assayed in triplicates by quantitative real-time PCR).Fold change was calculated as log2 (SCC/ADC). (b) Representative photos showing YAP immunohistochemical staining in KL mice treated with BAPN or saline. Scale bar, 150 μm. (c) Cell detachment from ECM resulted in YAP phosphorylation and inactivation in A549 cells. A549 cells were either trypsinized (T), or trypsinized and then attached onto collagen I-coated petri dishes for 2 h (A), or trypsinized and then cultured on Poly-HEMA coated dishes in suspension for 2h (S), or further trypsinized after attachment (ret). Samples were lysed and analyzed by Western blots. Representative data of western blot were shown from three independent experiments.
Supplementary Figure 8 Screen for YAP downstream repressors regulating DNp63 transcription. (a-b) Realtime PCR analysis of relative expression of 5 common E-box binding factors including SNAIL, SLUG, ZEB1, ZEB2 and MYC following YAP S127A ectopic expression in A549 (a) and HTB-182 (b) cells. Each sample was assayed in triplicates by quantitative real-time PCR. Representative data of real-time PCR were shown from three independent experiments. Data are shown as mean±sem. t-test, *, P<0.05; **, P<0.01.
Supplementary Figure 9 YAP up-regulates ZEB2 transcription in human lung cancer cell lines. (a-b) YAP S127A ectopic expression up-regulated ZEB2 mrna and protein levels in A549 (a) and CRL-1848 (b) cells. (c-d) Knockdown of YAP down-regulated ZEB2 mrna and protein levels in A549 (c) and CRL-1848 (d) cells. (e-f) Ectopic expression of YAP 5SA but not YAP 5SA with C-terminal transactivation domain deletion dramatically increased ZEB2 mrna and protein levels in A549 (e) and CRL-1848 (f) cells. The asterisk represents the YAP 5SA-ΔC mutant form with its c-terminal trans-activation domain deletion. (g) Knockdown of TEADs blocked the increase of ZEB2 mrna levels caused by YAP S127A ectopic expression in A549 cells. Each sample was assayed in triplicates by quantitative real-time PCR. Representative data of real-time PCR and western blot were shown from three independent experiments. Data are shown as mean±sem. t-test, *, P<0.05; **, P<0.01; ***, P<0.005.
Supplementary Figure 10 ZEB2 represses DNp63 transcription in human lung cancer cell lines. (a-b) ZEB2 ectopic expression down-regulated DNp63 mrna and protein levels in A549 (a) and CRL-1848 (b) cells. (c-d) Knockdown of ZEB2 up-regulated DNp63 mrna and protein levels in A549 (c) and CRL-1848 (d) cells. Each sample was assayed in triplicates by quantitative real-time PCR. Representative data of real-time PCR and western blot were shown from three independent experiments. Data are shown as mean±sem. t-test, *, P<0.05; **, P<0.01.
Supplementary Figure 11 YAP suppresses DNp63 expression in a ZEB2 dependent manner. (a-b) Knockdown of ZEB2 reversed the decrease of DNp63 mrna and protein levels caused by YAP ectopic expression in A549 (a) and CRL-1848 (b) cells. (c-d) Re-introduction of ZEB2 abolished the increase of DNp63 mrna and protein levels resulted from YAP knockdown in A549 (c) and CRL-1848 (d) cells. Each sample was assayed in triplicates by quantitative real-time PCR. Representative data of real-time PCR and western blot were shown from three independent experiments. Data are shown as mean±sem. t-test, **, P<0.01; ***, P<0.005.
Supplementary Figure 12 Estrogen receptor (ER)-mediated DNp63 inducible expression validation in vitro. Western blot using cytoplasmic and nuclear fractions showed that TAM treatment increased nuclear DNp63 expression with a concomitant decrease of cytoplasmic DNp63 level. LAMIN B and TUBLIN served as loading controls for nuclear and cytoplasmic fractions, respectively. Representative data of western blot were shown from three independent experiments.
Supplementary Figure 13 Original photos of western blots and gels shown in the main text.
Supplementary Figure 14 Original photos of western blots shown in Supplementary Information.
Supplementary Table 1. Clinical correlation between YAP expression and key oncogenic drivers in human NSCLC. YAP Characteristics Low High P EGFR Mut. Negative 86 (74.8%) 29 (25.2%) <0.001*** Positive 22 (43.1%) 29 (56.9%) KRAS Mut. Negative 103 (64.8%) 56 (35.2%) 1.000 Positive 5 (71.4%) 2 (28.6%) HER2 Mut Negative 105 (64.8%) 57 (35.2%) 1.000 Positive 3 (75.0%) 1 (25.0%) BRAF Mut Negative 105 (64.8%) 57 (35.2%) 1.000 Positive 3 (75.0%) 1 (25.0%) EML4-ALK fusions Negative 100 (63.3%) 58 (36.7%) 0.051 Positive 8 (100%) 0 (0%) FGFR fusions Negative 104 (64.6%) 57 (35.4%) 0.659 Positive 4 (80%) 1 (20%) 2-test and Fisher s Exact Test (n<5) were used to generate the P value.
Supplementary Table 2. Clinical correlation between YAP expression and key oncogenic drivers in human lung ADC. YAP Characteristics Low High P EGFR Mut. Negative 26 (74.3%) 9 (25.7%) 0.004** Positive 20 (42.6%) 27 (57.4%) KRAS Mut. Negative 44 (55.7%) 35 (44.3%) 1.000 Positive 2 (66.7%) 1 (33.3%) HER2 Mut Negative 43 (55.1%) 35 (44.9%) 0.627 Positive 3 (75.0%) 1 (25.0%) BRAF Mut Negative 44 (55.7%) 35 (44.3%) 1.000 Positive 2 (66.7%) 1 (33.3%) EML4-ALK fusions Negative 38 (51.4%) 36 (48.6%) 0.008** Positive 8 (100%) 0 (0%) 2-test and Fisher s Exact Test (n<5) were used to generate the P value.
Supplementary Table 3. Clinical correlation between YAP expression and FGFR fusions in human lung SCC. YAP Characteristics Low High P FGFR fusions Negative 58 (73.4%) 21 (26.6%) 1.000 Positive 4 (80.0%) 1 (20.0%) Fisher s Exact Test (n<5) was used to generate the P value.
Supplementary Table 4. Correlation analysis between Zeb2 and p63 expression in lung tumors from KL mice. Zeb2 Low High P Low 11 45 <0.001*** p63 19.6% 80.4% High 16 7 69.6% 30.4% 2-test was used to generate the P value.
Supplementary Table 5. The list of primers used in this study. Gene Forward primer Reverse primer p63 GGAAAACAATGCCCAGACTC GTGGAATACGTCCAGGTGGC CTGF CCTGGTCCAGACCACAGAGT TGGAGATTTTGGGAGTACGG Ctgf AGCAGCTGGGAGAACTGTGT GCTGCTTTGGAAGGACTCAC CYR61 ACCGCTCTGAAGGGGATCT ACTGATGTTTACAGTTGGGCTG Cyr61 GATGACCTCCTCGGACTCGAT CGTGCAGAGGGTTGAAAAGAA ITGB2 TGCGTCCTCTCTCAGGAGTG GGTCCATGATGTCGTCAGCC Itgb2 CAGGAATGCACCAAGTACAAAGT GTCACAGCGCAAGGAGTCA GLI2 CCCCTACCGATTGACATGCG GAAAGCCGGATCAAGGAGATG Gli2 CAACGCCTACTCTCCCAGAC GAGCCTTGATGTACTGTACCAC FGF1 ATACGGCTCACAGACACCAA CGTTTGCAGCTCCCATTCTT Fgf1 CTCGCAGACACCAAATGAGG ACAGCTCCCGTTCTTCTTGA AREG GAGCCGACTATGACTACTCAGA TCACTTTCCGTCTTGTTTTGGG Areg GGGGACTACGACTACTCAGAG TCTTGGGCTTAATCACCTGTTC AXL CCGTGGACCTACTCTGGCT CCTTGGCGTTATGGGCTTC Axl ATGGCCGACATTGCCAGTG CGGTAGTAATCCCCGTTGTAGA YAP CACAGCATGTTCGAGCTCAT GATGCTGAGCTGTGGGTGTA GAPDH CAGGTGGTCTCCTCTGACTT CCAAATTCGTTGTCATACCA β-actin TGAGCGCAAGTACTCTGTGTGGAT ACTCATCGTACTCCTGCTTGCTGA SNAIL CTGCCCTGCGTCTGCGGAAC GGAGCGGTCAGCGAAGGCAC SLUG CCGCGCTCCTTCCTGGTCAA TGGAGCAGCGGTAGTCCACACA ZEB1 AGCAGTGAAAGAGAAGGGAATGC GGTCCTCTTCAGGTGCCTCAG ZEB2 CAAGAGGCGCAAACAAGCC GGTTGGCAATACCGTCATCC MYC TCAAGAGGCGAACACACAAC GGCCTTTTCATTGTTTTCCA Col1a1 GCTCCTCTTAGGGGCCACT CCACGTCTCACCATTGGGG Col3a1 CTGTAACATGGAAACTGGGGAAA CCATAGCTGAACTGAAAACCACC Col4a1 CTGGCACAAAAGGGACGAG ACGTGGCCGAGAATTTCACC Col4a2 GACCGAGTGCGGTTCAAAG CGCAGGGCACATCCAACTT Col4a3 CAAAGGCATCAGGGGAATAACT ATCCGTTGCATCCTGGTAAAC Col4a5 GGAGAACGGGGGTTTCCAG CTCCCTTGGTTCCATTGCATC Col5a2 ACAGGTGAAGTGGGATTCTCA CCATAGCACCCATTGGACCA Col6a1 CTGCTGCTACAAGCCTGCT CCCCATAAGGTTTCAGCCTCA Col7a1 GCCCAGAGATAGAGTGACCTG CGCACTTCTCGAAAGTTGCTG Col18a1 GTGCCCATCGTCAACCTGAA GACATCTCTGCCGTCAAAAGAA Timp1 GCAACTCGGACCTGGTCATAA CGGCCCGTGATGAGAAACT Timp2 TCAGAGCCAAAGCAGTGAGC GCCGTGTAGATAAACTCGATGTC Timp3 CTTCTGCAACTCCGACATCGT GGGGCATCTTACTGAAGCCTC Timp4 TGTGGCTGCCAAATCACCA TCATGCAGACATAGTGCTGGG Mmp9 TGTCTGGAGATTCGACTTGAAGTC TGGTGTGCCCTGGAACTCA Mmp14 CAGTATGGCTACCTACCTCCAG GCCTTGCCTGTCACTTGTAAA Lox TCTTCTGCTGCGTGACAACC GAGAAACCAGCTTGGAACCAG Loxl1 TGCCCGACAACTGGAGAGA TGCGGATAGGGGAACTGCT
Loxl2 ATTAACCCCAACTATGAAGTGCC CTGTCTCCTCACTGAAGGCTC GAPDH promoter ChIP CCTTCTTGCCTTGCTCTTGCTAC GCCTGCCTGGTGATAATCTTTG ZEB2 promoter ChIP AATATCATGCTCTCCCTTCC TGGCATTTTAATAAAGGGTC DNp63 promoter ChIP TCCATTGGAGTGGAGGAGTC GCTGCTGTCCTTTCTTCTGG