KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
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| Antibodies | ||
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| anti-G3BP1 | Proteintech | Cat#13057-2-AP; RRID:AB_2232034 |
| anti-GFP | Thermo Fisher Scientific | Cat#33-2600; RRID:AB_2533111 |
| anti-G3BP1 | BD Biosciences | Cat#611126; RRID:AB_398437 |
| anti-mCherry | Novus biologicals | Cat#NBP1-96752; RRID:AB_11034849 |
| anti-m6A | Synaptic Systems (SySy) | Cat#202 003; RRID:AB_2279214 |
| anti-GFP | Millipore | Cat#MAB3580; RRID:AB_94936 |
| anti-ALKBH5 | Proteintech | Cat#16837-1-AP; RRID:AB_2242665 |
| anti-YTHDF1 | Proteintech | Cat#17479-1-AP; RRID:AB_2217473 |
| anti-YTHDF3 | Proteintech | Cat#25537-1-AP; RRID:AB_2847817 |
| anti-V5 | Thermo Fisher Scientific | Cat#R960-25; RRID:AB_2556564 |
| anti-Flag | Sigma | Cat#F1804; RRID:AB_262044 |
| anti-GAPDH | Meridian Life Science | Cat#H86504M; RRID:AB_151542 |
| anti-eIF3η | Santa Cruz | Cat#sc-137214; RRID:AB_2277705 |
| anti-Ataxin 2 | BD Biosciences | Cat#611378; RRID:AB_398900 |
| anti-TIA-1 | Abcam | Cat#ab40693; RRID:AB_2201438 |
| anti-GR | Millipore | Cat#MABN778; RRID:AB_2728664 |
| anti-eIF3η | Santa Cruz | Cat#sc-16377; RRID:AB_671941 |
| Anti-GA | Millipore | Cat#MABN889; RRID:AB_272866 |
| anti-GR (Rb7810) | Gendron, T.F., et al.31 | N/A |
| BIOTIN-Anti-C9RANTV2. 9259 Purified antibody | Zhang, Y.J., et al.4 | N/A |
| Sulfo-Affinity Purified 7810 (Anti-GR) | Zhang, Y.J., et al.4 | N/A |
| Peroxidase AffiniPure F(ab’)2 Fragment Donkey Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | Cat#715-036-150; RRID:AB_2340773 |
| Peroxidase AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG (H+L) |
Jackson ImmunoResearch | Cat#711-036-152; RRID:AB_2340590 |
| AlexaFluor 488 (Donkey anti-Rabbit IgG (H+L)) | Invitrogen | Cat#A32790; RRID:AB_2762833 |
| AlexaFluor 488 (Donkey anti-Rat IgG (H+L)) | Invitrogen | Cat#A48269; RRID:AB_2893137 |
| AlexaFluor 568 (Donkey anti-Mouse IgG (H+L)) | Invitrogen | Cat#A10037; RRID:AB_2534013 |
| AlexaFluor 568 (Goat anti-Rabbit IgG (H+L)) | Invitrogen | Cat#A11034; RRID:AB_2576217 |
| AlexaFluor 647 (Donkey anti-Mouse IgG (H+L)) | Invitrogen | Cat#A31571; RRID:AB_162542 |
| AlexaFluor 647 (Donkey anti-Rabbit IgG (H+L)) | Invitrogen | Cat#A31573; RRID:AB_2536183 |
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| Bacterial and virus strains | ||
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| RosettaTM(DE3)pLysS competent cells | Millipore | Cat#70956 |
| rAAV9-(GA)100-V5 | Shao W., et al.32 | N/A |
| rAAV9-GFP-(GR)200 | Cook, C.N., et al.9 | N/A |
| rAAV9-(G4C2)2 | Chew, J., et al.20 | N/A |
| rAAV9-(G4C2)149 | Chew, J., et al.20 | N/A |
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| Biological samples | ||
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| Postmortem mid-frontal cortex tissues from c9FTD patients | Mayo clinic Florida Brain Bank | Table S1 |
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| Chemicals, peptides, and recombinant proteins | ||
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| DNase | Sigma-Aldrich | Cat#11284932001 |
| RNase | Sigma-Aldrich | Cat#R6748 |
| Protease inhibitor | Roche | Cat#A32965 |
| Glutathione Sepharose 4B resin | GE Health | Cat#17-0756-01 |
| PreScission Protease | Cytiva | Cat#27084301 |
| TRIzol | Thermo Fisher Scientific | Cat#15596026 |
| RNeasy Plus Mini Kit | Qiagen | Cat#74034 |
| SecureSeal Imaging Spacers | Grace Bio-laboratories | Cat#470352 |
| Opti-MEM | Thermo Fisher Scientific | Cat#51985034 |
| Lipofectamine 2000 | Thermo Fisher Scientific | Cat#11668500 |
| FuGENE HD | Promega | Cat#E2311 |
| Lipofectamine RNAimax | Thermo Fisher Scientific | Cat#13778500 |
| Sodium Deoxycholate | Sigma-Aldrich | Cat#D6750-100G |
| Benzonase | Sigma-Aldrich | Cat#E8263 |
| Polyethylenimine | Polyscience, Inc. | Cat#23966 |
| Isopropyl β-d-1-thiogalactopyranoside (IPTG) | Thermo Fisher Scientific | Cat#BP1755 |
| Triton X-100 | Sigma-Aldrich | Cat#T9284-1L |
| Phosphate Buffered Saline | Thermo Fisher Scientific, | Cat#BP3994 |
| Dulbecco’s Phosphate Buffered Saline | Thermo Fisher Scientific, | Cat#14190144 |
| Dithiothreitol (DTT) | Genesee | Cat#18-203 |
| Coomassie Brilliant Blue R-250 | Bio-Rad | Cat#1610400 |
| Direct-zol RNA microprep kit | Zymo Research | Cat#R2062 |
| Dynabead mRNA purification kit | Thermo Fisher Scientific | Cat#61006 |
| G418 Sulfate | Gemini Bioproducts | Cat#400-111P |
| Protease inhibitor tablets | Roche | Cat#A32965 |
| RNase inhibitor | Invitrogen | Cat#AM2696 |
| Wash buffer A for sequential staining | Biosearch Technologies | Cat#SMF-WA1-60 |
| Hybridization buffer for sequential staining | Biosearch Technologies | Cat#SMF-HB1-10 |
| Cy5-Oligo d(T)20 | Genelink | Cat#26-4420-02 |
| Wash buffer B for sequential staining | Biosearch Technologies | Cat#SMF-WB1-20 |
| Dako Peroxidase Block | DAKO | Cat#S2001 |
| Dako Protein Block Serum-Free | DAKO | Cat#X0909 |
| Dako Envision-Plus anti-rabbit | DAKO | Cat#K4003 |
| Liquid DAB+ substrate Chromogen System | DAKO | Cat#K3468 |
| Gill’s Hematoxylin | Statlab (SLMP LLC) | Cat#HXGHE1LT |
| (GR)20 peptide | Peptide 2.0 Inc | N/A |
| G3BP1 FL | This study | N/A |
| G3BP1 ΔIDR1 | This study | N/A |
| G3BP1ΔIDR3 | This study | N/A |
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| Critical commercial assays | ||
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| Duolink In Situ PLA® Probe Anti-Rabbit PLUS | Sigma-Aldrich | Cat#DUO92002 |
| Duolink In Situ PLA® Probe Anti-Mouse MINUS | Sigma-Aldrich | Cat#DUO92004 |
| Duolink In Situ Detection Reagents FarRed | Sigma-Aldrich | Cat#DUO92013 |
| Duolink In Situ Wash Buffers, Fluorescence | Sigma-Aldrich | Cat#DUO82049 |
| EpiQuik m6A RNA methylation quantification Kit | EpigenTek | Cat#P-9005 |
| Coomassie Plus (Bradford) Assay Kit | Thermo Fisher Scientific | Cat#23236 |
| Pierce™ BCA Protein Assay Kit | Thermo Fisher Scientific | Cat#23227 |
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| Experimental models: Cell lines | ||
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| HEK293T cell line | ATCC | Cat#CRL-3216 |
| G3BP1/2 KO U2OS cell line | Sanders, D.W. et al.10 | N/A |
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| Experimental models: Organisms/strains | ||
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| Mouse: C57BL/6J | The Jackson Laboratory | Cat#000664; RRID:IMSR_JAX:000664 |
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| Oligonucleotides | ||
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| siALKBH5 ON-TARGETplus human ALKBH5 | Dharmacon | Cat#L-004281-01 |
| siYTHDF1 ON-TARGETplus human YTHDF1 | Dharmacon | Cat#L-018095-02 |
| siYTHDF3 ON-TARGETplus human YTHDF3 | Dharmacon | Cat#L-017080-01 |
| siYTHDF1 ON-TARGETplus human YTHDF1 3’UTR | Dharmacon | Cat#J-018095-19 |
| siYTHDF3 ON-TARGETplus human YTHDF3 3’UTR | Dharmacon | Cat#J-017080-10 |
| Primers for site-directed mutagenesis of YTHDF1 | Li, Q., et al.26 | Table S2 |
|
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| Recombinant DNA | ||
|
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| pmCherry-C1 | Clontech Laboratories | Cat#632524 |
| pmCherry-C1 G3BP1 FL | This study | N/A |
| pmCherry-C1 G3BP1 ΔNTF2L | This study | N/A |
| pmCherry-C1 G3BP1 ΔIDR1 | This study | N/A |
| pmCherry-C1 G3BP1 ΔIDR3 | This study | N/A |
| pmCherry-C1 G3BP1 ΔRRM | This study | N/A |
| pGEX-6P-1 | GE Health | Cat#28-9546-48 |
| pGEX-6P-1 G3BP1 FL | This study | N/A |
| pGEX-6P-1 G3BP1 ΔNTF2L | This study | N/A |
| pGEX-6P-1 G3BP1 ΔIDR1 | This study | N/A |
| pGEX-6P-1 G3BP1 ΔIDR3 | This study | N/A |
| pGEX-6P-1 G3BP1 ΔRRM | This study | N/A |
| pGEX-4T-1 YTHDF1 | Wang, X. et al.33 | Addgene Cat#70087 |
| pcDNA6 V5-His A | Invitrogen | Cat#V22120 |
| mTagBFP2-ER-5 | Subach, O.M., et al.34 | Addgene Cat#55294 |
| pAAV-tagBFP | This study | N/A |
| pcDNA6 V5-tagBFP | This study | N/A |
| pcDNA6 tagBFP-YTHDF1-WT-V5 | This study | N/A |
| pcDNA6 tagBFP-YTHDF1-mut-V5 | This study | N/A |
| pCMV-Flag-ALKBH5 | SinoBiological | Cat#HG24078-CF |
| pEGFP-(GR)100 | Cook, C.N.,etal.9 | N/A |
| pEGFP-(GA)100 | Cook, C.N.,etal.9 | N/A |
| pFDelta6 | Chakrabarty, P., et al.35 | N/A |
| pCap9 | Chakrabarty, P., et al.35 | N/A |
| pAM/CBA-pl-WPRE-BGH (pAAV) | Chakrabarty, P., et al.35 | N/A |
| pAAV-(GA)100-V5 | Shao W., et al.32 | N/A |
| pAAV-GFP-(GR)200 | Cook, C.N.,etal.9 | N/A |
| pAAV-(G4C2)2 | Chew, J., et al.20 | N/A |
| pAAV-(G4C2)149 | Chew, J., et al.20 | N/A |
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| Software and algorithms | ||
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| Graphpad Prism v9 | GraphPad Software, LLC | https://www.graphpad.com/scientificsoftware/prism/ |
| ImageScope® software | Leica Biosystems | https://www.leicabiosystems.com/us/digital-pathology/manage/aperioimagescope/ |
| Zeiss Zen 2.3 | Carl Zeiss | https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html |
| CellProfiler | Lamprecht, M.R., et al.36 | https://cellprofiler.org/ |
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| Others | ||
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| Zeiss AxioObserver A1 microscope | Zeiss | https://www.zeiss.com/microscopy/en/products/light-microscopes/widefieldmicroscopes/axio-observer-for-lifescience-research.html |
| Zeiss LSM980 laser scanning confocal microscope | Zeiss | https://www.zeiss.com/microscopy/en/products/light-microscopes/confocalmicroscopes/lsm-980-withairyscan-2.html |
| MESO QUICKPLEX SQ 120 | Meso Scale Diagnostics | https://www.mesoscale.com/en/products_and_services/instrumentation/quickplex_sq_120mm |
| Amersham ImageQuant 800 | Cytiva | https://www.cytivalifesciences.com/en/us/shop/protein-analysis/molecular-imagingfor-proteins/imaging-systems/amershamimagequant-800-systems-p-11546 |