protocols in WDR81 paper v1
Authors: Li Yang 
Published: January 2021

Experimental Procedures Cell culture, reagents, and transfection Cells were cultured at 37°C with 5% CO2in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, Novato, CA), 100 U/ml penicillin and 100 mg/ml streptomycin. Transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturers’ instructions. MG132 and doxycycline were obtained commercially. Fibroblasts from control individuals and HD patients with the Htt47Q and Htt68Q expansions were obtained from Dr. Boxun Lu’s lab in Fudan University. In brief, primary cortex neurons were isolated from appropriately timed pregnant female (16.5 days). Using forceps, we isolated cortex from each hemisphere of mouse brains under microscope. Once the cortex regions were isolated, they were cut into approximately 10 smaller pieces for digestion (1 ml prewarmed Trypsin, at 37℃ for 20 min). Then serum was added to inactivate trypsin. After aspirating the supernatant, we added 1 ml prewarmed DNase solution for 10 min at 37℃. Then, the cells were dissociated by pipetting up and down, and all supernatant were transferred to new tubes for subsequent centrifugation (800xg for 5 min). We resuspended cell pellet gently and plated them on coated dishes (Poly-D-Lysine, BD, #354210). The neuronal culture medium was changed every 2 days. After about 14 days of neuronal culture, we performed transient transfection of Htt97Q-EGFP (1 mg) or/and siWDR81 (100 pmol, three times at an interval of 24 h) using Lipofectamine MessengerMAX (Thermo Fisher Scientific) as manufacturing introduction. Immunostaining and confocal microscopy Cells grown on coverslips were fixed in 4% paraformaldehyde followed by permeabilization with 0.05% saponin. After extensive washing with phosphate buffered saline (PBS), cells were incubated with primary antibodies in PBS containing 5% BSA at 4ºC overnight. Cells were washed extensively again and incubated with secondary antibodies for 1 h at room temperature. Following another round of thorough washing, cells were sealed on slides for microscopy analysis. For live cell imaging, cells were grown in confocal dishes (Glass Bottom Dish, In Vitro Scientific, Sunnyvale, California, USA). Samples were examined with an inverted Olympus FV1000 confocal microscope. Images were analyzed with FV10-ASW 4.0a Viewer. Generation of cell lines stably expressing Tet-on Htt97Q-GFP HeLa cells stably expressing pcDNA6/TR (kindly provided by Dr. Quan Chen) were transfected with pcDNA4-Htt97Q-GFP. 24 h later, cells were subjected to selection for 2-3 weeks in medium supplemented with blasticidin (5 mg/ml) and zeocin (100 mg/ml). Single colonies were picked and propagated further. The expression of Htt97Q-GFP was determined by adding doxycycline (1 mg/ml) and observed by fluorescence microscopy. Htt polyQ clearance assay To assess the effect of siRNA knockdown of WDR81 on clearance of Htt polyQ inclusions, HeLa cells stably expressing Tet-on Htt97Q-EGFP were transfected twice at an interval of 24 h with siRNA oligos against WDR81. 12 h afterthe second siRNA transfection, Htt97Q-EGFP expression was induced with doxycycline (1 mg/ml). The medium was changed 12 h later to remove the doxycycline and the cells were split into several dishes for the remaining siRNA treatments. Time-course quantification of polyQ foci was performed 12 h after the induction of Htt97Q-EGFP expression. To rescue WDR81 siRNA-induced accumulation of polyQ foci, cells were first treated with control or WDR81 siRNA, then 3 mg of vector expressing siRNA-resistant WDR81 were transfected into the same cells simultaneous with the induction of Htt97Q-EGFP expression by doxycycline. siRNA treatments were performed twice more as above and polyQ foci were scored 48 h post induction of Htt97Q-EGFP expression. C. elegans genetics C. elegans Bristol strain N2 was used as wild type.sorf-2(tm5210) deletion mutants were provided by Dr. Shohei Mitani (Tokyo Women’s Medical University, Japan). The integrated arraysbpIs267and baIn1 were kindly provided by Dr. Hong Zhang (Institute of Biophysics, CAS, China) and Dr. Guy Caldwell (University of Alabama, Tuscaloosa). C. elegans cultures, genetic crosses, and generation of transgenic animals were performed according to standard procedures (Brenner 1974). Human subjects and IHC procedure Brain samples from 12 control individuals and 12 patients with indicated neurodegenerative diseases (AD, n=4; HD, n=3; PD, n=5) were obtained from theChinese Brain Bank of Zhejiang University (Table 1). The samples are paraffin-embedded sections of hippocampus and cortex of frontal lobe. The controls and patients were strictly paired according to parameters including age, gender, reason of death and phase of disease. All procedures were performed according to protocols approved by Human Ethics Committees at both Fudan University and Zhejiang University. Paraffin-embedded sections were dewaxed, rehydrated, and rinsed in PBS. After being boiled for 15 min in 0.01 mol/liter sodium citrate buffer (pH 6.0), sections were blocked in 5% guinea pig (Gp) or rabbitpre-immune serum in PBS for 1 h at room temperature and then incubated overnight with WDR81 antibody (Gp, 1:200) or p62 antibody (rabbit, 1:1000). Sections were then incubated in horseradish peroxidase-conjugated secondary antibody (1:200) at room temperature for 3 hours. After 4 washes in PBS, 3,3-diaminobenzidine (DAB) was added for 1 min at room temperature. Sections were counterstained with hematoxylin for 2 min. The IHC pictures were obtained by Nikon microscope (Ts2R). Results were calculated by software Image J and NIS-Element, BR. 3.00 (Nikon). For quantification measurements, images were randomly acquired from the hippocampus and cortex of frontal lobe and analyzed by researchers in a double-blind manner. Expression vectors The mammalian, bacterial and C. elegans expression vectors listed below were constructed using standard protocols. Expression vectorsGene(cDNA)Insertion siteBackbonepmCherry-c1-WDR81WDR81XhoI/BamHIpmCherry-c1pEGFP-c1-WDR81WDR81XhoI/XbaIpEGFP-c1pEGFP-c1-WDR81(1-650)WDR81 XhoI/BamHIpEGFP-c1pEGFP-c1-WDR81(730-1440)WDR81(730-1440)XhoI/BamHIpEGFP-c1pEGFP-c1-WDR81(1637-1940)WDR81(1637-1940)XhoI/BamHIpEGFP-c1pcDNA4-Htt97Q-GFPHtt97Q-GFPEcoRI/XhoIpcDNA4-TO-myc-HisPY37A1B.5Myc-sorf-2sorf-2ORF with MycXmaIpPD49.26PY37A1B.5WDR81WDR81KpnI/EcoRVpPD49.26Pdat-1sorf-2dat-1 promoter andsorf-2ORFPstI/XmaI for Pdat-1XmaI /NcoI forsorf-2pPD95.77Pdat-1WDR81dat-1 promoterSphI/BamHIPY37A1B.5WDR81pmCherry-c1-ATG5Atg5XhoI/BamHIpmCherry-c1pmCherry-c1-ATG12Atg12XhoI/BamHIpmCherry-c1 The following vector were kindly provided by other scientists: pcDNA3.1-Htt97Q-EGFP (Dr. Xiaojiang Li, Institute of Genetics and Developmental Biology, CAS, China). Small interfering RNAs (siRNAs) The oligos used for siRNA knockdown of WDR81 were as described previously [14]. Other oligos were as follows: p62: 5’-GCATTGAAGTTGATATCGAT-3’[37]; ATG12: 5’-AUGAGCUUCAAUUGCAUCCtt-3’ [38]; Control siRNA: 5’-UUCUCCGAACGUGUCACGUTT-3’. Cells were transfected with 100 pmol siRNA oligos twice at an interval of 24 h. Cells were subjected to further analysis 24 h after the last transfection. For the transfection of primary neurons, we performed the assay using Lipofectamine MessengerMax reagent (Invitrogen). Quantitative reverse transcription–polymerase chain reaction (qPCR) Total RNA was extracted using Trizol (Invitrogen) and chloroform. 2 μg of RNA was used as template to generate cDNAs using the ImProm-II Reverse Transcription system (Promega, Madison, Wisconsin, USA). qPCR reactions were carried out on an MX3000P system (Agilent Technologies, Santa Clara, CA). Antibodies WDR81 antibodies were generated in guinea pigs and rabbits by injecting purified recombinant GST-WDR81(332-604) and purchased from ABclonal (#A12780). GFP and mCherry antibodies were generated in guinea pigs or rabbits by injecting recombinant proteins. Rabbit polyclonal antibodiesto p62 werepurchased from Medical & Biological Laboratories (MBL, Nagoya, Japan). Mouse monoclonal antibodies against ATG12 werepurchased from MBL. Mouse monoclonal antibodies to b-actin and GFP were purchased from Sigma-Aldrich (St. Louis, MO). HRP-, Cy3-, and FITC-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Western blotting and immunoprecipitation To analyze levels of proteins of interest, cells were lysed in ice-cold Triton X-100 buffer (20 mM Tris-HCl, pH7.5, 100 mM NaCl, 1% TritonX-100, 1 mM phenylmethanesulfonyl fluoride (PMSF)) or RIPA buffer (20 mM Tris-HCl pH7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate, 1 mM PMSF) containing one Complete Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland). Cell lysates were spun down at 12000 rpm for 10 min. 50 g of supernatants were resolved onsodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and blotted with the indicated antibodies. b-actin was used as the loading control. To determine the interaction of endogenous WDR81 with autophagy factors or Htt polyQ-associated proteins, HeLa cells with or without Htt97Q-EGFP expression were lysed in ice-cold Triton X-100 buffer containing one Complete Protease Inhibitor Cocktail Tablet (Roche). In the case of Htt97Q-EGFP IP, the cell lysates were cleared by centrifugation at 500xg as described by Filimonenko et al. [12]. Cleared cell lysates were first mixed with antibodies to WDR81 or GFP (~5 mg) for 4 h followed by incubation with protein A agarose beads (10 ml) (GE Healthcare) overnight at 4°C. Analysis of precipitated proteins was performed as above. MTT assay for cell viability The MTT kit was purchased from Promega (#G4002). Cells were cultured in 96-well plates with DMEM containing 10% FBS. After various treatments, 15 mL of dye solution was added into each well. Then the plates were incubated at 37℃for 1.5 hours in a humidified CO2 incubator. Stop solution (100 mL) was added into each well, and the absorbance was recorded at 570 nm using plate reader. 630 nm was used as a reference wavelength. Transmission electron microscopy Cellswere fixed for 2.5h at 4°C with 0.1 M PBS containing 2.5% glutaraldehyde. Then, the fixed samples were rinsed with PBS and 1% OsO4 for 0.5 h at 4°C. The samples were rinsed with distilled water and dehydrated by sequential incubation with an acetone series (30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100%, 5 min each). After that, samples were infiltrated with Araldite 502/Embed 812 by gradually increasing the concentration of acetone (25% and 50%, 20 min; 75%, 30 min; 100%, 20 h) and then polymerized at 60°C for 70h. Embedded samples were sectioned using an UC6 ultramicrotome (Leica Biosystems) equipped with a 45° diamond knife (Diatome) to obtain 70-nm ultrathin sections. The grids were stained at room temperaturewith 2% aqueous uranyl acetate (10 min) and Reynolds lead citrate (5 min) before imaging. Imaging was performed at 80 kV on a JEM-1400 (JEOL) transmission electron microscope. Statistics and reproducibility Data were analyzed with Prism (GraphPad software). Statistical analyses were performed using t-tests or ANOVA. *P<0.05 was considered statistically significant. **P<0.01 was considered significant. ***P<0.001 was considered extremely significant. P>0.05 was considered not significant (NS). Experimental Procedures Cell culture, reagents, and transfection Cells were cultured at 37°C with 5% CO2in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, Novato, CA), 100 U/ml penicillin and 100 mg/ml streptomycin. Transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturers’ instructions. MG132 and doxycycline were obtained commercially. Fibroblasts from control individuals and HD patients with the Htt47Q and Htt68Q expansions were obtained from Dr. Boxun Lu’s lab in Fudan University. In brief, primary cortex neurons were isolated from appropriately timed pregnant female (16.5 days). Using forceps, we isolated cortex from each hemisphere of mouse brains under microscope. Once the cortex regions were isolated, they were cut into approximately 10 smaller pieces for digestion (1 ml prewarmed Trypsin, at 37℃ for 20 min). Then serum was added to inactivate trypsin. After aspirating the supernatant, we added 1 ml prewarmed DNase solution for 10 min at 37℃. Then, the cells were dissociated by pipetting up and down, and all supernatant were transferred to new tubes for subsequent centrifugation (800xg for 5 min). We resuspended cell pellet gently and plated them on coated dishes (Poly-D-Lysine, BD, #354210). The neuronal culture medium was changed every 2 days. After about 14 days of neuronal culture, we performed transient transfection of Htt97Q-EGFP (1 mg) or/and siWDR81 (100 pmol, three times at an interval of 24 h) using Lipofectamine MessengerMAX (Thermo Fisher Scientific) as manufacturing introduction. Immunostaining and confocal microscopy Cells grown on coverslips were fixed in 4% paraformaldehyde followed by permeabilization with 0.05% saponin. After extensive washing with phosphate buffered saline (PBS), cells were incubated with primary antibodies in PBS containing 5% BSA at 4ºC overnight. Cells were washed extensively again and incubated with secondary antibodies for 1 h at room temperature. Following another round of thorough washing, cells were sealed on slides for microscopy analysis. For live cell imaging, cells were grown in confocal dishes (Glass Bottom Dish, In Vitro Scientific, Sunnyvale, California, USA). Samples were examined with an inverted Olympus FV1000 confocal microscope. Images were analyzed with FV10-ASW 4.0a Viewer. Generation of cell lines stably expressing Tet-on Htt97Q-GFP HeLa cells stably expressing pcDNA6/TR (kindly provided by Dr. Quan Chen) were transfected with pcDNA4-Htt97Q-GFP. 24 h later, cells were subjected to selection for 2-3 weeks in medium supplemented with blasticidin (5 mg/ml) and zeocin (100 mg/ml). Single colonies were picked and propagated further. The expression of Htt97Q-GFP was determined by adding doxycycline (1 mg/ml) and observed by fluorescence microscopy. Htt polyQ clearance assay To assess the effect of siRNA knockdown of WDR81 on clearance of Htt polyQ inclusions, HeLa cells stably expressing Tet-on Htt97Q-EGFP were transfected twice at an interval of 24 h with siRNA oligos against WDR81. 12 h afterthe second siRNA transfection, Htt97Q-EGFP expression was induced with doxycycline (1 mg/ml). The medium was changed 12 h later to remove the doxycycline and the cells were split into several dishes for the remaining siRNA treatments. Time-course quantification of polyQ foci was performed 12 h after the induction of Htt97Q-EGFP expression. To rescue WDR81 siRNA-induced accumulation of polyQ foci, cells were first treated with control or WDR81 siRNA, then 3 mg of vector expressing siRNA-resistant WDR81 were transfected into the same cells simultaneous with the induction of Htt97Q-EGFP expression by doxycycline. siRNA treatments were performed twice more as above and polyQ foci were scored 48 h post induction of Htt97Q-EGFP expression. C. elegans genetics C. elegans Bristol strain N2 was used as wild type.sorf-2(tm5210) deletion mutants were provided by Dr. Shohei Mitani (Tokyo Women’s Medical University, Japan). The integrated arraysbpIs267and baIn1 were kindly provided by Dr. Hong Zhang (Institute of Biophysics, CAS, China) and Dr. Guy Caldwell (University of Alabama, Tuscaloosa). C. elegans cultures, genetic crosses, and generation of transgenic animals were performed according to standard procedures (Brenner 1974). Human subjects and IHC procedure Brain samples from 12 control individuals and 12 patients with indicated neurodegenerative diseases (AD, n=4; HD, n=3; PD, n=5) were obtained from theChinese Brain Bank of Zhejiang University (Table 1). The samples are paraffin-embedded sections of hippocampus and cortex of frontal lobe. The controls and patients were strictly paired according to parameters including age, gender, reason of death and phase of disease. All procedures were performed according to protocols approved by Human Ethics Committees at both Fudan University and Zhejiang University. Paraffin-embedded sections were dewaxed, rehydrated, and rinsed in PBS. After being boiled for 15 min in 0.01 mol/liter sodium citrate buffer (pH 6.0), sections were blocked in 5% guinea pig (Gp) or rabbitpre-immune serum in PBS for 1 h at room temperature and then incubated overnight with WDR81 antibody (Gp, 1:200) or p62 antibody (rabbit, 1:1000). Sections were then incubated in horseradish peroxidase-conjugated secondary antibody (1:200) at room temperature for 3 hours. After 4 washes in PBS, 3,3-diaminobenzidine (DAB) was added for 1 min at room temperature. Sections were counterstained with hematoxylin for 2 min. The IHC pictures were obtained by Nikon microscope (Ts2R). Results were calculated by software Image J and NIS-Element, BR. 3.00 (Nikon). For quantification measurements, images were randomly acquired from the hippocampus and cortex of frontal lobe and analyzed by researchers in a double-blind manner. Expression vectors The mammalian, bacterial and C. elegans expression vectors listed below were constructed using standard protocols. Expression vectorsGene(cDNA)Insertion siteBackbonepmCherry-c1-WDR81WDR81XhoI/BamHIpmCherry-c1pEGFP-c1-WDR81WDR81XhoI/XbaIpEGFP-c1pEGFP-c1-WDR81(1-650)WDR81 XhoI/BamHIpEGFP-c1pEGFP-c1-WDR81(730-1440)WDR81(730-1440)XhoI/BamHIpEGFP-c1pEGFP-c1-WDR81(1637-1940)WDR81(1637-1940)XhoI/BamHIpEGFP-c1pcDNA4-Htt97Q-GFPHtt97Q-GFPEcoRI/XhoIpcDNA4-TO-myc-HisPY37A1B.5Myc-sorf-2sorf-2ORF with MycXmaIpPD49.26PY37A1B.5WDR81WDR81KpnI/EcoRVpPD49.26Pdat-1sorf-2dat-1 promoter andsorf-2ORFPstI/XmaI for Pdat-1XmaI /NcoI forsorf-2pPD95.77Pdat-1WDR81dat-1 promoterSphI/BamHIPY37A1B.5WDR81pmCherry-c1-ATG5Atg5XhoI/BamHIpmCherry-c1pmCherry-c1-ATG12Atg12XhoI/BamHIpmCherry-c1 The following vector were kindly provided by other scientists: pcDNA3.1-Htt97Q-EGFP (Dr. Xiaojiang Li, Institute of Genetics and Developmental Biology, CAS, China). Small interfering RNAs (siRNAs) The oligos used for siRNA knockdown of WDR81 were as described previously [14]. Other oligos were as follows: p62: 5’-GCATTGAAGTTGATATCGAT-3’[37]; ATG12: 5’-AUGAGCUUCAAUUGCAUCCtt-3’ [38]; Control siRNA: 5’-UUCUCCGAACGUGUCACGUTT-3’. Cells were transfected with 100 pmol siRNA oligos twice at an interval of 24 h. Cells were subjected to further analysis 24 h after the last transfection. For the transfection of primary neurons, we performed the assay using Lipofectamine MessengerMax reagent (Invitrogen). Quantitative reverse transcription–polymerase chain reaction (qPCR) Total RNA was extracted using Trizol (Invitrogen) and chloroform. 2 μg of RNA was used as template to generate cDNAs using the ImProm-II Reverse Transcription system (Promega, Madison, Wisconsin, USA). qPCR reactions were carried out on an MX3000P system (Agilent Technologies, Santa Clara, CA). Antibodies WDR81 antibodies were generated in guinea pigs and rabbits by injecting purified recombinant GST-WDR81(332-604) and purchased from ABclonal (#A12780). GFP and mCherry antibodies were generated in guinea pigs or rabbits by injecting recombinant proteins. Rabbit polyclonal antibodiesto p62 werepurchased from Medical & Biological Laboratories (MBL, Nagoya, Japan). Mouse monoclonal antibodies against ATG12 werepurchased from MBL. Mouse monoclonal antibodies to b-actin and GFP were purchased from Sigma-Aldrich (St. Louis, MO). HRP-, Cy3-, and FITC-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Western blotting and immunoprecipitation To analyze levels of proteins of interest, cells were lysed in ice-cold Triton X-100 buffer (20 mM Tris-HCl, pH7.5, 100 mM NaCl, 1% TritonX-100, 1 mM phenylmethanesulfonyl fluoride (PMSF)) or RIPA buffer (20 mM Tris-HCl pH7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate, 1 mM PMSF) containing one Complete Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland). Cell lysates were spun down at 12000 rpm for 10 min. 50 g of supernatants were resolved onsodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and blotted with the indicated antibodies. b-actin was used as the loading control. To determine the interaction of endogenous WDR81 with autophagy factors or Htt polyQ-associated proteins, HeLa cells with or without Htt97Q-EGFP expression were lysed in ice-cold Triton X-100 buffer containing one Complete Protease Inhibitor Cocktail Tablet (Roche). In the case of Htt97Q-EGFP IP, the cell lysates were cleared by centrifugation at 500xg as described by Filimonenko et al. [12]. Cleared cell lysates were first mixed with antibodies to WDR81 or GFP (~5 mg) for 4 h followed by incubation with protein A agarose beads (10 ml) (GE Healthcare) overnight at 4°C. Analysis of precipitated proteins was performed as above. MTT assay for cell viability The MTT kit was purchased from Promega (#G4002). Cells were cultured in 96-well plates with DMEM containing 10% FBS. After various treatments, 15 mL of dye solution was added into each well. Then the plates were incubated at 37℃for 1.5 hours in a humidified CO2 incubator. Stop solution (100 mL) was added into each well, and the absorbance was recorded at 570 nm using plate reader. 630 nm was used as a reference wavelength. Transmission electron microscopy Cellswere fixed for 2.5h at 4°C with 0.1 M PBS containing 2.5% glutaraldehyde. Then, the fixed samples were rinsed with PBS and 1% OsO4 for 0.5 h at 4°C. The samples were rinsed with distilled water and dehydrated by sequential incubation with an acetone series (30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100%, 5 min each). After that, samples were infiltrated with Araldite 502/Embed 812 by gradually increasing the concentration of acetone (25% and 50%, 20 min; 75%, 30 min; 100%, 20 h) and then polymerized at 60°C for 70h. Embedded samples were sectioned using an UC6 ultramicrotome (Leica Biosystems) equipped with a 45° diamond knife (Diatome) to obtain 70-nm ultrathin sections. The grids were stained at room temperaturewith 2% aqueous uranyl acetate (10 min) and Reynolds lead citrate (5 min) before imaging. Imaging was performed at 80 kV on a JEM-1400 (JEOL) transmission electron microscope. Statistics and reproducibility Data were analyzed with Prism (GraphPad software). Statistical analyses were performed using t-tests or ANOVA. *P<0.05 was considered statistically significant. **P<0.01 was considered significant. ***P<0.001 was considered extremely significant. P>0.05 was considered not significant (NS).