Supplementary Figure1-10, Table1-2 from REGγ Controls Hippo Signaling and Reciprocal NF-κB–YAP Regulation to Promote Colon Cancer

Supplementary Figure1-10, Table1-2 Figure S1. The relations of REGγ with Hippo-YAP signal pathway in colon cancer. A. The protein levels of Lats2 and p-Lats1 were unchanged in HCT116 and HT29 human colon cancer cells with REGγ knockdown. The expression of REGγ, Lats2 and p-Lats1 were measured by Western Blot. B. REGγ knockdown did not change the levels of p-YAP (S397) in HCT116 and HT29 human colon cancer cells. Figure S2. REGγ promotes degradation of Lats1. A. Silencing REGγ slowed down degradation of endogenous Lats1. REGγ sh-N or sh-R HCT116 cells were treated with cycloheximide (100μg/ml) for indicated time followed by Western Blotting. Quantitated results were plotted to indicate dynamic changes (Analysis of Variance, n=3, *p<0.05, **p<0.01, ***p<0.001). B. Ectopic expression of wild-type (WT), but not inactive mutant N151Y REGγ promoted the degradation of endogenous Lats1 in HEK293 cells. Figure S3. REGγ promotes YAP signaling via degradation of Lats1. A Silencing Lats1 restored the expressions of YAP target genes in HCT116 sh-R cells similar to those in HCT116 cells by RT-PCR analysis. Data are presented as the means {plus minus} SEM (Analysis of Variance, n=3, **p<0.01, ***p<0.001). B. RNAi efficiently depleted Lats1 in HCT116 cells for experiments in A. HCT116 cells were harvested 72h after transient transfection of a control (Ctrl) or Lats1 small interfering RNA (siRNA) followed by Western blot analysis. C. Overexpression of Lats1 in HCT116 sh-N cells changed the expression of YAP target genes similar to the levels in HCT116 sh-R cells by RT-PCR analysis. Data are presented as the means {plus minus} SEM (Analysis of Variance, n=3, *p<0.05, **p<0.01, ***p<0.001). D. Western blot analysis validated successful expression of exogenous Lats1 in experiments shown in C. E Figure S4. REGγ promotes proliferation of human colon cancer cells. A. REGγ deletion and YAP silencing inhibited cell proliferation of HCT116 human colon cancer cells to a similar extent. After 72 h of transfection with a control- siRNA or YAP-siRNA, cell viability was measured on days 1, 2, 3, 4 and 5 by using MTT assays. Western Blot analysis showing marked silencing of YAP expression in HCT116 cells after 72h transfection with control (Ctrl) or YAP small interfering RNA (siRNA) oligos. B. Silencing REGγ or YAP alone or in combination inhibited cell proliferation of HT29 human colon cancer cells. Cell viability was measured on days 1, 2, 3, 4 and 5 by MTT assays following RNAi for 72h. Western blot analysis demonstrated the knockdown efficiency. C Figure S5. Constitutive YAP fully reversed the retardation of tumor growth induced by REGγ depletion. A. Xenograft tumors were generated by injecting HCT116 sh-N, HCT116 sh-N+YAP (S127A), HCT116 sh-R and HCT116 sh-R+YAP (S127A) cells into dorsal flanking sites of nude mice. B Tumors were dissected and volumes were measured. Values were presented as the means {plus minus} SEM (two-tailed Analysis of Variance, n=3, *p < 0.05, **p < 0.01). C. The xenografts were harvested 30 days post-injection and analyzed by Western blotting with antibodies against YAP or REGγ. Data in this figure are representatives of three independent repeats. Figure S6. P65 strengths YAP transcription and its signal pathway A. Quantitative PCR analysis of YAP expression in HT29 cells treated with TNFα (20 ng/ml) or IL-6 (20 ng/ml). Data are presented as the means {plus minus} SEM (Analysis of Variance, n=3, ***p<0.001). B. HCT116 cells were treated with or without TNFα (20 ng/ml) for 3 hours and processed for ChIP assay using anti-YAP antibodies. Immunoprecipitated chromatin was analyzed by RT-PCR using the specific primers for Cyr61 promoter. C. and D. TNFα or IL-6 treatment enhanced the expression of YAP downstream positive regulatory genes (Cyr61, AREG) and decreased the expression of negative regulatory gene (Trail, DDT4) in HCT116 and HT29 cells. Cells were treated were treated with TNFα (20 ng/ml) or IL-6 (20 ng/ml) with or without Verteporfin (VP) for 3 hours and were analyzed by real-time PCR. Figure S7. YAP enhances p65 transcription and its signal pathway A. Quantitative PCR analysis of p65 expression in HT29 cells treated with/without Verteporfin (VP) or transfected with S127A mutant YAP (S127A-YAP) compared with the empty vector. Data are presented as the means {plus minus} SEM (Analysis of Variance, n=3, **p<0.01). B. YAP activation increased the transcriptional activity of NF-κB in HT29 human colon cancer cells. NF-κB luciferase reporter activities were measured in sh-N, sh-N+YAP (S127A), sh-R and sh-R+YAP (S127A) cells. Data represent the means {plus minus} SEM (Analysis of Variance, n=3, ***P<0.001). C. and D. Figure S8. Clinical implication of REGγ in human colon cancer. A. A significant correlation among REGγ, YAP1 and RELA mRNA expression in 53 patients with ulcerative colitis. Pearson correlation coefficient was 0.737, 0.917 and 0.734, respectively. P<0.0001. B. The correlation of survival rate with p-p65 overexpression in 172 CRC patients. p=0.379. C. Diagrams summarizing the percentage of positive staining for each marker examined. Results were evaluated in a double-blinded fashion. D. High expression of REGγ was not related with patient age, patient gender, tumor size, tumor grade and tumor metastasis (p>0.05) Figure S9. A Model of crosstalk among REGγ, Hippo-Yap, and NF-κB. In human colon cancer cells, overexpression of REGγ promotes the degradation of Lats1, thus activating YAP signaling. YAP activation empowers a reciprocal positive regulation with NF-κB, leading to aberrant cell proliferation and development of inflammationassociated colon cancer. In contrast, cells with REGγ depletion are resistant to Lats1 inactivation, inhibiting YAP activation and its crosstalk to NF-κB to prevent tumor formation Figure S10. P53 was not affected by REGγ deficiency in human colon cancer cells. A. Western Blot analysis for REGγ and p53 in HCT116 sh-N and sh-R human colon cancer cells. β-actin served as a loading control. B. Western Blot analysis of p53 from mouse normal colon and tumor tissues of REGγ+/+ and REGγ-/- mice showed no marked changes. All experiments were repeated three times. Table 1. Sequences of primers used for Q-PCR Table 2. Sequences of primers used for ChIP