Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

doi: 10.1073/pnas.2306381120

Figure Lengend Snippet: Fig. 1. Protein-splicing activity of an Anaeramoeba singleton intein. In vitro splicing of (A and B) A. ignava intein Aig g14419 and (C and D) Synechocystis sp. strain PCC6803 (Ssp) DnaX mini-intein. Metal ion affinity-purified precursor from inclusion bodies produced by E. coli BL21 (DE3) transformed with plasmid GFPuv-Aig g14419 or GFPuv-Ssp DnaX in buffer B was renatured by dilution in modified buffer C as follows: (−TCEP) A small amount of purified protein (30 µL) was diluted in buffer C without tris(2-carboxyethyl)phosphine (TCEP), (+TCEP) was diluted into buffer C with 1 mM TCEP, (+TCEP, Zn2+) was diluted into buffer C supplemented with 1 mM TCEP, 2 mM ZnCl2 but without Ethylenediaminetetraacetic acid (EDTA), and (+TCEP, Zn2+, +EDTA) was diluted in buffer C with 1 mM TECP, 2 mM ZnCl2, and 4 mM EDTA. After dilution, the samples were incubated for 14 h at 20 °C and analyzed (A and C) for fluorescence upon excitation at 395 nm (B and D) for protein splicing by SDS-PAGE. Sizes of expected precursors, spliced GFPs, and excised inteins are indicated. Fluorescence intensity is in arbitrary units. The size markers are PageRuler Unstained Protein Ladder (#26614, Thermo Scientific).

Article Snippet: Curiously, multiple Anaeramoeba proteins belonging to these categories were found to be invaded by inteins: two types of Dynein (Cytoplasmic dynein 1 heavy chain 1 and Dynein heavy chain 10, axonemal isoform) and one Kinesin for Cytoskeleton (Z); and two different Helicases in category B (Chromatin remodeling protein HARP/SMARCAL1 and Chromatin remodeling complex SWI/SNF, component SWI2) (Fig. 2).

Techniques: Activity Assay, In Vitro, Affinity Purification, Produced, Transformation Assay, Plasmid Preparation, Modification, Purification, Incubation, Fluorescence, SDS Page

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

doi: 10.1073/pnas.2306381120

Figure Lengend Snippet: Fig. 2. Host proteins for Anaeramoeba inteins are functionally diverse. KOG-based functional categories of intein-containing proteins in three strains of Anaeramoeba. INFORMATION STORAGE AND PROCESSING: [J] Translation, ribosomal structure and biogenesis; [A] RNA processing and modification; [K] Transcription; [L] Replication, recombination and repair; [B] Chromatin structure and dynamics. CELLULAR PROCESSES AND SIGNALING: [D] Cell cycle control, cell division, chromosome partitioning; [Y] Nuclear structure; [V] Defense mechanisms; [T] Signal transduction mechanisms; [M] Cell wall/membrane/envelope biogenesis; [N] Cell motility; [Z] Cytoskeleton; [W] Extracellular structures; [U] Intracellular trafficking, secretion, and vesicular transport; [O] Posttranslational modification, protein turnover, chaperones. METABOLISM: [C] Energy production and conversion; [G] Carbohydrate transport and metabolism; [E] Amino acid transport and metabolism; [F] Nucleotide transport and metabolism; [H] Coenzyme transport and metabolism; [I] Lipid transport and metabolism; [P] Inorganic ion transport and metabolism; [Q] Secondary metabolites biosynthesis, transport and catabolism. POORLY CHARACTERIZED: [R] General function prediction only; [S] Function unknown.

Article Snippet: Curiously, multiple Anaeramoeba proteins belonging to these categories were found to be invaded by inteins: two types of Dynein (Cytoplasmic dynein 1 heavy chain 1 and Dynein heavy chain 10, axonemal isoform) and one Kinesin for Cytoskeleton (Z); and two different Helicases in category B (Chromatin remodeling protein HARP/SMARCAL1 and Chromatin remodeling complex SWI/SNF, component SWI2) (Fig. 2).

Techniques: Functional Assay, Modification, Control, Transduction, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

doi: 10.1073/pnas.2306381120

Figure Lengend Snippet: Fig. 3. Anaeramoeba inteins are evolutionarily diverse. Sequence Similarity Network (SSN) of inteins in A. ignava strain BMAN, A. flamelloides strains BUSSELTON2 and SCHOONER1, and related inteins found in public databases. The edges between the nodes are a measure of the similarity between nodes (score threshold corresponding to 30% identity on average). Anaeramoebae inteins belong to 18 different clusters corresponding to at least as many acquisition events. Some inteins do not show obvious sequence similarity with any other known inteins and are singleton or “pseudo-singleton” clusters (see main text). Inteins in viral-like sequences in eukaryotic genomes are designated EVEs (Endogenous Viral Elements). Note: “+”: HE domain detected in Anaeramoeba intein using one or both methods (HEs in the inteins of other organisms are not highlighted); *In orthologous site; **In homologous site.

Article Snippet: Curiously, multiple Anaeramoeba proteins belonging to these categories were found to be invaded by inteins: two types of Dynein (Cytoplasmic dynein 1 heavy chain 1 and Dynein heavy chain 10, axonemal isoform) and one Kinesin for Cytoskeleton (Z); and two different Helicases in category B (Chromatin remodeling protein HARP/SMARCAL1 and Chromatin remodeling complex SWI/SNF, component SWI2) (Fig. 2).

Techniques: Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

doi: 10.1073/pnas.2306381120

Figure Lengend Snippet: Fig. 4. Modalities of intein spread in Anaeramoeba are diverse. (A) Phylogenetic tree of cluster 17 inteins inferred under the C20+G4 mixture model and 1,000 ultrafast bootstraps from 191 sites. A. ignava BMAN inteins are in blue, A. flamelloides inteins are in pink, viral inteins are in red, and eukaryotic inteins are in green. Viral inteins are interspersed among inteins from diverse eukaryotes including Anaeramoeba, suggesting that viruses are involved in the spread of inteins between eukaryotes. The expanded tree (SI Appendix, Fig. S4) shows that viruses could also be involved in the transfer of inteins between nuclear and chloroplast homologs (SI Appendix, section 9). (B) Phylogenetic tree of cluster 15 inteins inferred under the C20+G4 mixture model and 1,000 ultrafast bootstraps. The sequences were aligned with mafft-linsi and 300 sites with <20% gaps were selected. An expanded tree is presented in SI Appendix, Fig. S5. Color-coding is the same as in part A. A potential intragenic intein transfer between site b and site d in A. flamelloides Prp8 is marked in pink. An intragenomic intein transfer appears to have taken place between the Signal Recognition Particle Receptor (SRP) and Translation initiation factor IF-2P (TIF) in a Rhizophagus ancestor. *The source of the intein labeled “Anthurium amnicola” is unclear.

Article Snippet: Curiously, multiple Anaeramoeba proteins belonging to these categories were found to be invaded by inteins: two types of Dynein (Cytoplasmic dynein 1 heavy chain 1 and Dynein heavy chain 10, axonemal isoform) and one Kinesin for Cytoskeleton (Z); and two different Helicases in category B (Chromatin remodeling protein HARP/SMARCAL1 and Chromatin remodeling complex SWI/SNF, component SWI2) (Fig. 2).

Techniques: Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

doi: 10.1073/pnas.2306381120

Figure Lengend Snippet: Fig. 5. Location of inteins and introns in paralogous proteins in Anaeramoeba. (A) Msh family proteins (Msh2, 3, 4, 5, and 6). (B) Family B-DNA polymerases (DNApol epsilon, alpha, and delta). (C) MCM family proteins (MCM3, 4, 5, 6, 7, 8, and 9; note that in the lower half of the panel, only MCMs with introns near intein insertion sites are shown; all such introns are in A. ignava). *Novel site described in this study. (D) Phylogenetic reconstruction of MCM site b inteins inferred under the C20+G4 mixture model and 1,000 ultrafast bootstraps. Sequences were aligned with mafft-linsi, and 145 sites with <10% gaps were selected. A. ignava BMAN inteins are in blue, A. flamelloides inteins for both strains are in pink.

Article Snippet: Curiously, multiple Anaeramoeba proteins belonging to these categories were found to be invaded by inteins: two types of Dynein (Cytoplasmic dynein 1 heavy chain 1 and Dynein heavy chain 10, axonemal isoform) and one Kinesin for Cytoskeleton (Z); and two different Helicases in category B (Chromatin remodeling protein HARP/SMARCAL1 and Chromatin remodeling complex SWI/SNF, component SWI2) (Fig. 2).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

doi: 10.1073/pnas.2306381120

Figure Lengend Snippet: Fig. 6. Intragenomic intein transfers have occurred in Anaeramoeba. (A) Inteins c and c’ form their own network cluster (cluster 14) and are inserted at the same site in the BRB2 helicase domain of activating signal cointegrator 1 complex subunit 3 (ASC1S3). Inteins b and b’ are singletons but are best matches to each other, suggesting that they have a common origin. They are also inserted at the same site in the BRB2 domain. DNApol theta/eta (POLQ) inteins are next to each other in the BRB2 domain, while the ASC1S3 inteins are each present in one of the two BRB2 domains encoded by ASC1S3. (B) All Anaeramoeba cluster 1 inteins are inserted in P-loop motifs (shown at right) and branch together in the phylogenetic tree. The ML phylogeny was inferred under the C20+G mixture model with 1,000 ultrafast bootstrap replicates. The sequences were aligned with MUSCLE (40) (default parameters) and 115 amino acid sites manually selected. These data suggest intein transfers between P-loops in unrelated proteins.

Article Snippet: Curiously, multiple Anaeramoeba proteins belonging to these categories were found to be invaded by inteins: two types of Dynein (Cytoplasmic dynein 1 heavy chain 1 and Dynein heavy chain 10, axonemal isoform) and one Kinesin for Cytoskeleton (Z); and two different Helicases in category B (Chromatin remodeling protein HARP/SMARCAL1 and Chromatin remodeling complex SWI/SNF, component SWI2) (Fig. 2).

Techniques:

Journal: bioRxiv

Article Title: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

doi: 10.1101/2023.12.12.571328

Figure Lengend Snippet: (a) Schematic of (top) the C9ORF72 gene and (bottom) the three main mRNA transcript variants expressed from it. V1 produces the short protein isoform (C9-S), while V2 and V3 produce the long protein isoform (C9-L). (b, c) Schematic of the dual-reporter system used to evaluate crRNAs. The platform consists of a (b) Renilla luciferase-encoding plasmid, pSV40-RLuc, whose 3’ untranslated region (UTR) carries a fragment of the C9ORF72 gene with 20 copies of the hexanucleotide repeat and 250-and 98-base pairs (bps) of the flanking upstream and downstream gene sequences, respectively, and (c) a firefly luciferase-encoding plasmid, pHSV-TK-FLuc, which was used as a proxy for collateral cleavage. (d) Normalized Renilla and firefly luciferase expression in HEK293T cells transfected with pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d and one of the 15 candidate crRNAs. All values were normalized to cells transfected with pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d with a non-targeted (NTG) crRNA ( n = 3). (e, f) Relative all-V and V3 mRNA in (e) HEK293T and (f) SH-SY5Y cells transfected with RfxCas13d and crRNAs 13, 7, and 1 or a NTG crRNA or one of two ASOs ( n = 3). All values from HEK293T and SH-SY5Y cells were normalized to untreated cells. Values indicate means and error bars indicate SD. **P < 0.01, ***P < 0.001, ****P < 0.0001; one-tailed unpaired t-test comparing each crRNA to the NTG crRNA. All data points are biologically independent samples.

Article Snippet: Neural progenitor cells (NPCs) derived from a 74-year-old female ALS patient with >145 copies of the hexanucleotide repeat expansion in the C9ORF72 gene were obtained from AXOL Bioscience (ax0073).

Techniques: Luciferase, Plasmid Preparation, Expressing, Transfection, One-tailed Test

Journal: bioRxiv

Article Title: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

doi: 10.1101/2023.12.12.571328

Figure Lengend Snippet: (a) Cartoon illustrating the injection scheme. (b) Overview of the experimental plan to analyze C9ORF72 mRNA in EGFP-KASH + nuclei isolated by fluorescence-activated cell sorting (FACS). (c ) Representative immunofluorescent staining of the hippocampus (HPC) and motor cortex (MC) in C9-BACexp mice two-months after injection with 2 x 10 10 GCs each of AAV-PHP.eB-RfxCas13d-crRNA and AAV-PHP.eB-EGFP-KASH. Scale bar, 20 µm. (d, e) Relative all-V and V3 mRNA in (d) the HPC and (e) MC of EGFP-KASH + nuclei from C9-BACexp injected with AAV-PHP.eB-RfxCas13d-crRNA-13, -7, -1, or -NTG with AAV-PHP.eB-EGFP-KASH ( n ≥ 7). (f) Representative RNA FISH to detect the G 4 C 2 repeat RNA (purple) from the HPC of C9-BACexp mice injected with AAV-PHP.eB-RfxCas13d-crRNA-13 or -NTG and AAV-PHP.eB-EGFP-KASH. Arrowheads indicate representative cells. Scale bar, 15 µm. (g, h) Quantification of (g) the number of RNA foci per EGFP-KASH + cell in the HPC and (h) the percentage of EGFP-KASH + cells with 0, 1-2, 3-4, or >5 foci ( n ≥ 4). (g, h) 58-455 cells were counted per animal. 398, 845, and 467 cells total were counted for AAV-PHP.eB-RfxCas13d-crRNA-7, crRNA-13, and -NTG respectively ( n ≥ 4). RNA foci measurements were conducted by a blinded investigator. Values indicate means and error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001; one-tailed unpaired t-test comparing each crRNA to the NTG crRNA. All data points are biologically independent samples.

Article Snippet: Neural progenitor cells (NPCs) derived from a 74-year-old female ALS patient with >145 copies of the hexanucleotide repeat expansion in the C9ORF72 gene were obtained from AXOL Bioscience (ax0073).

Techniques: Injection, Isolation, Fluorescence, FACS, Staining, One-tailed Test

Journal: Nature communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking.

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: Fig. 9 CXCL12 mediates ATF4 upregulation via CXCR4. a Cultured DRG neurons were treated with CXCL12 (1 μg/mL) for 120, 240 or 360 min, and then ATF4 expression was measured. n = 6. F(3,20) = 4.642, P = 0.0462 in 120 min, P = 0.0154 in 240 min, P = 0.0128 in 360 min. *P < 0.05 versus 0 min. b CXCL12 (1 μg in 10 μL PBS + 0.5% BSA) was intrathecally injected three times every 3 h, and ATF4 expression was measured in the DRG 2 h after the last injection. n = 6 mice per group. t10 = 5.151, P = 0.0004. **P < 0.01. c ATF4 siRNA significantly relieved CXCL12-induced thermal hyperalgesia. n = 12 mice per group. F(2,33) = 15.68, P < 0.0001 in vehicle vs. CXCL12, P = 0.0101 in CXCL12 vs. CXCL12 + ATF4-siRNA. *P < 0.05, **P < 0.01. d CXCR4 siRNA but not CXCR7 siRNA abolished the increase in ATF4 expression in the DRG induced by CXCL12 in vivo. n = 6 mice per group. F(3,20) = 54.47, P < 0.0001 in CXCL12, CXCL12 + CXCR4-siRNA and CXCL12 + CXCR7-siRNA. **P < 0.01 versus the vehicle group, ##P < 0.01 versus the CXCL12 group. e Hypothetical model illustrating that ATF4 interacts with TRPM3 and KIF17 to form a complex to regulate the membrane trafficking of TRPM3 in sensory neurons and thus contributes to thermal sensitivity. a, c, d One-way ANOVA followed by Tukey’s multiple comparisons test. b Two-tailed Independent Student’s t test. The error bars indicate the SEMs.

Article Snippet: To evaluate the direct interaction between ATF4 and TRPM3 or KIF17, 5 μg purified ATF4 protein (Proteintech, Ag16665) was incubated with 5 μg TRPM3-GST-Flag or KIF17-GST-Flag purified protein in 400 μl RIPA buffer.

Techniques: Cell Culture, Expressing, Injection, In Vivo, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: Cytotoxicity of SB743921 against mitotic inhibitors in cells with the wild type (WT) and knocked out p53 ( TP53-/- ). A , IC50 values (mean ± SD, n=3) of A549, MCF7 and HCT116 exposed to various mitotic inhibitors for 7 days were obtained based on the SRB assay. B , Proliferative and colony formation ability of A549 WT and A549 TP53-/- after 7 d or 14 d from the removal of SB743921 (SB) or Ixabepilone (Ixa). Cells were exposed to drugs for 72 h and then incubated in fresh media the following 7 d or 14 d. C, Cell cycle distribution of A549 WT and A549 TP53-/- ( left panels ) and MCF7 WT and MCF7 TP53-/- ( right panels ) treated with SB (10 nM) or Ixa (25 nM) for 24 h ( upper panels ) and 72 h ( bottom panels ).

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Sulforhodamine B Assay, Incubation

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: p53-mediated cell cycle inhibitory and proapoptotic pathways are activated in response to the exposure of SB743921 (SB) or Ixabepilone (Ixa). A, Immunoblotting analysis of MCF7 and A549 treated with SB or Ixa in different concentrations (SB 0.5 nM and 10 nM, Ixa 2 nM and 25 nM) for 24 h. B, C , qPCR analysis of MCF7 and A549 respectively treated with SB (10 nM for MCF7, 3 nM for A549) or Ixa (25 nM) for 16 h (A549) or 24 h (MCF7).

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: SB743921 induces irreversible proliferative arrest and predominant death of A549 TP53-/- cells. A , B , Cell cycle distribution after 6 days after removal of SB743921 (SB) or Ixabepilone (Ixa). Cells were exposed to the drugs for 24 h, washed and incubated for 3 or 6 d in fresh media. C , Confocal microscopy images of EdU-treated samples after a week since the removal of SB or Ixa. Cells were exposed to drugs for 72 h before the washout. D , Single cell concentrations in flow cytometry analysis as a percentage of the untreated control samples. *, P < 0.05, **, P < 0.01; ns – not significant.

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Incubation, Confocal Microscopy, Single Cell, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: SB743921 depletes pro-replicative signaling causing irreversible arrest, while p53 is crucial for G1/S arrest in WT cells. A , Immunoblotting analysis in A549 WT and A549 TP53-/- cells 6 days after removal of SB743921 (SB) or Ixabepilone (Ixa). Cells were exposed to drugs for 24 h, then washed and incubated for 6 d in fresh media. C , qPCR analysis of A549 WT and A549 TP53-/- expression after 6 days since the removal of SB or Ixa. Cells were exposed to drugs for 24 h before the washout.

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Western Blot, Incubation, Expressing

Journal: bioRxiv

Article Title: Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

doi: 10.1101/2025.01.23.634373

Figure Lengend Snippet: SB743921 induces irreversible senescence in A549 WT and SB743921-treated senescent cells can be targeted by senolytics, such as ABT-263. A , Light microscopy images of A549 WT 6 days after the removal of SB743921 (SB) or Ixabepilone (Ixa). Cells were fixed and then stained with solution for the SA-β-Gal detection. B, Quantitative analysis of stained A549 WT and A549 TP53-/- cells as demonstrated in A . Data were compared using the ANOVA test; **, P < 0.01, ****, P < 0.0001; ns – not significant. C, Crystal violet staining of A549 WT and A549 TP53-/- treated with SB or Ixa alone or together with 3 uM ABT-263.

Article Snippet: The following drugs were used: KSP (Eg5) inhibitor SB743921 (SB, Medchem Express), Ixabepilone (Ixa, Swords laboratories), Paclitaxel (Pacl, Fujian South Pharmaceutical), Vincristine (Vinc, Teva), Nocodazole (Noc, Calbiochem), MDM2 inhibitor Nutlin-3 (SelleckChem), Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 (ApexBio) and Doxorubicin (SelleckChem).

Techniques: Light Microscopy, Staining

Journal: Scientific reports

Article Title: A directional 3D neurite outgrowth model for studying motor axon biology and disease.

doi: 10.1038/s41598-021-81335-z

Figure Lengend Snippet: Figure 1. Motor neuron progenitors form 3D networks in the OrganoPlate. (A) Image of an OrganoPlate 3-lane. The OrganoPlate is a microfluidic platform based on a 384-well plate design containing 40 individual tissue culture chips. The image is copyright of MIMETAS BV. (B) Each tissue culture chip contains 3 microfluidic lanes that join in the center of the chip. The 3-lane architecture in the chips’ center is assured by phaseguides that enable patterning of extracellular matrix gel. Inlet and outlet wells allow access to each lane: top inlet (TI) and top outlet (TO) wells allow access to top lane; middle inlet (MI) and outlet (MO) wells to middle lane; and bottom inlet (BI) and outlet (BO) wells to the bottom lane. Matrigel-GFR without cells is added to the middle lane. Following gelation, motor neuron progenitors embedded in matrigel-GFR are seeded in the top lane. (C) Schematic overview of cell seeding procedure. Matrigel-GFR is seeded in the middle lane and MNPs embedded in matrigel-GFR are seeded in the top lane (panel 1). MNPs form networks in the top lane (panel 2), after which neurites protrude into the middle and eventually bottom lane (panel 3). Blue color indicates presence of gel, red color indicates presence of medium. (D) Schematic overview of the spatial segregation of motor neuron somata (top lane) and axons (middle and bottom lanes) in OrganoPlate 3-lane cultures.

Article Snippet: Cell culture. iPSC-derived motor neuron progenitors (MNPs) (Axol, ax0078) were expanded in T75 flasks (734-2705, Corning, NY, USA) coated with 1:100 matrigel-GFR (Corning, 356341).

Techniques:

Journal: Scientific reports

Article Title: A directional 3D neurite outgrowth model for studying motor axon biology and disease.

doi: 10.1038/s41598-021-81335-z

Figure Lengend Snippet: Figure 2. Progenitors differentiate into motor neurons and extend their axons into an adjacent gel lane. (A) MNPs were differentiated in the OrganoPlate for 17 days and immunostained for motor neuron markers SMI- 32, ISL1, and CHAT. Maximum projection images of neuronal networks in the top lane are shown, the scale bar is 200 µm. (B) mRNA expression levels of induced pluripotent stem cells (iPSC), motor neuron progenitors (MNP) and motor neurons differentiated for 17 days (MN d17) in the OrganoPlate. Expression of markers of neural stem cells (NANOG, SOX2, PAX6), motor neuron progenitors (OLIG2), motor neurons (ISL1, CHAT, RBFOX3, VACHT, NFH), mature neurons (SYN1/SYNAPSIN, MAP2) and glial cells (S100β) was evaluated. Heatmap shows fold-change values as compared to expression in iPSCs, and the graphic representation was created using GraphPad Prism, version 8.3.1 (https://www.graphpad.com/scientific-software/prism/). (C) Motor neurons (day 17) were immunostained for dendritic marker MAP2 (red) and axonal marker TAU (green). Neurites protruding into the middle lane are mostly TAU-positive and MAP2-negative, indicating a predominantly axonal nature. Maximum projection images, the scale bar is 200 µm.

Article Snippet: Cell culture. iPSC-derived motor neuron progenitors (MNPs) (Axol, ax0078) were expanded in T75 flasks (734-2705, Corning, NY, USA) coated with 1:100 matrigel-GFR (Corning, 356341).

Techniques: Expressing, Software, Marker