The BioID2 was amplified by PCR from the Myc-BioID2-MCS plasmid (Addgene #74223, Watertown, MA), fused with human ANO5, mouse Ano6 or mouse Mg53 cDNA, and inserted into pLVX-puro (Clontech, San Jose, CA) to obtain pLVX-hANO5-BioID2, pLVX-mAno6-BioID2, and pLVX-BioID2-mMG53, respectively. BVES-myc, POPDC2-myc and POPDC3 were constructed by inserting the corresponding cDNA into pCDNA3.1-myc backbone. ANO5 related plasmids were described previously (39). The truncation or deletion mutant BVES constructs were generated by overlapping PCR. The Ano5-targeting TALEN pairs were assembled using the Golden Gate TALEN kit (Addgene # 1000000016) as previously described (25) and the TALEN pairs were coupled with 2A peptide (40). The protein sequences of Ano5-TALENs are provided in the Supplementary Fig. S6. pCMV-AncBE4max plasmid was obtained from Addgene (#112094). The pCMV-AncBE4-GFP was generated by ligation of SacI-EcoR1 fragment from pCMV-AncBE4max and the EcoRI/AgeI-digested GFP fragment into SacI/AgeI linearized pCMV-AncBE4max backbone. The annealed gRNA oligos (targeting mouse Bves) were cloned into pLenti-OgRNA-Zeo plasmid as previously described (41, 42). All plasmids used in this study are listed in Supplementary Table S2.
Cell culture and transfection
HEK293 and COS-1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, GIBCO) containing 1 g/l glucose and supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma) and 1% (v/v) penicillin–streptomycin (Pen-Strep, Sigma-Aldrich). C2C12 and human myoblasts were grown in DMEM with 20% FBS and differentiated in DMEM with 2% horse serum after 70-80% confluency. All cells were cultured in a 37 oC incubator with a humidified 5% CO2 atmosphere. Transfection of HEK293 or COS-1 cells were performed using X-tremeGENETM HP DNA transfection reagent (#6366244001, Sigma-Aldrich, St. Louis, MO). For C2C12 and human myoblasts, plasmids were transfected with the Neon electroporation system (Thermo Scientific, NY). For confocal imaging, the aforementioned cells were plated onto collagen-coated 35-mm glass-bottom dishes post transfection and grown for 24-48 h.
Generation of Bves-KO and Ano5-KO C2C12 cells
To generate Ano5-KO C2C12 cells, the C2C12 cells were electroporated with pTAL9-Ano5E5A and individual cell clones were screened by PCR and T7E1 assay. To generate Bves-KO C2C12 cell line, the C2C12 cells were sorted for GFP into single cells in a 96-well plate after electroporation with the pCMV-AncBE4-GFP and pLenti-Bves-gRNA-Zeo. The expanded individual cell clones were screened by PCR and Sanger sequencing.
For BioID2 pull down, C2C12 myotubes stably expressing hANO5-BioID2, mAno6-BioID2 or BioID2-mMG53 were incubated with 50 µM biotin for 16 h. After washing the cells with PBS twice very gently, the cells were lysed in 2.4 ml lysis buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 0.4% SDS, 1 mM dithiothreitol, and 1x complete protease inhibitor). Triton X-100 was added to 2% final concentration. After sonication, an equal volume of 50 mM Tris (pH 7.4) was added. After centrifugation at 16,500x g for 10 min, the supernatant was collected to a 15-mL tube and incubated with 300 ul magnetic streptavidin beads (#88816, Thermo Scientific, NY) overnight at 4°C. Beads were washed according to the following steps: twice with 2% SDS, once with wash buffer containing 0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM HEPES, pH 7.5, once with wash buffer containing 250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8, and once with 50 mM Tris pH 8. Ten percent of the samples were saved for Western blot analysis. The other 90% of the samples were subjected to mass spectrometry analysis at the Ohio State University Comprehensive Cancer Center Proteomic Shared Resources.
Cell proliferation assay
The cell proliferation was examined by the CCK-8 cell counting kit (Dojindo Molecular Technologies, MD, USA). WT, BVES-KO or Ano5-KO C2C12 cells were prepared in 96-well plates at the initial density of 5 x 103 cells/well. The 450nm absorbance was measured at 12-84 hrs according to the manufacturer’s instructions.
The cells were lysed with cold RIPA buffer supplemented with protease inhibitors, and the extracted proteins were quantified by DCTM Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA). The extracted protein samples were separated by stain-free SDS-PAGE gels (Bio-Rad Laboratories, 4-15%) and transferred onto Nitrocellulose Membranes (0.45 µm). Primary antibodies include the rabbit polyclonal anti-BVES (1:1000, HPA014788, Sigma-Aldrich, St. Louis, MO), mouse monoclonal anti-Ano5 (1:1000, N421A/85, UC Davis/NIH NeuroMab Facility, Davis, CA), anti-GAPDH (1:4000, MAB374, Cell Signaling Technology, Danvers, MA), anti-GFP antibody (1:1000, A01388, Genscript, Piscataway, NJ), streptavidin-HRP (1:20, DY998, R&D Systems, Minneapolis, MN), myc (1:1000, #2276, Cell Signaling Technology, Danvers, MA), FLAG (1:1000, #F3165, Sigma-Aldrich, St. Louis, MO) and HA (1:1000, #3724, Cell Signaling Technology, Danvers, MA). Secondary HRP-conjugated goat anti-mouse (1:4000), goat anti-rabbit (1:4000) antibodies were obtained from Cell Signaling Technology. The membranes were developed using ECL western blotting substrate (Pierce Biotechnology, Rockford, IL) and images were scanned with the ChemiDoc XRS+ system (Bio-Rad Laboratories). Western blots were quantified using Image Lab 6.0.1 software (Bio-Rad Laboratories) according to the manufacturer’s instructions.
Cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the slides were blocked with 5% BSA with 0.3% Triton X-100 for 1 h. The slides were incubated with primary antibodies at 4°C overnight. The primary antibodies include myc (1:200, #2276, Cell Signaling Technology, Danvers, MA), calnexin (1:200, #2679, Cell Signaling Technology, Danvers, MA), and myosin heavy chain (1:200, MF20, Developmental Studies Hybridoma Bank, Iowa City, IA). The cells were then washed extensively with PBS and incubated with Alexa Fluor 568 (donkey anti-rabbit IgG, Invitrogen) or Alexa Fluor 488 (goat anti-rabbit IgG, Invitrogen) for 1 h at room temperature. The stained cells were sealed with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratory, Burlingame, CA). ER-tracker red (E34250, Thermo Scientific) was used for labeling endoplasmic reticulum in COS-1 cells after 24h transfection with GFP-Ano5. All images were taken with a Zeiss 780 confocal microscope (Jena, Germany).
Cells were transfected with the indicated plasmids and lysed in RIPA lysis buffer [25 mM Tris-HCl (pH 7.4), 150mM NaCl, 5% Glycerol, 1% Triton X-100, 2 mM EDTA, and 1 mM DTT supplemented with protease inhibitor cocktail (Roche)] at 48 h after transfection. Immunoprecipitation was performed by incubation with the indicated primary antibodies for 4 h and protein A/G agarose beads (#20423, Thermo Scientific) overnight at 4°C. The beads were washed at least three times with RIPA lysis buffer. Lysates and immunoprecipitates were examined by using the indicated primary antibodies followed by the related secondary antibodies and the SuperSignal Chemiluminescence Kit (Thermo Fisher Scientific, Waltham, MA, USA).
RNA extraction and quantitative RT-PCR analysis
Total RNA was extracted from C2C12 myoblasts or myotubes with Trizol. First-strand cDNA was synthesized using RevertAid RT Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). Real-time PCR was performed using PerfeCTa SYBR Green FastMix (QuantaBio, USA) in CFX384 Real-time PCR Detection Systems (Bio-Rad). Samples were normalized for expression of GAPDH and analyzed by the 2−ΔΔCt method.
All in vitro experimental data were repeated a minimum of three times. Data are expressed as mean ± the standard error of the mean (S.E.M.). Statistical differences were determined by two-tailed, unpaired Student’s t test for two groups or one-way ANOVA with Turkey’s post tests for multiple group comparisons using Prism 8 (Graphpad Software, La Jolla, California). A P value < 0.05 was considered to be significant.