Enterovirus D68 2A protease causes nuclear pore complex dysfunction and independently contributes to motor neuron toxicity
- Hugo W. Moser Research Institute, Michael V. Johnston Center for Developmental Neuroscience, Kennedy Krieger Institute, United States
- Department of Neurology, Johns Hopkins School of Medicine, United States
Figures
Figure 1 with 1 supplement
Enterovirus D68 proteases cleave several nucleoporins.
(A) HEK cells were transfected with Enterovirus D68 2Apro, Poliovirus 1 2Apro, or empty vector control, and the indicated nucleoporins were quantified by western blot, normalized to control = 100%. EIF4G was included as a non-nucleoporin positive control. Mean ± SD of three independent replicates. Statistical comparison was by multiple t-tests of the EV-D68 2Apro vs control, with multiple comparisons correction by the false discovery rate (FDR) method. Red asterisks represent significance with FDR<10%, blue asterisks represent samples for which a cleavage product was visible on the western blot irrespective of significance. (B) As in A, except transfecting with 3Cpro. The positive control was CREB. (C) Nuclear lysates from HEK cells were incubated at 37°C with recombinant EV-D68 2Apro at a ratio of 1:50 (protease:lysate by mass) for the indicated time. Gray bars represent lysate incubated for 4 hr at 37°C in the absence of protease as a negative control. Mean ± SD of three independent replicates. Statistical comparison was by t-test between the with- and without-protease samples at 4 hr. (D) As in C, except with EV-D68 3Cpro at a ratio of 1:200. *p<0.05, **p<0.01, ***p<0.001.
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Figure 1—source data 1
Raw data from quantifications of western blot data by densitometry for Figure 1A–D, reported as percent of control.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data1-v1.zip
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Figure 1—source data 2
Labeled uncropped western blots for Figure 1A–D.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data2-v1.zip
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Figure 1—source data 3
Original western blot images for Figure 1A.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data3-v1.zip
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Figure 1—source data 4
Original western blot images for Figure 1B (part 1).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data4-v1.zip
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Figure 1—source data 5
Original western blot images for Figure 1B (part 2).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data5-v1.zip
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Figure 1—source data 6
Original western blot images for Figure 1B (part 3).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data6-v1.zip
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Figure 1—source data 7
Original western blot images for Figure 1C (part 1).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data7-v1.zip
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Figure 1—source data 8
Original western blot images for Figure 1C (part 2).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data8-v1.zip
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Figure 1—source data 9
Original western blot images for Figure 1C (part 3).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data9-v1.zip
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Figure 1—source data 10
Original western blot images for Figure 1C (part 4).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data10-v1.zip
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Figure 1—source data 11
Original western blot images for Figure 1D (part 1).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data11-v1.zip
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Figure 1—source data 12
Original western blot images for Figure 1D (part 2).
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-data12-v1.zip
Figure 1—figure supplement 1
Western blots demonstrating evidence of 2Apro and 3Cpro protease activity on well-established substrates following transfection with each of the expression vectors used in this study.
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Figure 1—figure supplement 1—source data 1
Labeled uncropped western blots for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original western blot images for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig1-figsupp1-data2-v1.zip
Figure 2 with 2 supplements
Enterovirus D68 2A protease impairs nucleocytoplasmic transport of protein cargoes and disrupts the nuclear pore complex permeability barrier.
(A) tdTomato reporter assay showing nuclear-cytoplasmic ratios at steady state 20 hr after transfection of IRES-GFP, IRES-GFP-2Apro, or IRES-GFP-3Cpro. Mean values of three independent replicates n=29,404 GFP+ cells in total, error bars are SD. Statistical comparison was by two-way ANOVA with Šídák’s multiple comparisons test. (B) Example output of image analysis protocol. In the overlay image, yellow represents the nuclei of transfected cells, defined by the area of expression of H2A-iRFP670 in GFP+ cells only. The surrounding dark blue ring is the perinuclear cytoplasm. The intensity of the reporter construct was measured in the red channel and reported as a ratio of nuclear to perinuclear cytoplasmic signal. (C) Example images from A. Dotted outlines mark the nuclei. (D) Kinetics of nuclear import (left) and export (right) measured by photoinducible LINus and LEXY reporter assays, respectively, in HeLa cells 20 hr after transfection with IRES-GFP, IRES-GFP-2Apro, or IRES-GFP-3Cpro. Mean values of three biologically independent replicates, n=999 GFP+ cells for LINus and 1059 for LEXY in total. Error bars are SEM. Statistical comparison was by two-way ANOVA. (E) Example images from D. Dotted outlines mark GFP+ cells. (F) Dextran exclusion assay was performed in HeLa cells 15 hr after transfection with IRES-iRFP670-H2A, IRES-iRFP670-H2A-(2A)-2Apro, or IRES-iRFP670-H2A-(2A)-3Cpro. n=45–46 cells per group pooled from three biologically independent replicates. Statistical comparison was by two-way ANOVA. (H) Propidium iodide viability assay. Mean values of three independent replicates, n=8042 cells in total. Error bars are SD. Statistical comparison was by one-way ANOVA with Tukey’s multiple comparison test.
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Figure 2—source data 1
Raw data used to generate Figure 2A.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig2-data1-v1.zip
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Figure 2—source data 2
Raw data used to generate Figure 2D.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig2-data2-v1.zip
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Figure 2—source data 3
Raw data used to generate Figure 2F.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig2-data3-v1.zip
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Figure 2—source data 4
Raw data used to generate Figure 2H.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig2-data4-v1.zip
Figure 2—figure supplement 1
Source data for Figure 2A, showing individual cell-level data for three independent replicates.
Bars are mean ± SD.
Figure 2—figure supplement 2
Source data for Figure 2D, showing mean ± SD values from three independent replicates for LINus (A) and LEXY (C) nucleocytoplasmic transport reporters, individual cell-level data is also presented at t=10 min for LINus (B) and LEXY (D).
Bars are mean ± SD.
Figure 3 with 1 supplement
Enterovirus D68 proteases do not significantly alter RNA export.
(A) Steady-state mRNA levels were measured by poly-A FISH in HeLa cells 20 hr after transfection with IRES-GFP, IRES-GFP-2Apro, or IRES-GFP-3Cpro. Data represent the mean ± SD of four independent replicates, n=55,177 GFP+ cells in total. Statistical comparison was by one-way ANOVA with Šídák’s multiple comparisons test. (B) Nuclear export of EU-labeled RNAs in HEK cells by 1 hr pulse followed by 0–8 hr chase. EU labeling began 20 hr after transfection with IRES-GFP, IRES-GFP-2Apro, or IRES-GFP-3Cpro. Pulse labeling of total RNA or primarily the products of the listed RNA polymerases was performed in the presence of pairs of combinations of actinomycin D, α-amanitin, and CAS 577784-91-9 as described in Materials and methods. Data represent the mean ± SEM of three independent replicates. n=253,015 GFP+ cells analyzed in total. Statistical comparison was by two-way ANOVA. (C) Representative images from the experiment described in B. (D) Nuclear export of EU-labeled RNAs in RD cells by 1 hr pulse followed by 0–4 hr chase. EU labeling began 24 hr after infections with EV-D68 strains at MOI 5 or mock infection as indicated. Data represent the mean ± SEM of three independent replicates. n=125,401 cells analyzed in total. Statistical comparison was by two-way ANOVA. (E) Representative images from the experiment described in D.
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Figure 3—source data 1
Raw data used to generate Figure 3A.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig3-data1-v1.zip
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Figure 3—source data 2
Raw data used to generate Figure 3B.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig3-data2-v1.zip
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Figure 3—source data 3
Raw data used to generate Figure 3D.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig3-data3-v1.zip
Figure 3—figure supplement 1
Source data for Figure 3A, demonstrating individual cell-level data across four independent replicates.
Bars are mean ± SD.
Figure 4 with 1 supplement
Effects of 2A protease during Enterovirus D68 (EV-D68) infection on nuclear pore complex (NPC) function and motor neuron toxicity.
(A) Western blots of direct-induced motor neuron (diMN) lysates collected 24 hr after infection with the indicated strains of EV-D68 at MOI 5 followed by treatment with telaprevir at 0.3, 1, 3, and 10 µM. Total protein loading control was Bio-Rad stain-free imaging. One representative example out of three biologically independent replicates. (B) tdTomato reporter assays in RD cells showing nuclear-cytoplasmic ratios 24 hr after mock infection or infection with EV-D68 US/MO/2014-18947. Telaprevir was added following infection at the indicated concentrations. Data are mean of three independent replicates, n=11,847 cells in total for NLS-tdTomato and n=8941 for tdTomato-NES. (C) Representative images of tdTomato signal from B. (D) tdTomato reporter assays in diMN showing nuclear-cytoplasmic ratios 24 hr after mock infection or infection with EV-D68 US/MO/2014-18947, treated with DMSO or 3 µM telaprevir. Means of three replicates from independent iPSC lines. n=3388 cells in total for NLS-tdTomato and n=3371 for tdTomato-NES. (E) Representative images from panel D. (F) Survival of diMNs following infection with EV-D68 at MOI 5 with the indicated EV-D68 strains in the presence of varying concentrations of telaprevir or DMSO control. diMNs were generated from four independent iPSC lines. n=8512 and 7046 cells counted for US/MO/2014-18947 and US/MD/2018-23209, respectively. Statistical comparisons were by Cox proportional hazard regression. (G) Representative images of diMNs from the experiment presented in panel F. Note the fragmentation of neurites and loss of cell body shape, signifying cell death following EV-D68 infection, and the relative preservation following treatment with 3 µM telaprevir. (H) Growth curve of EV-D68 in the presence of varying concentrations of telaprevir or DMSO control. Infections were performed with the indicated strains at MOI 0.5, using diMNs generated from four independent iPSC lines for each virus. Error bars are SEM. Statistical comparisons were by two-way ANOVA with Dunnett’s multiple comparisons test between DMSO and telaprevir-treated groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 4—source data 1
Labeled uncropped western blots for Figure 4A.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig4-data1-v1.zip
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Figure 4—source data 2
Original western blot images for Figure 4A.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig4-data2-v1.zip
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Figure 4—source data 3
Raw data used to generate Figure 4B.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig4-data3-v1.zip
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Figure 4—source data 4
Raw data used to generate Figure 4D.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig4-data4-v1.zip
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Figure 4—source data 5
Raw data used to generate Figure 4F.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig4-data5-v1.zip
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Figure 4—source data 6
Raw data used to generate Figure 4H.
- https://cdn.elifesciences.org/articles/108672/elife-108672-fig4-data6-v1.zip
Figure 4—figure supplement 1
Source data for Figure 4D, showing individual cell-level data for the nuclear/cytoplasmic ratio of NLS-tdTomato (A) or tdTomato-NES (B) for three independent replicates in direct-induced motor neurons (diMNs) derived from induced pluripotent stem cell (iPSC) lines CS0002, CS0003, and CS88.
Bars are mean ± SD.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Enterovirus D68) | 2A | GenBank | KM851225.1 | Nucleotides 3280–3720 of genomic cDNA |
| Gene (Enterovirus D68) | 3C | GenBank | KM851225.1 | Nucleotides 5341–5979 of genomic cDNA |
| Strain, strain background (Enterovirus D68) | US/MO/2014-18947 | BEI Resources | NR-49129 | |
| Strain, strain background (Enterovirus D68) | US/MD/2018-23209 | Gift of Andrew Pekosz, Johns Hopkins School of Public Health | ||
| Cell line (Homo sapiens) | RD | ATCC | CCL-136 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216 RRID:CVCL_0063 | |
| Cell line (Homo sapiens) | HeLa | ATCC | CRM-CCL-2 RRID:CVCL_0030 | |
| Cell line (Homo sapiens) | iPSC | Cedars Sinai Biomanufacturing Center | CS0002 | |
| Cell line (Homo sapiens) | iPSC | Cedars Sinai Biomanufacturing Center | CS0003 | |
| Cell line (Homo sapiens) | iPSC | Cedars Sinai Biomanufacturing Center | CS88 | |
| Cell line (Homo sapiens) | iPSC | Cedars Sinai Biomanufacturing Center | CS0YX7 | |
| Cell line (Homo sapiens) | iPSC | Cedars Sinai Biomanufacturing Center | CS8VTR | |
| Antibody | Aladin (rabbit polyclonal) | Bethyl Laboratories | A304-514A RRID:AB_2620708 | (1:500) |
| Antibody | CG1 (NUPL2; Nup42) (rabbit polyclonal) | Thermo | PA5-88035 RRID:AB_2804606 | (1:1000) |
| Antibody | CREB (rabbit polyclonal) | Cell Signaling | 9197 RRID:AB_331277 | (1:1000) |
| Antibody | eIF4G (rabbit polyclonal) | Cell Signaling | 2498S RRID:AB_2096025 | (1:1000) |
| Antibody | ELYS (rabbit polyclonal) | Bethyl Laboratories | A300-166A RRID:AB_2225879 | (1:1000) |
| Antibody | GAPDH (mouse monoclonal) | Abcam | ab8245 RRID:AB_2107448 | (1:10,000) |
| Antibody | GFP (mouse monoclonal) | Takara Bio | 632375 RRID:AB_2756343 | (1:20,000) |
| Antibody | Gle1 (rabbit polyclonal) | Novus | NBP2-47449 RRID:AB_3311113 | (1:250) |
| Antibody | GP210 (rabbit polyclonal) | Bethyl Laboratories | A301-795A RRID:AB_1211450 | (1:100) |
| Antibody | mAb414 (rabbit polyclonal) | Abcam | Ab24609 RRID:AB_448181 | (1:1000) |
| Antibody | NDC1 (TMEM48) (rabbit polyclonal) | Novus Biologicals | NBP1-91603 RRID:AB_11030742 | (1:1000) |
| Antibody | Nucleolin (rabbit polyclonal) | Cell Signaling | 14574S RRID:AB_2798519 | (1:10,000) |
| Antibody | Nup107 (rabbit polyclonal) | Thermo Fisher Scientific | 19217-1-AP RRID:AB_10597702 | (1:1000) |
| Antibody | Nup133 (mouse monoclonal) | Santa Cruz | sc-376699 RRID:AB_11149388 | (1:1000) |
| Antibody | Nup153 (mouse monoclonal) | Abcam | Ab24700 RRID:AB_2154467 | (1:1000) |
| Antibody | Nup155 (rabbit polyclonal) | Abcam | ab199528 RRID:AB_3751167 | (1:1000) |
| Antibody | Nup160 (rabbit polyclonal) | Bethyl Laboratories | A301-790A RRID:AB_1211264 | (1:100) |
| Antibody | Nup188 (rabbit polyclonal) | Bethyl Laboratories | A302-323A-T RRID:AB_1850233 | (1:2500) |
| Antibody | Nup205 (rabbit polyclonal) | Abcam | ab157090 RRID:AB_3751168 | (1:1000) |
| Antibody | Nup214 (rabbit polyclonal) | Abcam | ab70497 RRID:AB_1269607 | (1:1000) |
| Antibody | Nup35 (Nup53) (rabbit polyclonal) | Proteintech | 19819–1-AP RRID:AB_2878611 | (1:1000) |
| Antibody | Nup37 (rabbit polyclonal) | Thermo Fisher Scientific | PA5-66007 RRID:AB_2664134 | (1:200) |
| Antibody | Nup43 (mouse polyclonal) | Abcam | ab69447 RRID:AB_1269608 | (1:1000) |
| Antibody | Nup50 (mouse monoclonal) | Santa Cruz | sc-398993 RRID:AB_2941760 | (1:1000) |
| Antibody | Nup54 (rabbit polyclonal) | Sigma-Aldrich | HPA035929 RRID:AB_10671236 | (1:200) |
| Antibody | Nup62 (rabbit polyclonal) | Millipore | MABE1043 RRID:AB_3751169 | (1:2000) |
| Antibody | Nup85 (rabbit polyclonal) | Proteintech | 19370–1-AP RRID:AB_10859826 | (1:100) |
| Antibody | Nup88 (mouse monoclonal) | Santa Cruz | sc-365868 RRID:AB_10842170 | (1:200) |
| Antibody | Nup93 (mouse monoclonal) | Santa Cruz | sc-374399 RRID:AB_10990113 | (1:200) |
| Antibody | Nup96 (rabbit polyclonal) | Bethyl | A301-785A RRID:AB_1211486 | (1:1000) |
| Antibody | Nup98 (rat polyclonal) | Abcam | ab50610 RRID:AB_881769 | (1:5000) |
| Antibody | POM121 (rabbit polyclonal) | GeneTex | GTX102128 RRID:AB_10732546 | (1:1000) |
| Antibody | RAE1 (mouse monoclonal) | Santa Cruz | sc-393252 RRID:AB_3751170 | (1:200) |
| Antibody | RanBP2 (mouse monoclonal) | Santa Cruz | sc74518 RRID:AB_2176784 | (1:200) |
| Antibody | Sec13 (rabbit polyclonal) | Proteintech | 15397–1-AP RRID:AB_2186234 | (1:1000) |
| Antibody | SNAP29 (rabbit polyclonal) | Abcam | ab181151 RRID:AB_2687668 | (1:5000) |
| Antibody | TPR (rabbit polyclonal) | Bethyl Laboratories | A300-828A RRID:AB_597886 | (1:1000) |
| Antibody | Goat Anti-Mouse IgG H&L HRP | Abcam | ab205719 RRID:AB_2755049 | (1:20,000) |
| Antibody | Goat Anti-Rabbit IgG H&L HRP | Abcam | ab205718 RRID:AB_2819160 | (1:20,000) |
| Antibody | Goat Anti-Rat IgG H&L HRP | Abcam | ab205720 RRID:AB_2941939 | (1:20,000) |
| Antibody | Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher | A32723 RRID:AB_2633275 | (1:500) |
| Recombinant DNA reagent | pmTurquoise-H2A (plasmid) | Addgene (Goedhart et al., 2012) | 36207 | |
| Recombinant DNA reagent | FU-MAP2-Gateway (plasmid) | Addgene (Addis et al., 2011) | 43915 | |
| Recombinant DNA reagent | eBFP2-N1 (plasmid) | Addgene (Michael Davidson) | 54595 | |
| Recombinant DNA reagent | pDB22 (mCherry-LINus) (plasmid) | Addgene (Niopek et al., 2014) | 61342 | |
| Recombinant DNA reagent | pDN122 (NLS-mCherry-LEXY) (plasmid) | Addgene (Niopek et al., 2016) | 72655 | |
| Recombinant DNA reagent | pLenti-DsRed-IRES-GFP (plasmid) | Addgene (Rousseaux et al., 2016) | 92194 | |
| Recombinant DNA reagent | hMAP2-pGreenZero (plasmid) | System Biosciences | SR10047PA-1 | |
| Recombinant DNA reagent | Shuttle-tdTomato (plasmid) | Zhang et al., 2015 | ||
| Recombinant DNA reagent | peSUMOstar (plasmid) | LifeSensors, Inc | ||
| Recombinant DNA reagent | CMV-(EV-D68)2Apro | This paper | Backbone: NheI/AgeI digest of eBFP2-N1 Insert: Synthetic DNA 1 | |
| Recombinant DNA reagent | CMV-(EV-D68)3Cpro | This paper | Backbone: NheI/AgeI digest of eBFP2-N1 Insert: Synthetic DNA 7 | |
| Recombinant DNA reagent | CMV-(PV1)2Apro | This paper | Backbone: NheI/AgeI digest of eBFP2-N1 Insert: Synthetic DNA 2 | |
| Recombinant DNA reagent | CMV-(PV1)3Cpro | This paper | Backbone: NheI/AgeI digest of eBFP2-N1 Insert: Synthetic DNA 8 | |
| Recombinant DNA reagent | pLenti-IRES-GFP | This paper | Backbone: PCR amplification of pLenti-DsRed-IRES-GFP (primers MJE_69, MJE_71) Recircularization via In-Fusion Snap Assembly | |
| Recombinant DNA reagent | pLenti-IRES-GFP-2Apro | This paper | Backbone: BsrGI digest of pLenti-IRES-GFP Insert: Synthetic DNA 11 | |
| Recombinant DNA reagent | pLenti-IRES-GFP-3Cpro | This paper | Backbone: BsrGI digest of pLenti-IRES-GFP Insert: Synthetic DNA 12 | |
| Recombinant DNA reagent | pLenti-IRES-H2A-iRFP | This paper | Backbone: PCR amplification of pLenti-IRES-GFP (primers MTI_1, MTI_7) Insert: PCR amplificaion of H2A-iRFP from MAP2-H2A-iRFP670 with addition of P2A site (primers MTI_5, MTI_13) | |
| Recombinant DNA reagent | pLenti-IRES-H2A-iRFP(P2A)2Apro | This paper | Backbone: PCR amplification of pLenti-IRES-GFP-2A (primers MTI_2, MTI_8) Insert: PCR amplificaion of H2A-iRFP from MAP2-H2A-iRFP670 with addition of P2A site (primers MTI_5, MTI_13) | |
| Recombinant DNA reagent | pLenti-IRES-H2A-iRFP(P2A)3Cpro | This paper | Backbone: PCR amplification of pLenti-IRES-GFP-3C (primers MTI_12, MTI_9) Insert: PCR amplificaion of H2A-iRFP from MAP2-H2A-iRFP670 with addition of P2A site (primers MTI_5, MTI_13) Insert: Synthetic DNA 23 | |
| Recombinant DNA reagent | NLS-tdTomato | This paper | PCR amplification of Shuttle-tdTomato, followed by recircularization (primers MJE_10, MJE_11) | |
| Recombinant DNA reagent | tdTomato-NES | This paper | PCR amplification of Shuttle-tdTomato, followed by recircularization (primers MJE_8, MJE_53) | |
| Recombinant DNA reagent | MAP2-pmTurquoise-H2A | This paper | Backbone: PCR amplification of pmTurquoise-H2A (primers KEB_1, KEB_2) Insert: PCR amplification of FU-MAP2-Gateway (primers KEB_3, KEB_4) | |
| Recombinant DNA reagent | MAP2-H2A-iRFP670 | This paper | Backbone: AgeI/NotI digest of MAP2-pmTurqoise-H2A Insert: Synthetic DNA 22, digested with AgeI/NotI | |
| Recombinant DNA reagent | peSUMOstar-2Apro | This paper | Backbone: BsaI digest of peSUMOSTAR Insert: Synthetic DNA 23 | |
| Sequence-based reagent | KEB_1 | This paper | PCR primers | CGGAACTCCATATATGGGCTATGAACTAAT |
| Sequence-based reagent | KEB_2 | This paper | PCR primers | GGTTTAGTGAACCGTCAGATCCGCA |
| Sequence-based reagent | KEB_3 | This paper | PCR primers | ATATATGGAGTTCCGCAGCTGGCCTTTTTGGTTCTCAT |
| Sequence-based reagent | KEB_4 | This paper | PCR primers | GGTTTAGTGAACCGTTCGGTTAGAGACAAGCTGAAGAATCTACCG |
| Sequence-based reagent | MJE_10 | This paper | PCR primers | CTCGAGCAGAAACTCATCTCAGAAGAGGATC |
| Sequence-based reagent | MJE_11 | This paper | PCR primers | TGAGTTTCTGCTCGAGCTGTAGCTTGTACAGCTCGTCCATGC |
| Sequence-based reagent | MJE_53 | This paper | PCR primers | CATGCCCCCGCCTCCCATGGTGGCACGCGTCG |
| Sequence-based reagent | MJE_69 | This paper | PCR primers | CACCGGCGGCCTAGCCGCCCCTCTC |
| Sequence-based reagent | MJE_70 | This paper | PCR primers | CACCGGCGGCCTAGCAACCATGG |
| Sequence-based reagent | MJE_8 | This paper | PCR primers | GGAGGCGGGGGCATGGTGAG |
| Sequence-based reagent | MTI_1 | This paper | PCR primers | GAGAACCCTGGACCTTCTAGATAACTGATCCTCTCCC |
| Sequence-based reagent | MTI_12 | This paper | PCR primers | GAGAACCCTGGACCTGGTCCAGGCTTCGGAGGAGTTTTTG |
| Sequence-based reagent | MTI_13 | This paper | PCR primers | AGGTCCAGGGTTCTCCTCCACGTCGCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCGCGTTGGTGGTGGG |
| Sequence-based reagent | MTI_2 | This paper | PCR primers | GAGAACCCTGGACCTGGTCCAGGCTTCGG |
| Sequence-based reagent | MTI_5 | This paper | PCR primers | AATATGGCCACAACCATGTCGGGACGTGGC |
| Sequence-based reagent | MTI_7 | This paper | PCR primers | GAGAACCCTGGACCTGGACCAGG |
| Sequence-based reagent | MTI_8 | This paper | PCR primers | GAGAACCCTGGACCTGGACCAGGGTTCG |
| Sequence-based reagent | MTI_9 | This paper | PCR primers | GAGAACCCTGGACCTGGACCAGGGTTCGATT |
| Sequence-based reagent | 1 | This paper | Synthetic DNA | GTCAGATCCGCTAGCGCCACCATGGGTCCAGGCTTCGGAGGAGTTTTTGTAGGGTCTTTTAAAATAATCAACTATCACTTGGCCACTACAGAAGAGAGACAGTCAGCTATCTATGTGGATTGGCAATCAGACGTCTTGGTTACCCCCATTGCTGCTCATGGAAGGCACCAAATAGCAAGATGCAAGTGCAACACAGGGGTTTACTATTGTAGGCACAAAAACAGAAGTTACCCGATTTGCTTTGAAGGCCCAGGGATTCAATGGATTGAACAAAATGAATATTACCCAGCAAGGTACCAGACCAATGTACTTTTGGCAGTTGGTCCTGCGGAAGCAGGAGATTGCGGTGGTTTACTAGTTTGTCCACATGGGGTAATCGGTCTTCTTACAGCAGGAGGGGGTGGAATTGTAGCTTTCACTGATATCAGGAATTTGCTATGGTTAGATACTGATGCTATGGAACAATGGGATCCACCGGTCG |
| Sequence-based reagent | 2 | This paper | Synthetic DNA | GTCAGATCCGCTAGCGCCACCATGGGATTCGGACACCAAAACAAAGCGGTGTACACTGCAGGTTACAAAATTTGCAACTACCACTTGGCCACTCAGGATGATTTGCAAAACGCAGTGAACGTCATGTGGAGTAGAGACCTCTTAGTCACAGAATCAAGAGCCCAGGGCACCGATTCAATCGCAAGGTGCAATTGCAACGCAGGGGTGTACTACTGCGAGTCTAGAAGGAAATACTACCCAGTATCCTTCGTTGGCCCAACGTTCCAGTACATGGAGGCTAATAACTATTACCCAGCTAGGTACCAGTCCCATATGCTCATTGGCCATGGATTCGCATCTCCAGGGGATTGTGGTGGCATACTCAGATGTCACCACGGGGTGATAGGGATCATTACTGCTGGTGGCGAAGGGTTGGTTGCATTTTCAGACATTAGAGACTTGTATGCCTACGAAGAAGAAGCCATGGAACAATGGGATCCACCGGTCG |
| Sequence-based reagent | 7 | This paper | Synthetic DNA | GTCAGATCCGCTAGCGCCACCATGGGACCAGGGTTCGATTTTGCACAAGCCATAATGAAGAAAAATACCGTCATTGCAAGGACTGAAAAGGGTGAGTTCACCATGCTGGGTGTATATGATAGGGTAGCGGTCATCCCCACACACGCATCTGTTGGAGAAACCATTTACATTAATGATGTAGAGACTAAAGTTTTAGATGCGTGTGCACTTAGAGACTTGACTGATACAAACTTAGAGATAACCATAGTCAAATTAGACCGTAATCAAAAATTTAGAGATATCAGACATTTTCTGCCCAGATATGAGGATGATTACAATGACGCTGTGCTTAGCGTACATACATCAAAATTCCCAAATATGTATATCCCAGTTGGACAAGTCACCAATTATGGCTTCTTGAACCTAGGTGGTACACCGACGCACCGCATTTTAATGTATAACTTCCCAACAAGAGCTGGCCAGTGTGGTGGTGTGGTGACAACTACAGGTAAGGTGATAGGAATACATGTAGGTGGAAATGGAGCTCAAGGATTTGCAGCAATGCTACTACACTCTTACTTTTCCGATACACAAGGTGAGATAGTTAGTAGTGAAAAGAGTGGGGTGTGCATTAACGCACCGGCAAAGACTAAACTCCAACCTAGTGTTTTCCATCAAGTTTTTTAATGGGATCCACCGGTCG |
| Sequence-based reagent | 8 | This paper | Synthetic DNA | GTCAGATCCGCTAGCGCCACCATGGGACCAGGGTTCGATTACGCAGTGGCTATGGCTAAAAGAAACATTGTTACAGCAACTACTAGCAAGGGAGAGTTCACTATGTTAGGAGTCCACGACAACGTGGCTATTTTACCAACCCACGCTTCACCTGGTGAAAGCATTGTGATCGATGGCAAAGAAGTGGAGATCTTGGATGCCAAAGCGCTCGAAGATCAAGCAGGAACCAATCTTGAAATCACTATAATCACTCTAAAGAGAAATGAAAAGTTCAGAGACATTAGACCACATATACCTACTCAAATCACTGAGACAAATGATGGAGTCTTGATCGTGAACACTAGCAAGTACCCCAATATGTATGTTCCTGTCGGTGCTGTGACTGAACAGGGATATCTAAATCTCGGTGGGCGCCAAACTGCTCGTACTCTAATGTACAACTTTCCAACCAGAGCAGGACAGTGTGGTGGAGTCATCACATGTACTGGGAAAGTCATCGGGATGCATGTTGGTGGGAACGGTTCACACGGGTTTGCAGCGGCCCTGAAGCGATCATACTTCACTCAGAGTCAATAATGGGATCCACCGGTCG |
| Sequence-based reagent | 11 | This paper | Synthetic DNA | ATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGTCCAGGCTTCGGAGGAGTTTTTGTAGGGTCTTTTAAAATAATCAACTATCACTTGGCCACTACAGAAGAGAGACAGTCAGCTATCTATGTGGATTGGCAATCAGACGTCTTGGTTACCCCCATTGCTGCTCATGGAAGGCACCAAATAGCAAGATGCAAGTGCAACACAGGGGTTTACTATTGTAGGCACAAAAACAGAAGTTACCCGATTTGCTTTGAAGGCCCAGGGATTCAATGGATTGAACAAAATGAATATTACCCAGCAAGGTACCAGACCAATGTACTTTTGGCAGTTGGTCCTGCGGAAGCAGGAGATTGCGGTGGTTTACTAGTTTGTCCACATGGGGTAATCGGTCTTCTTACAGCAGGAGGGGGTGGAATTGTAGCTTTCACTGATATCAGGAATTTGCTATGGTTAGATACTGATGCTATGGAACAATAAGTACAAGTAATCTAG |
| Sequence-based reagent | 12 | This paper | Synthetic DNA | ATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGACCAGGGTTCGATTTTGCACAAGCCATAATGAAGAAAAATACCGTCATTGCAAGGACTGAAAAGGGTGAGTTCACCATGCTGGGTGTATATGATAGGGTAGCGGTCATCCCCACACACGCATCTGTTGGAGAAACCATTTACATTAATGATGTAGAGACTAAAGTTTTAGATGCGTGTGCACTTAGAGACTTGACTGATACAAACTTAGAGATAACCATAGTCAAATTAGACCGTAATCAAAAATTTAGAGATATCAGACATTTTCTGCCCAGATATGAGGATGATTACAATGACGCTGTGCTTAGCGTACATACATCAAAATTCCCAAATATGTATATCCCAGTTGGACAAGTCACCAATTATGGCTTCTTGAACCTAGGTGGTACACCGACGCACCGCATTTTAATGTATAACTTCCCAACAAGAGCTGGCCAGTGTGGTGGTGTGGTGACAACTACAGGTAAGGTGATAGGAATACATGTAGGTGGAAATGGAGCTCAAGGATTTGCAGCAATGCTACTACACTCTTACTTTTCCGATACACAATAAGTACAAGTAATCTAG |
| Sequence-based reagent | 22 | This paper | Synthetic DNA | ATGGCGCGTAAGGTCGATCTCACCTCCTGCGATCGCGAGCCGATCCACATCCCCGGCAGCATTCAGCCGTGCGGCTGCCTGCTAGCCTGCGACGCGCAGGCGGTGCGGATCACGCGCATTACGGAAAATGCCGGCGCGTTCTTTGGACGCGAAACTCCGCGGGTCGGTGAGCTACTCGCCGATTACTTCGGCGAGACCGAAGCCCATGCGCTGCGCAACGCACTGGCGCAGTCCTCCGATCCAAAGCGACCGGCGCTGATCTTCGGTTGGCGCGACGGCCTGACCGGCCGCACCTTCGACATCTCACTGCATCGCCATGACGGTACATCGATCATCGAGTTCGAGCCTGCGGCGGCCGAACAGGCCGACAATCCGCTGCGGCTGACGCGGCAGATCATCGCGCGCACCAAAGAACTGAAGTCGCTCGAAGAGATGGCCGCACGGGTGCCGCGCTATCTGCAGGCGATGCTCGGCTATCACCGCGTGATGTTGTACCGCTTCGCGGACGACGGCTCCGGGATGGTGATCGGCGAGGCGAAGCGCAGCGACCTCGAGAGCTTTCTCGGTCAGCACTTTCCGGCGTCGCTGGTCCCGCAGCAGGCGCGGCTACTGTACTTGAAGAACGCGATCCGCGTGGTCTCGGATTCGCGCGGCATCAGCAGCCGGATCGTGCCCGAGCACGACGCCTCCGGCGCCGCGCTCGATCTGTCGTTCGCGCACCTGCGCAGCATCTCGCCCTGCCATCTCGAATTTCTGCGGAACATGGGCGTCAGCGCCTCGATGTCGCTGTCGATCATCATTGACGGCACGCTATGGGGATTGATCATCTGTCATCATTACGAGCCGCGTGCCGTGCCGATGGCGCAGCGCGTCGCGGCCGAAATGTTCGCCGACTTCTTATCGCTGCACTTCACCGCCGCCCACCACCAACGCTAA |
| Sequence-based reagent | 23 | This paper | Synthetic DNA | GAACAGATTGGAGGTGGTCCTGGTTTTGGTGGCGTCTTTGTGGGTTCTTTCAAAATAATTAACTACCACCTGGCGACGACAGAGGAACGCCAATCAGCGATTTATGTTGACTGGCAAAGCGATGTCTTGGTTACCCCGATTGCAGCACACGGACGTCACCAAATTGCCCGGTGTAAATGCAATACGGGAGTATATTATTGTAGACATAAGAACCGTAGTTACCCCATTTGTTTTGAAGGACCAGGCATCCAATGGATCGAGCAGAACGAGTACTACCCAGCCAGATACCAAACGAACGTACTGCTGGCGGTGGGTCCTGCGGAAGCTGGCGATTGTGGGGGCTTGTTGGTGTGTCCGCACGGCGTAATTGGATTACTGACTGCCGGGGGTGGTGGTATCGTGGCGTTTACTGATATTCGCAATCTTCTGTGGTTAGATACTGATGCCATGGAACAATGATGACTAGAGGATCCGAAT |
| Other | Complete Media (CM) | Cell culture media | Dulbecco’s modified Eagle medium (Thermo Fisher 11995073) 10% fetal bovine serum (Thermo Fisher 16140071) 1% penicillin/streptomycin (Thermo Fisher 15140122) 2 mM L-glutamine (Thermo Fisher 25030081) 1 mM Sodium Pyruvate (Millipore Sigma S8636) | |
| Other | Infection Media (IM) | Cell culture media | Dulbecco’s modified Eagle medium (Thermo Fisher 11995073) 2.5% fetal bovine serum (Thermo Fisher 16140071) 1% penicillin/streptomycin (Thermo Fisher 15140122) 2 mM L-glutamine (Thermo Fisher 25030081) 1 mM Sodium Pyruvate (Millipore Sigma S8636) | |
| Other | Stage 1 diMN Media (S1) | Li et al., 2021 | Cell culture media | 50% Iscove’s Modified Dulbecco’s Medium (IMDM) (Thermo Fisher 12440053) 50% Ham’s F12 nutrient mix (Thermo Fisher 11765062) 1× Non-essential amino acids (Thermo Fisher 11140050) 1× Penicillin-Streptomycin (Thermo Fisher 15140122) 1× B27 Supplement (Thermo Fisher 17504044) 1× N2 Supplement (Thermo Fisher 17502048) 0.2 µM LDN-193189 (Stemgent 04-0074) 10 µM SB431542 (STEMCELL Technologies 72234) 3 µM CHIR99021 (Millipore Sigma SML1046) |
| Other | Stage 2 diMN Media (S2) | Li et al., 2021 | Cell culture media | 50% IMDM (Thermo Fisher 12440053) 50% Ham’s F12 nutrient mix (Thermo Fisher 11765062) 1× Non-essential amino acids (Thermo Fisher 11140050) 1× Penicillin-Streptomycin (Thermo Fisher 15140122) 1× B27 Supplement (Thermo Fisher 17504044) 1× N2 Supplement (Thermo Fisher 17502048) 0.2 µM LDN-193189 (Stemgent 04-0074) 10 µM SB431542 (STEMCELL Technologies 72234) 3 µM CHIR99021 (Millipore Sigma SML1046) 0.1 µM All-trans retinoic acid (Millipore Sigma R2625) 1 µM SAG (Cayman Chemicals 11914) |
| Other | Stage 3 diMN Media (S3) | Li et al., 2021 | Cell culture media | 50% IMDM (Thermo Fisher 12440053) 50% Ham’s F12 nutrient mix (Thermo Fisher 11765062) 1× Non-essential amino acids (Thermo Fisher 11140050) 1× Penicillin-Streptomycin (Thermo Fisher 15140122) 1× B27 Supplement (Thermo Fisher 17504044) 1× N2 Supplement (Thermo Fisher 17502048) 0.1 µM Compound E (Millipore Sigma 565790) 2.5 µM DAPT (Millipore Sigma D5942) 0.1 µM db-cAMP (Millipore Sigma 28745) 0.5 µM All-trans retinoic acid (Millipore Sigma R2625) 1 µM SAG (Cayman Chemicals 11914) 200 ng/mL Ascorbic acid (Millipore Sigma A4544) 10 ng/mL Brain-derived neurotrophic factor (PeproTech 450-02) 10 ng/mL Glial cell-derived neurotrophic factor (PeproTech 450-10) |
| Other | PBS+ | Cell culture solution | Phosphate-buffered saline (Gibco 10010023) 100 g/L MgCl2 (Sigma) 100 g/L CaCl2 (Sigma) |
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Enterovirus D68 2A protease causes nuclear pore complex dysfunction and independently contributes to motor neuron toxicity
eLife 14:RP108672.
https://doi.org/10.7554/eLife.108672.3
