Myc tag

DMEM, DMEM/F12, MEM and Opti-MEM media, TRIzol and Lipofectamine 2000, MitoSOX, and CellEvent Caspase-3/7 green detection kit were purchased from Invitrogen (Carlsbad, CA); Sorafenib from Enzo Life Sciences, Inc. (Farmingdale, NY), Ultra-low attachment plates and MTT from Millipore Sigma (Burlington, MA), FITC Annexin V Apoptosis Detection Kit from BD Biosciences (San Jose, CA), JC-1 dye, Doxycycline Hydrochloride, and Transwell inserts from Thermo-Fisher Scientific (Waltham MA). The antibodies utilized were obtained from the following sources: Ets-1 (Cell Signaling Technology, #14069), E-cadherin (BD Biosciences, #610181, N-cadherin (BD Biosciences, #610920), Vimentin (Cell Signaling Technology, #5741), Snail (Cell Signaling Technology, #3895), Slug (Cell Signaling Technology, #9585), Zeb2 (Cell Signaling Technology, #97885), GAPDH (Ambion, #AM4300), PARP (Cell Signaling Technology, #9542), Cleaved Caspase 3 (Cell Signaling Technology, #9664), Myc-tag (Cell Signaling Technology, #2276), GPX2 (Abcam, #137431), Cytochrome C (Cell Signaling Technology, #11940), COX IV (Abcam, #14744). RT2 profiler PCR Array Human Transcription Factors (PAHS-075Z) was from Qiagen (Germantown, MD).

Cells were washed twice with cold PBS and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, phosphatase and protease inhibitors). Sample proteins were separated by SDS-PAGE and transferred onto PVDF membranes. For protein turnover assay, cells were treated with 60 μg/mL cycloheximide (MDBio, Inc.) for the indicated times before collection. The following antibodies were used in the immunoblotting and immunoprecipitation experiments: CHD6 (1:500, Abcam, ab114095), CHD6 (1:1000, Santa Cruz, sc-393445), TMEM65 (1:500, Sigma, HPA025020), p-AKT (Ser473) (1:1000, Cell Signaling, 4060S), AKT (1:2000, Cell Signaling, 2920S), Vinculin (1:4000, Cell Signaling, 4650S), FBXW7 (1:5000, Abcam, ab109617), COX IV (1:5000, Cell Signaling, 4850), PPOX (1:2000, Santa Cruz, sc-271768), VDAC1 (1:2000, Santa Cruz, sc-390996), mtTFA (1:1000, Santa Cruz, sc-166965), p-Drp1 (Ser616) (1:800, Cell Signaling, 4494S), Drp1 (1:2000, Proteintech, 12957-1-AP), Parkin (1:1000, Proteintech, 14060-1-AP), GAPDH (1:4000, Proteintech, 60004-1-Ig), Flag-tag (1:5000, Sigma, F1804), HA-tag (1:5000, Cell Signaling, 3724S), Myc-tag (1:5000, Cell Signaling, 2276S), β-Catenin (1:4000, BD, 610153), TCF4 (1:1000, Santa Cruz, sc-166699), GSK3β (1:4000, Cell Signaling, 9832S).

The SARS-CoV-2 N gene was PCR-amplified from cDNA derived from the isolate SARS-CoV-2/human/USA/CA-CZB017/2020 (GenBank MT385497.1), nucleotides 28254 to 29513, and cloned into pCS2MT in frame with five N-terminal myc epitope tags (https://www.addgene.org/153201/). Priming site mutations were generated by site-directed mutagenesis to modify serine-188 and serine-206 to alanine based on prior observations with SARS-CoV-1 (8 (link)). Antibodies to the SARS-CoV-2 N protein were purchased from Invitrogen (#PA1-41386). Antibodies from Cell Signaling included phospho-Glycogen Synthase (#3891), phospho-S6 (#4858), β-catenin (#9562), phospho-β-catenin (#9561), GAPDH (#2118), PKCα (#2056), PKCδ (#2058), PKCε (#2683), Myc-tag (#2276), and phospho (Ser) substrate (#2261). Other antibodies included antibodies to GSK-3 (Calbiochem #368662), Tau (T14/46 antibodies provided by Virginia Lee, University of Pennsylvania, Philadelphia, PA), and β-actin (Sigma #A5441). Monoclonal anti–SARS-CoV S Protein (similar to 240C) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases, NIH (NR-616).

Most chemicals, including dexamethasone, 3-isobutyl-1-methylxanthine, insulin, saponin, cycloheximide (CHX), verteporfin, crystal violet, anti-FLAG M2 affinity gels, Dulbecco's modified Eagle medium (DMEM), and antibodies against vinculin (V4505), β-actin (A2228), α-tubulin (T5168), and FLAG-tag (F1804) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Opti-MEM, Lipofectamine 2000, Lipofectamine RNAiMAX, bovine serum albumin, goat serum, 4′,6-diamidino-2-phenylindole (DAPI), and Alexa Fluor-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against PIP5Kα (#9693), PIP5Kγ (#3296), phospho-YAP (Ser127; #4911), YAP (#4912), phospho-LATS1 (Ser909; #9157), LATS1(#9153), phospho-TAZ (Ser89; #59971), TAZ (#4883), Merlin (#6995), HA-tag (#3724), Myc-tag (#2278 and #2276), p44/42 mitogen-activated protein kinase (MAPK, #4695), phospho-p44/42 MAPK (Thr202/Tyr204; #8544), p38 MAPK (#9212), phospho-p38 MAPK (Thr180/Tyr182; #9211), c-Jun N-terminal kinase (JNK, #9258), phospho-JNK (Thr183/Tyr185; #4668), Akt (#9272), and phospho-Akt (Ser473; #9271) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against lamin B1 (sc-374015), GAPDH (sc-47724), and green fluorescent protein (GFP, sc-9996) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

All immunoblots represent at least two independent experiments. Adherent cells were washed and lysed with RIPA buffer supplemented with proteinase and phosphatase inhibitors. Proteins were separated by SDS-PAGE, transferred to Nitrocellulose membrane, and blotted with antibodies recognizing: CIC (Thermo Fisher Scientific), YAP, LATS1, LATS2, Myc-tag, total-ERK, phospho-ERK, HSP90, IgG (Cell Signaling), ETV4 (Santa Cruz).
Xenograft tumors harvesting. Subcutaneous xenografts were explanted on day 15 of treatment. Tumor explants were immediately immersed in liquid nitrogen and stored at −80 degrees. Tumors were disrupted with a mortar and pestle, followed by sonication in RIPA buffer supplemented with proteinase and phosphatase inhibitors. Proteins were separated as above. Antibodies to CIC (Thermo Fisher Scientific), YAP, and HSP90 (Cell Signaling) were used.
Quantification of immunoblots was performed using ImageJ software.

The antibodies used in these studies were mostly purchased from commercial sources. These included antibodies recognizing a Myc-tag (Cell Signalling), H3K9me3 (Active Motif), and H4K20me3 (Abcam). The VACV I3L 10D11 monoclonal antibody was from laboratory stocks. The Alexa-fluor 488 and Cy-5 conjugated secondary antibodies were purchased from Molecular Probes.