Supplemental Material DLG5 CONNECTS CELL POLARITY AND
Total Page：16
File Type：pdf, Size：1020Kb
Supplemental Material DLG5 CONNECTS CELL POLARITY AND HIPPO SIGNALING PROTEIN NETWORKS BY LINKING PAR-1 WITH MST1/2 Julian Kwan, Anna Sczaniecka, Emad Heidary Arash, Liem Nguyen, Chia-Chun Chen, Srdjana Ratkovic, Olga Klezovitch, Liliana Attisano, Helen McNeill, Andrew Emili and Valeri Vasioukhin Supplemental Experimental Procedures Immunoprecipitation and Western blot analysis HEK293T cells transfected with indicated plasmids were lysed in immunoprecipitation buffer containing 50mM Tris-HCl pH7.5, 100 mM NaCl, 1% Triton X-100, 10% glycerol, 0.1mM EDTA, 0.5mM MgCl2, phosphatase inhibitors (Roche 04906837001), and protease inhibitors (Roche 11836170001). V5-tagged proteins were immunoprecipitated using V5 Sepharose (Sigma A7345-1ML). nVA-tagged proteins were immunoprecipitated using either anti -FLAG M2 affinity beads (Sigma A2220) or Strep-Tactin Sepharose (IBA 2-1201-002). Protein complexes were washed four times in immunoprecipitation buffer and analyzed by Western blotting. For co-immunoprecipitation experiments with endogenously expressed proteins, NPC cells were lysed in immunoprecipitation buffer containing 50mM Tris, 100mM NaCl, 0.5% IGPAL, 0.1mM EDTA, 0.5mM MgCl2, phosphatase and protease inhibitors (Roche). Protein extracts from wild-type and Dlg5-/- NPCs were pre-cleared by incubation with Protein A Beads (Millipore 16-156) for 1 hour and supernatants were incubated overnight with either anti-Dlg5(Nechiporuk et al., 2013), anti-MARK3 (Cell Signaling, 9311) or anti-MST1/2 (Bethyl Laboratories A300-468A) antibodies. The protein complexes were incubated with Protein A Beads for 2 hours followed by 4 washes in the lysis buffer and western blot analyses. Western blot analysis was performed using NuPage Novex 4-12% gradient gel system, semi-dry protein transfer to Immobolon P membrane (Millipore) and ECL kit (Pierce), as previously described(Lien et al., 2008). Western blotting was performed using the flowing antibodies: Ms anti-YAP1 (Santa Cruz sc-101199), Rb anti- Phospho-YAP S127 (Cell Signaling 4911), Ms anti-TAZ (BD Pharmingen 560235), Rb anti-MST1 (abcam ab97399), Rb anti-MST1 (Millipore [Upstate] 07-061), Rb anti-MST2 (Thermo Scientific PA5-28567), Rb anti- MST1/2 (Bethyl Laboratories, A300-468A), Rb anti-Phospho-MST1/2 183/180 (Cell Signaling 3681), Rb anti- LATS1 (Cell Signaling 3477), Rb anti-LATS2 (Bethyl Labs A300-479A), Rb anti-LATS1/2 S909 (Cell Signaling 9157), Rb anti-phospho-LATS1/2 T1079 (Cell Signaling 9159), Rb anti-Dlg5 (PMID: 23466739) Primary antibodies were detected using HRP-labeled secondary antibodies (Jackson ImmunoResearch Laboratories). Plasmids and siRNA oligos All ORFs cloned from cDNA were fully sequence verified, ORFs from existing collections were partially or fully sequenced (Table S4) Most ORFs were expressed from pLD-puro-TnVA(Mak et al., 2010). Generation of expression constructs encoding V5-tagged full-length and individual domains of DLG5 was previously described(Nechiporuk et al., 2013). siGENOME Human DLG5 (9231) and control pools were obtained from Dharmacon(GE). Quantitative RT-PCR for mouse transcripts Total RNA was isolated using Trizol (ThermoFisher), purified using the RNeasy MinElute Cleanup Kit (Qiagen 74204), and cDNA was made using the SuperScript III First strand cDNA Synthesis System (Invitrogen 18080- 051). Relative cDNA levels were assessed using 7900 Real-Time PCR System (Applied Biosystems) and Universal Probes (Roche) or Invitrogen SuperMix-UDG (Invitrogen # 11730-025) kits. qPCR data were normalized to ribosomal protein Rps16. See Table S2 for primer sequences. Drosophila Dlg5 RNAi stocks used were: BDSC stock#: 30925, 30926, VDRC ID#: 46234, 101596, 22496. Expression of RNAi was induced using en-Gal4 at 25C and ey-Gal4 at 29C. Changes in expression were examined using wing imaginal discs from ex697/UAS-Dicer2; UAS-Dlg5RNAi30925/dpp-Gal4 and ex697/en-Gal4; UAS- Dlg5RNAi30925/UAS-Dicer2 at 25C. Quantification was performed for three discs from each condition, the mean staining intensity was measured using ImageJ in five areas of mutant tissue and adjacent non-mutant tissue areas. Chosen areas were representative of the heterogeneity of the tissue. siRNA knockdowns in MDA-MB-231 and HepG2 cells Cells were transfected with Dharmacon siGENOME pools of four individual siRNAs (GE) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's instructions. Cells were lysed in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM DTT containing phosphatase and protease inhibitors). Lysates were separated on SDS–PAGE gels, and immunoblotting was performed using standard protocols. Phos-Tag gels(Waco Chemicals), were prepared according to manufacturer's instructions. The antibodies used were as follows: pYAP (D9W2I; Cell Signaling #13008); YAP (Cell Signaling #4912), TAZ (Cell Signaling #2149), Lats1 (C66B5; Cell Signaling #3477). Immunofluorescence microscopy of cultured cells Cell were plated in 4-well Lab-Tek chambers (#154526) and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. Samples were washed three times with 0.01% PBS–Tween and then blocked in 2% BSA–PBS for 30 min before treatment with primary antibody. Samples were then incubated with mouse anti- YAP 1:300; Santa Cruz sc-101199 in 2% BSA–PBS overnight at 4°C. After washing three times with 0.01% PBS–Tween, slides were incubated with goat anti-mouse Alexa Fluor 546 (Invitrogen #A11029, 1:1,000 in 2% BSA–PBS) for 1–2 h at room temperature. Slides were washed three times with 0.01% PBS–Tween and once with PBS and mounted with ProLong Gold Antifade Reagent (Life Technologies #P36035). Cell nuclei were visualized by DAPI staining. Images were captured using a spinning disc confocal scanner (CSU10, Yokogawa) on Leica DMI6000B microscope, and Volocity software was used for image acquisition and processing. Quantitative RT-PCR for human transcripts Total RNA was purified using PureLink RNA Mini Kit (Life Technologies). cDNA was synthesized using 1 µg of purified RNA using oligo-dT primers and M-MLV Reverse Transcriptase (Invitrogen #28025-013). Real- Time PCR was performed using the SYBR Green master mix (Applied Biosystems) on the ABI Prism 7900 HT system (Applied Biosystems). Relative gene expression was quantified by ΔΔCt method and normalized to Gapdh. See Table S2 for primer sequences. Luciferase assay For luciferase reporter assays, cells were seeded in 96-well black/clear imaging plates (BD Falcon). Firefly Luciferase reporter, CMV-Renilla Luciferase, and indicated plasmids were co-transfected into HEK293 cells. 48 hours after transfection, cells were lysed and luciferase activity was assayed using the Dual Glo Luciferase assay system (Promega) by Biotek Synergy 2 multi-mode plate reader utilizing Gen5 software. CTGF- luciferase(Lai et al., 2011) or 8xGTIIC-luciferase (Dupont et al., 2011) were used as reporters of YAP1 transcriptional activity. CTGF-luciferase was a gift from Xiaolong Yang (Lai et al., 2011). 8xGTIIC-luciferase was a gift from Stefano Piccolo, Addgene plasmid # 34615 (Dupont et al., 2011). Firefly luciferase activities were normalized to Renilla Luciferase. In vitro pull down assay Mouse Dlg5 domains were cloned into pGEX vectors, expressed and purified from BL21 cells. FLAG tagged SAV1, MST1, and MST2 or MARK3 were immunoprecipitated from HEK 293T cells with M2 affinity agarose gel (Sigma) and incubated in high salt buffer 50mM Tris, 1.15M NaCl, 0.7% Nonidet P-40 with protease and phosphatase inhibitors for 4 hours to disrupt weaker protein interactions. Salt-washed-bait-bound affinity gel was then incubated with 100ug of purified GST-PDZ fusion protein overnight. Affinity gel was washed 3 times and pull-downs were analyzed by western blot. Supplemental Figures Figure S1. Interaction between DLG5 and MST2 proteins expressed in HEK293FT cells, related to Figure 1. (A) Western blot (WB) analysis of total (input) and immunoprecipitated using Strep-Tactin-sepharose (IP-nVA) proteins with anti-V5 (V5-DLG5) and FLAG (nVA-MST2) antibodies. Cells were transfected with indicated expression constructs two days before the analysis. (B) Western blot (WB) analysis of total (input) and immunoprecipitated using anti-V5-sepharose (IP-V5) proteins with anti-V5 (V5-DLG5) and FLAG (nVA-MST2) antibodies. Cells were transfected with indicated expression constructs two days before the analysis. Figure S2. Decreased YAP/TAZ levels and phosphorylation of YAP1 in Dlg5-/- embryos in vivo, related to Figure 2. Western blot analyses of total brain, lung, and skin protein extracts from E14.5 wild type (WT) and Dlg5-/- (KO) embryos with anti-phosphoS127-YAP1 (P-Yap), total YAP1, total TAZ, and b-actin antibodies. Figure S3. Negative genetic interaction between murine Dlg5 and Hippo pathway effectors Yap1 and Taz(Wwtr1), related to Figure 3. (A) Negative genetic interaction between murine Dlg5 and Yap1. Dlg5+/- females were crossed with Dlg5+/- /Yap1+/- males and the resulting progeny were genotyped at postnatal days 7-10. Table shows the expected and observed genotypes (Chi-squared analysis). (B) Negative genetic interaction between murine Dlg5 and Taz (Wwtr1). Dlg5+/-/Taz+/- females were crossed with Dlg5+/-/Taz+/- males and the resulting progeny were genotyped at postnatal days 7-10 (Chi-squared analysis). (C) Negative genetic interaction between murine Dlg5 and Taz (Wwtr1). Dlg5+/-/Taz+/- females were crossed with Dlg5+/-/Taz-/- males and the resulting