Knockdown of YAP inhibits autophagic flux in breast cancer cells.

<p><b>(A)</b> MCF7-shCtrl and shYAP cells were cultured in normal medium (ND 0 h or no treatment) or maintained in EBSS (ND) +vehicle control (VC) or EBSS +CQ or Bafilomycin A1 (BafA1) for 2,4, and 6 h. Total proteins were subjected to Western blotting for the indicated antibodies. Representative results are shown. <b>(B)</b> Quantified relative LC3-II flux according to the Western blot results of (A) is expressed as the normalized LC3-II ratio calculated by the levels of LC3-II in the presence of CQ divided by the levels of LC3-II in absence of CQ. Data are derived from three independent experiments and shown as the means±SEM. *<i>P</i><0.05. <b>(C)</b> Quantitative relative p62 levels according to results of (A) are expressed as p62 levels after different treatments divided by that of no treatment. Data are derived from three independent experiments and shown as the means±SEM. *<i>P</i><0.05. <b>(D)</b> MCF7-shCtrl, shYAP and YAP-rescue-overexpression cells were cultured in normal medium (ND 0 h or no treatment) or maintained in EBSS (ND)+Vehicle Control (VC) or EBSS +CQ for 6 h. Total proteins were assayed by Western blotting. Representative results are shown. <b>(E)</b> Quantitative relative LC3-II flux according to Western blot results of (D) was assayed as previous. Data are derived from three independent experiments and shown as the means±SEM. *<i>P</i><0.05. <b>(F)</b> Quantified relative p62 levels according to the results of (D) were assayed as previous. Data are derived from three independent experiments and shown as the means±SEM. *<i>P</i><0.05. <b>(G)</b> MDA-MB-231-shCtrl and shYAP cells were cultured for 4 h in normal medium (no treatment), EBSS+VC (ND) or EBSS+BafA1. Total proteins were assayed by Western blotting with indicated antibodies. <b>(H)</b> MDA-MB231-Control and YAP overexpression cells were cultured for 6 h in normal medium (no treatment), EBSS+VC (ND) or EBSS+BafA1. Total proteins were assayed by Western blotting.</p

YAP-increased autolysosome degradation is TEAD-dependent.

<p><b>(A)</b> TEAD1/3/4 was knocked down in normal and YAP-overexpressing MCF7 cells. Indicated cells were cultured for 6 h in normal medium (no treat), EBSS (ND) or EBSS +CQ. Total proteins were assayed by Western blotting. <b>(B)</b> MCF7-shYAP cells were stably infected with retrovirus expressing YAP-WT or YAP-S94A to generate WT Res and S94A Res cell lines. MCF7-shCtrl, shYAP, WT Ras and S94A Ras cells were cultured for 6 h in normal medium (no treatment), EBSS (ND) or EBSS +CQ. Total proteins were detected by Western blotting for indicated antibodies. <b>(C)</b> Images of normal cultured tfLC3-shYAP, tfLC3-WTr and tfLC3-S94Ar cells were taken with a confocal microscope. <b>(D)</b> YAP protein levels in MCF7-shCtrl-tfLC3, tfLC3-shYAP, tfLC3-WTr and tfLC3-S94Ar cell lines were determined. <b>(E)</b> tfLC3-shYAP, tfLC3-WTr and tfLC3-S94Ar cells were starved in EBSS for 2 h, and CQ was added to elevate lysosomal pH. Images of live cells were taken with a confocal microscope (Scale bar: 10 μm). <b>(F)</b> Average red and green fluorescent intensities per live cell in each well were valued by a high content cell assay microscope system. Green/red ratios were calculated and shown here. Data are shown as the means±SEM. *<i>P</i><0.05, n = 3.</p

The different sensitivity to nutrient deprivation (ND) caused by YAP is attenuated after autophagy inhibition.

<p><b>(A)</b> MCF7 cells were cultured under the indicated conditions for 36 h without chloroquine (CQ) or for 18 h with CQ, stained by Annexin V-FITC/PI, and analyzed with flow cytometry. Representative results are shown. <b>(B)</b> Quantitative analysis of percentages of apoptotic cells according to results of (A) are expressed as the percentage of Annexin V-positive cells out of the total number of cells in the gate. Data are shown as the means±SEM. *<i>P</i><0.05, n = 3. <b>(C)</b> After 38 h (without CQ) or 18 h (with CQ) of indicated treatments, MCF7-shCtrl, shYAP and YAP Res cell viability was determined by MTT assay. *<i>P</i><0.05, n = 3. <b>(D)</b> After 38 h (without CQ/NH₄Cl) or 18 h (with CQ/NH₄Cl) of indicated treatments, MCF7-shCtrl, shYAP and YAP Res cells were stained by Annexin V-FITC/PI and analyzed with flow cytometry. <b>(E)</b> Quantitative analysis of percentages of apoptotic cells according to the results of (D) was assayed as previously. Data are shown as the means±SEM. *<i>P</i><0.05, n = 3. <b>(F)</b> MCF7-shCtrl and shYAP cells were transfected with control (Ctrl) and LC3 siRNA for 48 h and treated with ND for 36 h. Cells were then stained by Annexin V-FITC/PI and analyzed with flow cytometry. Quantitative analysis of percentages of apoptotic cells is derived from triplicates. *<i>P</i><0.05, n = 3. <b>(G)</b> MCF7-shCtrl and shYAP cells were transfected with control (Ctrl) and LC3 siRNA for 48 h and cultured in normal medium or EBSS (ND) for 36 h. Cell viability was determined by MTT assay. *<i>P</i><0.05, n = 3. <b>(H)</b> MCF7-shCtrl and shYAP cells were transfected with the indicated siRNA for 48 h and cultured in normal medium or EBSS (ND) for 36 h. Representative blots are shown. The experiment was repeated twice.</p

YAP interferes with autophagic flux via enhancing autolysosome degradation.

<p><b>(A)</b> MCF7-shCtrl and shYAP cells were cultured for 6 h in normal medium (no treatment), EBSS (ND), EBSS+3-MA or EBSS+CQ. Total proteins were subjected to Western blotting. Representative results are shown. <b>(B)</b> Quantitative relative levels of LC3-II according to the results of (A) are expressed as LC3-II levels after different treatments (ND, ND+3-MA, ND+CQ) divided by that of no treatment. Data are derived from three independent experiments and shown as the means±SEM. *<i>P</i><0.05. <b>(C)</b> Quantitative relative p62 levels according to the results of (A) are expressed as p62 levels after different treatments divided by that of no treatment. Data are derived from three independent experiments and shown as the means±SEM. *<i>P</i><0.05. <b>(D)</b> MCF7-shCtrl, shYAP and YAP Res cells were treated with rapamycin (Rapa) or Rapa +CQ for 4 h. Total proteins were subjected to western blotting. Increased LC3-II levels were quantified as LC3-II levels after different Treats (Rapa, Rapa+CQ) subtract that of no treat. Quantitative data are derived from three independent experiments and shown as the means±SEM. *<i>P</i><0.05. <b>(E)</b> Lysosomal acidification levels of MCF7-shCtrl, shYAP and YAP Res cells were assayed by flow cytometry after staining with LysoTracker Red. <b>(F)</b> ACP activities of MCF7-shCtrl, shYAP and YAP Res cells were assayed. <b>(G)</b> MCF7-shCtrl-tfLC3 and MCF7-shYAP-tfLC3 cells were starved in EBSS for 2 h. CQ was then added to elevate the lysosomal pH. Images of live cells were taken with a confocal microscope (Scale bar: 10 μm). <b>(H)</b> Percentages of cells with red puncta per field were counted and calculated. Data are shown as the means±SEM. *<i>P</i><0.05, n = 3. <b>(I)</b> Percentages of cells with more than half the puncta yellow per field were counted and calculated. Data are shown as the means±SEM. *<i>P</i><0.05, n = 3.</p

Nutrient deprivation-induced apoptosis in MCF7 cells is increased by YAP knockdown.

<p><b>(A)</b> Expression of YAP in MCF7-shCtrl, shYAP and YAP Res cells was detected by Western blotting. <b>(B)</b> MCF7-shCtrl, shYAP and YAP Res cells were cultured in normal medium or EBSS for 36 h. Cell viability was determined by MTT assay. Data are shown as the means±SEM. *<i>P</i><0.05, n = 3. <b>(C)</b> After 36 h of ND, cells were stained by Annexin V-FITC/PI and analyzed with flow cytometry. Representative results are shown. <b>(D)</b> Quantitative analysis of the percentage of surviving cells according to the results of (C) is shown. The numbers of surviving cells are expressed as the percentage of Annexin V-FITC/PI double negative cells out of the total number of cells in the gate. Data are shown as the means±SEM. *<i>P</i><0.05, n = 3. <b>(E)</b> Quantitative analysis of the percentages of apoptotic cells, according to the results of (C) is shown. The numbers of apoptotic cells are expressed as the percentage of Annexin V-positive cells out of the total number of cells in the gate. Data are shown as the means±SEM. *<i>P</i><0.05, n = 3. <b>(F)</b> Cells were maintained in normal medium or EBSS (ND) for 36 h. Cell morphology was examined by inverted phase contrast microscopy. Representative morphologic images are shown. <b>(G)</b> Nuclear morphology was investigated using Hoechst staining after treatment as in (F). <b>(H)</b> MCF7-shCtrl and shYAP cells were cultured in the indicated conditions for the indicated time. Total proteins were subjected to Western blotting for cleaved PARP. The results are representative of two independent experiments.</p

Additional file 6: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S6. Hakai promotes BEL7402 cells invasion and growth. (A) Representative images and quantification of invasion in GFP-tagged Hakai-overexpressing BEL7402 cells by adenovirus. Scale bar = 200 μm. (B) Analysis of the ability of Hakai-overexpressing BEL7402 cells by adenovirus to form colonies. Data are presented as Mean ± SEM from three independent experiments (**p < 0.01, ***p < 0.001). (JPG 146 kb

Additional file 2: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S2. Ajuba was co-localized with Hakai in HepG2 cells. (A) HepG2 cells were co-transfected with Myc-Ajuba or Myc-Vector and GFP-Hakai for 24 h. Cells were analyzed for GFP-Hakai/Myc-Ajuba co-localization, Scale bar = 25 μm. (JPG 97 kb

Additional file 1: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S1. The regulation of β-Catenin and cell growth in HCC cells. (A, B) HCC cells were transfected with specific siRNAs to silence GSK3β protein in Ajuba-depleted HCC cell lines. The expression of GSK3β, Ajuba, CyclinD1 and GAPDH were tested by immunoblot assay (A). β-Catenin translocation were tested by confocal assay, Scale bar = 25 μm (B). (C, D) HCC cells were transfected with specific siRNAs to silence β-Catenin protein in Ajuba-depleted HCC cell lines. The expression of β-Catenin, Ajuba, CyclinD1 and GAPDH were tested by immunoblot assay (C). Cell growth was tested by colony formation (D). (E) HCC cells were transfected with specific siRNAs to silence YAP protein in Ajuba-depleted HCC cell lines. Cell growth was tested by colony formation. Data are presented as Mean ± SEM from three independent experiments (***p < 0.001). (JPG 515 kb

Additional file 5: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S5. Ajuba knockdown-mediated β-catenin translocation into nucleus is not dependent on Hakai. (A, B) HepG2 cells were transfected with specific siRNAs to silence Ajuba protein in Hakai-depleted HepG2 cells. β-catenin translocation were tested by confocal assay, Scale bar = 25 μm (A). The expression of Ajuba, Hakai and β-catenin were tested by immunoblot assay, GAPDH was used as a loading control (B). (C) Immunoblot analysis of Ajuba and Hakai in BEL7402 and HepG2 cell lysis. GAPDH was used as a loading control. (JPG 617 kb

Additional file 4: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S4. Hakai mediates Ajuba degradation via neddylation. (A) Immunoblot analysis and quantification of the half-life of Ajuba in the presence of cycloheximide (CHX, 80 μg/ml), and in the presence or absence of MLN4924 (5 μM) in BEL7402 and HepG2 cells. GAPDH was used as a loading control. (B) Ubiquitination (Ub) assay of Ajuba in 293 T cells transfected with the indicated plasmids. (C) Neddylation assay of Ajuba in 293 T cells transfected with the indicated plasmids. IB, immnoblot. IP, immunoprecipitation. WCL, Whole-cell lysates. (JPG 103 kb