Our original manuscript erroneously listed the concentration of SB431542 as 10 mM instead of 10 μM in the dopamine neuron differentiation section of the Method details section. We apologize for this error, which has been corrected in the original version.
The corrected manuscript also includes additional changes to comply with the open access policy requirements of the funder (Aligning Science Across Parkinson’s disease). These include more detailed information listed in the key resource table, links to detailed protocols and code used in the study, and access to source data for all figures and supplemental information (which was added as Data S1 in the supplemental files). None of the changes made in this correction affect the conclusions of the manuscript.
Supplementary Material
Data S1 (Raw Data)
NIHMS2134384-supplement-2.xlsx (61KB, xlsx)
Data S1 (Readme file)
NIHMS2134384-supplement-1.rtf (3KB, rtf)
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
|
| ||
| Mouse anti β-ACTIN | Sigma | Cat# A2228–100UL RRID: AB_476697 |
| Mouse anti BAG1 | Santa Cruz Biotech | Cat# sc-33704 RRID: AB_626720 |
| Rabbit anti BAG3 | Abcam | Cat# ab47124, RRID: AB_867788 |
| Mouse anti Cas9 | Cell Signaling Tech | Cat# 14697S, RRID: AB_2750916 |
| Mouse anti Ki67 | Dako | Cat# M7240, RRID: AB_2142367 |
| Mouse anti GAPDH-HRP | Santa Cruz Biotech | Cat# sc-47724 RRID: AB_627678 |
| Rabbit anti LMNB1 | Abcam | Cat# ab16048, RRID: AB_443298 |
| Rabbit anti NAE1 | Cell Signaling Tech | Cat# 14321, RRID: AB_2798448 |
| Rabbit anti NEDD8 | Abcam | Cat# ab81264, RRID: AB_1640720 |
| Rabbit anti p21 | Cell Signaling Tech | Cat# 2947, RRID: AB_823586 |
| Mouse anti TAU | Thermo Fisher | Cat# MN1000 RRID: AB_2314654 |
| Mouse anti p-TAU(S202/T205) | Thermo Fisher | Cat# MN1020, RRID: AB_223647 |
| Rabbit anti UBA3 | Abcam | Cat# ab124728, RRID: AB_10974248 |
| Mouse anti phospho-ATM(S1981) | Thermo Fisher | Cat# MA1–2020, RRID: AB_1086244 |
| Rabbit anti Cleaved Caspase-3 (Asp175) | Cell Signaling Tech | Cat# 9661, RRID: AB_2341188) |
| Mouse anti p53 | Cell Signaling Tech | (Cat# 48818, RRID: AB_2713958) |
| Rabbit anti FOXG1 | TaKaRa | Cat# M227 RRID: AB_2827749 |
| Chicken anti MAP2 | Thermo Fisher | Cat# PA1–16751 RRID: AB_2138189 |
| Rabbit anti NANOG | Thermo Fisher | Cat# 4903, RRID: AB_10559205 |
| Rabbit anti PAX6 | Biolegend | Cat# 901301 RRID: AB_2749901 |
| Mouse anti SENP8 | Santa Cruz Biotech | Cat# sc-271498 RRID: AB_10649498 |
| Mouse anti p62 Lck Ligand | BD Bioscience | Cat# 610832 RRID: AB_398151 |
| Rabbit anti LC3A/B XP | Cell Signaling Tech | Cat# 12741S RRID: AB_2617131 |
| Rabbit p-TAU(S235) | Thermo Fisher | Cat# PA5–104785, RRID: AB_2816258 |
| Mouse anti TAU | Dako | Cat# A0024, RRID: AB_10013724 |
| Mouse anti Caveolin 1 (CAV1) | Thermo Fisher | Cat# MA3–600, RRID: AB_779568 |
| Rabbit anti H3K9me3 | Abcam | Cat# ab176916 RRID: AB_2797591 |
| Mouse anti Lap2 | BD Bioscience | Cat# 611000 RRID: AB_398313 |
| Rabbit anti H3k9me3-PE | Cell Signaling Tech | Cat# 13969S RRID: AB_2798355 |
| Anti-mouse IgG, HRP-linked | Cell Signaling Tech | Cat#7076S RRID: AB_330924 |
| Anti-rabbit IgG, HRP-linked | Cell Signaling Tech | Cat#7074S, RRID: AB_2099233 |
| Donkey anti-mouse IgG H&L (Alexa Fluor®488) | Invitrogen | Cat# R37114, RRID: AB_2556542 |
| Donkey anti-rabbit IgG H&L (Alexa Fluor®488) | Invitrogen | Cat# A21206, RRID: AB_2535792 |
| Donkey anti-mouse IgG H&L (Alexa Fluor®555) | Invitrogen | Cat# A31570, RRID: AB_2536180 |
| Donkey anti-rabbit IgG H&L (Alexa Fluor®555) | Invitrogen | Cat# A31572 RRID: AB_162543 |
| Donkey anti-chicken IgG H&L (Alexa Fluor®647) | Jackson ImmunoResearch | Cat# 703–605-155 RRID: AB_2340379 |
| Goat anti-mouse IgG H&L (Alexa Fluor®488) | Invitrogen | Cat# A11029 RRID: AB_2534088 |
| Goat anti-rabbit IgG H&L (Alexa Fluor®488) | Invitrogen | Cat# A11034 RRID: AB_2576217 |
| Goat anti-mouse IgG H&L (Alexa Fluor®555) | Invitrogen | Cat# A21422 RRID: AB_141822 |
| Goat anti-rabbit IgG H&L (Alexa Fluor®555) | Invitrogen | Cat# A27039 RRID: AB_2536100 |
| Goat anti-chicken IgG H&L (Alexa Fluor®647) | Invitrogen | Cat# A21449 RRID: AB_2535866 |
|
| ||
| Bacterial and virus strains | ||
|
| ||
| One Shot™ Stbl3™ Chemically Competent E. coli | Thermo Fisher | C737303 |
|
| ||
| Chemicals, peptides, and recombinant proteins | ||
| Halt Protease Inhibitor | Thermo Fisher | 87786 |
| Halt Phosphatase inhibitor | Thermo Fisher | 78420 |
| Y-27632 | R&D Systems | 1254 |
| SB431542 | R&D Systems | 1614 |
| LDN193189 (LDN) | Stemgent | 04–0074 |
| CHIR99021 | R&D Systems | 4432 |
| SHH C25II | R&D Systems | 464-SH |
| Poly-L-Ornithine (PO) | Sigma Aldrich | P3655 |
| Mouse Laminin I (LAM) | R&D Systems | 3400–010-1 |
| Fibronectin (FN) | Thermo Fisher Scientific | 356008 |
| 0.5M EDTA, pH 8.0 | Thermo Fisher Scientific | 15575020 |
| Accutase | Innovative Cell Technologies | AT104–500 |
| Trypsin-EDTA 0.05% | Thermo Fisher Scientific | 25300–054 |
| GDNF | Peprotech | 450–10 |
| AA | Sigma | 4034–100g |
| cAMP | Sigma | D0627 |
| BDNF | R&D Systems | 248-BD |
| TGFβ3 | R&D Systems | 243-B3 |
| DAPT | R&D Systems | 2634 |
| DMEM:F12 w/high glucose | Thermo Fisher Scientific | 11320–033 |
| E8 medium | Thermo Fisher | A1517001 |
| E6 medium | Thermo Fisher | A1516401 |
| DMEM/F12 | Thermo Fisher | 21331020 |
| Neurobasal medium | Life Technologies | 21103–049 |
| 100X N2 supplement | Life Technologies | 17502–048 |
| 50X B27 supplement w/o Vit A | Life Technologies | 12587–010 |
| L-Glutamine (100X) | Thermo Fisher Scientific | 25030–081 |
| Penicillin Streptomycin | Thermo Fisher Scientific | 15140–122 |
| 4’,6-diamidino-2-phenylindole (DAPI) | Sigma | D9542 |
| Matrigel | BD Biosciences | 356234 |
| Vitronectin (VTN-N) | Thermo Fisher Scientific | A14700 |
| Geltrex | Life Technologies | A1413201 |
| Trizol | Thermo Fisher Scientific | 15596026 |
| STEM-CELLBANKER | Amsbio | 11890 |
| 4% paraformaldehyde | Affymetrix | MFCD00133991 |
| Neuron Isolation Enzyme | Thermo Fisher Scientific | 88285 |
| Precision Red Advanced Protein Assay | Cytoskeleton, Inc | ADV02-A |
| DPBS | Thermo Fisher Scientific | 14190–136 |
| CellEvent Senescence Green | Thermo Fisher Scientific | C10840 |
| X-tremeGENE HP | Sigma | 6366236001 |
| Doxycycline hyclate | Sigma | D9891 |
| Proteostat | Enzo Life Sciences | ENZ-51023-KP050 |
| MLN4924 (Pevonedistat) | MedChemExpress | HY-70062 |
| CSN5i3 | Tocris | 7089 |
| AggreSure ß-Amyloid (1–42), human | AnaSpec | AS-72216 |
| MG132 | Selleck | S2619 |
| Bafilomycin A1 | Cayman Chemical | 11038 |
| N-Lauroylsarcosine sodium salt solution | Sigma | L7414–10ML |
|
| ||
| Critical commercial assays | ||
|
| ||
| Direct-zol RNA Microprep | Zymo Research | R2062 |
| QiAquick gel extraction kit 250 | Qiagen | 28706 |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 |
| NucleoBond Xtra Midi Plus EF | Macherey-Nagel | 740422.50 |
| iScript™ Reverse Transcription Supermix for RT-qPCR | Biorad | 1708841 |
| SsoFast™ EvaGreen® Supermix | Biorad | 1725201 |
| NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.5 mm, 15-wells | Thermo Scientific | NP0336BOX |
| NuPAGE® Novex 4–12% Bis-Tris Gel 1.5 mm, 10 well | Thermo Scientific | NP0335Box |
| NuPAGE® Novex® 4–12% Bis-Tris Gels, 1.0 mm, 12 wel | Thermo Scientific | NP0322BOX |
| Zombie UV™ Fixable Viability Kit | Biolegend | 423107 |
| Cell Counting Kit - 8 | Sigma Aldrich | 96992–100TESTS-F |
| PrestoBlue Cell Viability Reagent | Thermo | A13261 |
| V-PLEX Aβ Peptide Panel 1 (6E10) Kit | Meso Scale Discovery | K15200E-2 |
| Pierce Reversible Protein Stain Kit for PVDF Membranes | Thermo Fisher Scientific | 24585 |
| Cell-Based Proteasome-Glo™ Assay kit | Promega | G8660 |
|
| ||
| Deposited data | ||
|
| ||
| RNAseq data. Young and Old Cortex | Zhang et al.35 | PRJNA1107241 |
| Tabula Muris Senis trajectory analysis | Schaum et al.66 | GEO: GSE132040) https://twc-stanford.shinyapps.io/maca/ |
| Young and Old Protein expression | Keele et al.67 | Table S3; DOI: https://doi.org/10.1016/j.celrep.2023.112715 |
| Essential genes in iNeurons | Tian et al.19 | Table S1; DOI: https://doi.org/10.1038/s41593-021-00862-0 |
| Essential genes in striatal neurons | Wertz et al.18 | Table S2; DOI: https://doi.org/10.1016/j.neuron.2020.01.004 |
| Essential gene catalogue | Project Achilles | CRISPRGeneEffect.csv; https://depmap.org/portal/download/all/ |
|
| ||
| Experimental models: Cell lines | ||
|
| ||
| H9 derived iCas9 cell line | This study | RRID: CVCL_E7G4 |
| H9 derived iCas9 cell line with APPswe/swe mutation | This study | RRID: CVCL_E7G5 |
| 7889SA iCas9 cell line | This study | RRID: CVCL_E7GB |
| 7889SA iCas9 cell line with PSENM146V/M146V mutation | This study | RRID: CVCL_E7GC |
| H9 derived Nurr1::H2B-GFP iCas9 | Kim et al.68 | RRID: CVCL_E7GJ |
| H9 derived Nurr1::H2B-GFP iCas9 with the LRRK2G2019S/G2019S mutation | This study | RRID: CVCL_E7GK |
|
| ||
| Oligonucleotides | ||
|
| ||
| qPCR Primers: See Table S2 | This study | N/A |
| Cloning Primers: See Table S2 | This study | N/A |
| Oligonucleotides for Cloning: See Table S2 | This study | N/A |
| sgRNAs: See Table S3 | Doench et al69 | Table S3; DOI: https://doi.org/10.1038/nbt.3437 |
|
| ||
| Recombinant DNA | ||
|
| ||
| Hygro-Cas9 donor | Shi et al., 201770 | RRID: Addgene_86883 |
| Neo-M2rtTA donor | Gonzalez et al.12 | Danwei Huangfu Lab; MSKCC |
| AAVS1-TALEN-L | Gonzalez et al.12 | RRID: Addgene_59025 |
| AAVS1-TALEN-R | Gonzalez et al.12 | RRID: Addgene_59026 |
| lentiGuide-Puro | Sanjana et al.71 | RRID: Addgene_52963 |
| Brunello sgRNA library | Doench et al.69 | RRID: Addgene_73178 |
| psPAX2 | Didier Trono lab | RRID: Addgene_12260 |
| pMD2.G | Didier Trono lab | RRID: Addgene_12259 |
| pLV-EF1a-IRES-Puro | Hayer et al.72 | RRID: Addgene_85132 |
| UBA3 ORF | Sino Biological | Cat# HG16320-G |
| NAE1 ORF | Sino Biological | Cat # HG14282-G |
| pLV-EF1aNAE1-IRES-Puro | This study | RRID: Addgene_#235659 |
| pLV-EF1aUBA3-IRES-Puro | This study | RRID: Addgene_#235660 |
|
| ||
| Software and algorithms | ||
|
| ||
| FIJI | Schindelin et al.73 |
https://fiji.sc/ RRID: SCR_003070 |
| Volocity Imaging Software | Perkin Elmer |
http://www.perkinelmer.com/pages/020/cellularimaging/products/volocity.xhtml RRID: SCR_002668 |
| FlowJo | FlowJo |
https://www.flowjo.com/ RRID: SCR_008520 |
| R |
https://cran.r-project.org/ RRID: SCR_001905 |
|
| Prism 9 | GraphPad |
www.graphpad.com RRID: SCR_002798 |
| Biorender | Biorender |
http://biorender.com RRID: SCR_018361 |
| MAGECK-MLE | Wang et al.17 |
https://mageck.sourceforge.net/ RRID: SCR_025016 |
| Python |
https://www.python.org RRID: SCR_008394 |
|
| Image Lab Software | Bio-Rad |
http://www.bio-rad.com/en-us/sku/1709690-image-lab-software RRID: SCR_014210 |
| DAVID (v6.8) | Huang da et al.74 |
https://david.ncifcrf.gov/summary.jsp RRID: SCR_001881 |
| Harmony | Revvity |
https://www.revvity.com/product/harmony-5-2-office-revvity-hh17000019 RRID: SCR_023543 |
| ggplot2 |
https://cran.r-project.org/web/packages/ggplot2/index.html RRID: SCR_014601 |
|
| GSEA 3.0 |
http://software.broadinstitute.org/gsea/index.jsp RRID: SCR_003199 |
|
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data S1 (Raw Data)
NIHMS2134384-supplement-2.xlsx (61KB, xlsx)
Data S1 (Readme file)
NIHMS2134384-supplement-1.rtf (3KB, rtf)