pkg0743 ppv1 zf43x6 c comet addgene  (Addgene inc)

Journal: Cell & Bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: HOIP specifically binds to p31 comet . A . Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates (IP) derived from asynchronous 293 T cells transfected with Flag-HOIP and the indicated HA-tagged constructs. EV: empty vector. B . IB analysis of WCL, anti-HA, and anti-Flag IP derived from asynchronous 293 T cells transfected with HA-p31 comet and the indicated Flag-tagged constructs. C . Domain structures of p31 comet and HOIP. The ubiquitinated lysines are indicated in the p31 comet domain structure. Functional domains are indicated in the HOIP domain structure. Catalytically inactive HOIP mutants are indicated at the bottom. D . IB analysis of WCL, anti-HA, and anti-Flag IP derived from asynchronous 293 T cells transfected with HA-p31 comet and the indicated Flag-HOIP constructs. E . IB analysis of WCL and anti-Flag IP derived from asynchronous 293 T cells transfected with Flag-HOIP and the indicated HA-p31 comet constructs. F . p31 comet bound to HOIP in vitro. Immunopurified Flag-HOIP was incubated with the indicated recombinant GST proteins before washing and being resolved by SDS-PAGE and IB. G . Immunofluorescent staining of HeLa cells using anti-HOIP and anti-p31 comet antibodies, DAPI was used for DNA staining. Representative images shown here are from a mitotic HeLa cell. (See also Figure S2)

Article Snippet: pcDNA5 FRT TO myc p31 Comet , Addgene , 59,833.

Techniques: Western Blot, Derivative Assay, Transfection, Construct, Plasmid Preparation, Functional Assay, In Vitro, Incubation, Recombinant, SDS Page, Staining

Journal: Cell & Bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: HOIP promotes linear ubiquitination of p31 comet . A. Immunoblot (IB) analysis of whole cell lysates (WCL) and Ni–NTA (Ni-nitrilotriacetic acid) affinity precipitates derived from 293T cells transfected with HA-p31 comet , Flag-HOIP and the indicated His-UB constructs. The indicated lysine residue denotes that all but the indicated lysine (K) was mutated to arginine (R). K0: all the seven lysines of the ubiquitin were mutated to arginine. B . IB analysis of WCL and Ni–NTA affinity precipitates derived from 293T cells transfected with HA-p31 comet , Flag-HOIP and the indicated His-UB constructs. K0-ΔGG denotes the K0-UB with the two C-terminal glycine (G) residues deleted. C . IB analysis of WCL and Ni–NTA affinity precipitates derived from 293T cells transfected with HA-p31 comet , His-UB, and the indicated Flag-HOIP constructs. D . Bacterially purified recombinant His-p31 comet proteins were incubated with E1, E2, ubiquitin (UB), immune-purified WT, and catalytic inactive (R2-3 CS + HA) Flag-HOIP as indicated at 30 °C for 60 min before resolved by SDS-PAGE and IB analysis. E . IB analysis of WCL and Ni–NTA affinity precipitates derived from control (Ctrl) and sgHOIP-293T cells transfected with HA-p31 comet and His-UB constructs as indicated. 293T cells were treated with 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off. F . Crystal structure of p31 comet (PDBID 2QYF) was superimposed with the AlphaFold predicted structure to highlight the localization of the C-terminal lysine residues. G . IB analysis of WCL and Ni–NTA affinity precipitates derived from 293 T cells transfected with Flag-HOIP, His-UB, and the indicated HA-p31 comet constructs. (See also Figure S3)

Article Snippet: pcDNA5 FRT TO myc p31 Comet , Addgene , 59,833.

Techniques: Ubiquitin Proteomics, Western Blot, Derivative Assay, Transfection, Construct, Residue, Purification, Recombinant, Incubation, SDS Page, Control

Journal: Cell & Bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: Delayed mitotic exit is observed in HOIP-depleted cells. A–B Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from control (sgGFP) or HOIP-deleted (sgHOIP) HeLa ( A ) and HCT116 ( B ) cells. Cells were arrested in mitosis using 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off and released back into the cell cycle in media without nocodazole. Cells were harvested at the indicated time points after nocodazole release. C–D . IB analysis of WCL derived from HCT116 ( C ) and HeLa ( D ) cells transduced with shScr (as the negative control) or the indicated lentiviral sh p31 constructs. E. IB analysis of WCL derived derived from control (sgGFP) or p31 comet -deleted (sgp31) HCT116 cells. Cells were arrested in mitosis using 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off and released back into the cell cycle in media without nocodazole. Cells were harvested at the indicated time points after nocodazole release. F–G. Time-lapse microscopy of sgGFP and sgHOIP HeLa cells stably expressing a H2B-YFP retroviral construct. F Representative images showing that sgHOIP-HeLa took a significantly longer time to complete the mitosis. G Quantification of the recorded mitotic duration times (minutes) of sgGFP and sgHOIP cells, n = 200, Student’s t-test. H–I. Time-lapse microscopy of sgGFP and sgp31 H2B-YFP-HeLa cells. H Representative images showing that sgp31-HeLa took a significantly longer time to complete the mitosis. I Quantification of the recorded mitotic duration times (minutes) of sgGFP and sgHOIP cells, n = 50, Student’s t-test. (See also Figure S4)

Article Snippet: pcDNA5 FRT TO myc p31 Comet , Addgene , 59,833.

Techniques: Western Blot, Derivative Assay, Control, Transduction, Negative Control, Construct, Time-lapse Microscopy, Stable Transfection, Expressing, Retroviral

Journal: Cell & Bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: Cells expressing a linear ubiquitination-deficient p31 comet exhibit prolonged mitosis. A. Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-ubiquitin immunoprecipitates (IP) derived from HEK293 cells. Cells were treated with 50 ng∙ml −1 TNFα for 10 min as indicated before harvest. B . IB analysis of WCL and anti-HA IP derived from HEK293 cells. C . IB analysis of WCL and anti-HA IP derived from HEK293 cells. Cells were treated with 50 ng∙ml −1 TNFα for 10 min as indicated before harvest. D . IB analysis of WCL and anti-HA IP derived from 293 T cells transfected with Myc-p31 comet , HA-PLK1, His-UB and Flag-HOIP as indicated. E–F . Time-lapse microscopy of sgp31-H2B-YFP-HeLa cells stably expressing WT or 5KR-p31 comet . E Representative images showing that 5KR-p31-HeLa cells took a significantly longer time to complete the mitosis. F Quantification of the recorded mitotic duration times (minutes) of WT- and 5KR-p31 comet expressing cells, n = 50, Student’s t-test. (See also Figure S5)

Article Snippet: pcDNA5 FRT TO myc p31 Comet , Addgene , 59,833.

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Derivative Assay, Transfection, Time-lapse Microscopy, Stable Transfection

Journal: Cell & bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation.

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: Fig. 5 Cells expressing a linear ubiquitination-deficient p31comet exhibit prolonged mitosis. A. Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-ubiquitin immunoprecipitates (IP) derived from HEK293 cells. Cells were treated with 50 ng∙ml−1 TNFα for 10 min as indicated before harvest. B. IB analysis of WCL and anti-HA IP derived from HEK293 cells. C. IB analysis of WCL and anti-HA IP derived from HEK293 cells. Cells were treated with 50 ng∙ml−1 TNFα for 10 min as indicated before harvest. D. IB analysis of WCL and anti-HA IP derived from 293 T cells transfected with Myc-p31comet, HA-PLK1, His-UB and Flag-HOIP as indicated. E–F. Time-lapse microscopy of sgp31-H2B-YFP-HeLa cells stably expressing WT or 5KR-p31comet. E Representative images showing that 5KR-p31-HeLa cells took a significantly longer time to complete the mitosis. F Quantification of the recorded mitotic duration times (minutes) of WT- and 5KR-p31comet expressing cells, n = 50, Student’s t-test. (See also Figure S5)

Article Snippet: HOIL-1L MilliporeSigma clone 2E2 MABC576 TUBULIN MilliporeSigma T-5168 VINCULIN MilliporeSigma V-4505 Flag (polyclonal) MilliporeSigma F-2425 Flag (monoclonal) MilliporeSigma F-3165 anti-Flag agarose beads MilliporeSigma A-2220 anti-HA agarose beads MilliporeSigma A-2095 peroxidase-conjugated anti-mouse secondary antibody MilliporeSigma A-4416 peroxidase-conjugated anti-rabbit secondary antibody MilliporeSigma A-4914 Plasmids pCMV3flag8HOIP Addgene 50,015 pCMV3flag8HOIL-1L Addgene 50,016 pCMV3-flag8-SHARPIN Addgene 50,014 pcDNA5 FRT TO myc p31 Comet Addgene 59,833 pERB160-hMAD1 Addgene 58,285 pcDNA5-EGFP-AIDBubR1 Addgene 47,330 VL3 (Flag-Mad2) Addgene 16,047 pcDNA3 HA human NEMO Addgene 13,512 pLKO.1-puro Addgene 8453 lentiCRISPRv2-puro Addgene 98,290 pHK-mTRIP13 Dr. Kevin D. Corbett, UCSD pcDNA3-6His-ubiquitin and mutants PMID: 31,015,455

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Derivative Assay, Transfection, Time-lapse Microscopy, Stable Transfection

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Body weight support system enabling overground walking and wireless implantable pulse generator operating in closed loop, connected to a paddle lead targeting the dorsal roots that innervate lumbosacral segments. b , Chronophotography showing the transitioning from sitting to walking in a representative participant. c , 18 FDG-PET projected onto a personalized model of the spinal cord elaborated from high-resolution MRI (participant ID DM002), showing the metabolic activity of the spinal cord—expressed as standardized uptake value (SUV bw )—in response to walking before and after EES REHAB . d , Bar plots reporting the relative change in normalized FDG-PET metabolic activity during walking before and after EES REHAB , the lower limb motor scores, and the distance covered during the 6-min walk test ( n = 9; metabolic activity mixed-effects model: t = −3.2, P = 0.002; lower limb motor scores, paired samples two-tailed t -test: t = 3.7, P = 0.0063; distance covered, paired samples two-tailed t -test: t = 3.5; P = 0.0076). e , Left, body weight support system enabling overground walking in mice, with implantable electrodes to deliver EES. Right, spinal cord visualization of projections from neurons in the motor cortex and glutamatergic (vGluT2 ON ) neurons in the reticular formation, traced with AAV5-CAG-COMET-GFP and AAV5-CAG-DIO-COMET-tdTomato, respectively. Scale bars, 1 mm. f , Chronophotography of representative mice with SCI only (SCI, EES OFF ) or SCI with EES REHAB (EES REHAB , EES OFF ). g , Lumbar spinal cord expression of cFos following walking with EES ON following SCI or SCI with EES REHAB . Scale bars, 500 μm. h , Walking performance of uninjured mice ( n = 3), mice with SCI ( n = 10), and mice with SCI and EES REHAB tested with EES OFF ( n = 10) or EES ON ( n = 10) (one-way ANOVA; Tukey’s honest significant difference for SCI versus EES REHAB →EES OFF : P = 3.3 × 10 –11 ). i , The number of neurons expressing cFos(cFos ON ) (mice with SCI with EES ON , n = 4; mice with EES REHAB and EES ON , n = 4; independent samples two-tailed t -test: t = –5.7; P = 0.001). h , i , Bars show mean ± s.e.m. with individual points overlaid. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following AAV plasmids were used and detailed sequence information is available as detailed or upon request: AAVDj-hSyn-flex-mGFP-2A-synaptophysin-mRuby (Stanford Vector Core Facility, reference AAV DJ GVVC-AAV-100), AAV5-CAG-DIO-COMET-tdTomato and AAV5CAG-COMET-GFP (a gift from M. Tuszynski), AAV5-Syn-flex-ChrimsonR-tdT (Addgene 62723), AAV5-CAG-flex-Jaws-KGC-GFP-ER2 (Addgene 84445), AAV5-hSyn-DIO-hm4D (Gi)-mCherry (Addgene 44362), AAV5-hSyn-DIO-hm3D (Gq)-mCherry (Addgene 44361), AAV5-CAG-flex-tdTomato (a gift from S. Arber), AAV5CAG-flex-human diphtheria toxin receptor (DTR) (a gift from S. Arber), AAV5-DIO-TC66T-2A-eGFP-2A-oG (GT3) (Salk Institute) and AAV5-hSyn-DIO-TVAP2A-EGFP-2A-oG (a gift from T. Karayannis).

Techniques: Activity Assay, Two Tailed Test, Expressing

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Three-dimensional visualization and quantification of the contusion SCI. Photographs show multiple 3D views of a contused spinal cord that has been cleared using uDISCO . The extent of spinal cord damage was quantified from coronal sections immunolabeled against glial fibrillary acidic protein (GFAP). The relative amount of spared tissues was quantified at the lesion epicentre and at regular intervals from the lesion epicentre in the rostral and caudal directions, as reported in the line plots. Sparing was consistent across groups (mean = 10.6%, n = 5 mice per group; independent samples two-tailed t-test on lesion epicenter, t = 0.5, P = 0.61). The impact of the contusion SCI on descending pathways compared to an uninjured spinal cord was visualized in whole brain-spinal cord preparations cleared with CLARITY. Projections from neurons located in the leg region of the motor cortex (AAV5-CAG-COMET-GFP) and from GluT2 ON neurons located in the reticular formation (AAV5-CAGDIO-COMET-tdTomato) were labeled with virus infusions, as indicated in the photograph. Insets show the complete interruption of corticospinal fibers and partial preservation of reticulospinal fibers. b , Adaptations of the technological features of EES REHAB in mice, including a new robotic body weight support interface with 1g accuracy and a pair of electrodes attached to L2 and S1 spinal segments to deliver EES. Kinematic recordings of whole-body movements were mapped onto a 3D model of the mouse including bones and skin contours to generate realistic visualization of leg movements . The line shows the trajectory of the lower limb endpoint (toe) while circles indicate the maximum step height. Representative leg movements are shown concomitantly to the electromyographic (EMG) activity of the tibialis anterior, recorded from chronically implanted bipolar electrodes into the muscle. At one week, EES was insufficient to reactivate the spinal cord to a level that enabled walking in mice. Consequently, a small bolus of agonists to 5H1A/7 (8-OH-DPAT, 0.05-0.2 mg/kg) and 5HT2A receptors (quipazine 0.2-0.3 mg/kg) was administered to augment the response of the spinal cord . The combination of EES and 5-HT agonists enabled all the mice to walk overground as early as one week after the contusion SCI. 5-HT agonists were used to enable sustained walking during EES REHAB , but the amount of drug was progressively decreased over the course of the recovery, and eventually suppressed. Final testing was performed without 5-HT agonists. c , We noticed that compared to rats and humans, EES was not as effective to enable walking in mice with SCI. Inspection of muscle responses to EES revealed that the stimulation recruited motor nerves (efferents) at relatively low amplitudes. This off-target stimulation of the ventral roots is due to the relatively small size of the mouse spinal cord. We previously showed that high-frequency bursts are effective to maximize the activation of large-diameter afferent fibers targeted by EES . To adapt this stimulation protocol to mice, we elaborated a computational model of the mouse spinal cord . The model includes a spiking neural network model of muscle spindle feedback circuits for a pair of antagonistic muscles. Response in motor neurons are shown for two amplitudes of EES that correspond to the recruitment of 20% and 60% of the entire afferent population. Simulations suggested that, compared to conventional protocols (40 Hz), high-frequency burst stimulation (carrier frequency 600 Hz, modulating frequency 30 Hz) leads to a summation of synaptic events into motor neurons, which augment the probability to activate muscles via the recruitment of afferents compared to efferents. To confirm these findings experimentally, we compared conventional stimulation protocols to high-frequency burst stimulation during stepping on a treadmill in mice with contusion SCI. Reconstructed leg movements including foot trajectories are shown for each experimental condition at two amplitudes of EES. High frequency burst stimulation increased the therapeutic window through which stimulation could be delivered. Concretely, step height scaled linearly up to 1.5× motor threshold using burst stimulation protocols, and stimulation could be delivered effectively up to 2.5× motor threshold. In contrast, conventional stimulation at 40 Hz facilitated stepping around motor threshold, but additional increase in amplitude that would be necessary to promote robust stepping led to tonic activation of leg muscle activation and thus cessation of stepping. d , To model the volitional control of stepping observed in humans during EES ON , we manipulated cortical activity with optogenetics. We expressed channelrhodopsin in the neurons of the leg motor cortex using targeted injections of AAV5-hSyn-ChR2 ( n = 4 mice). Mice with contusion SCI were recorded during stepping on a treadmill with EES ON (no 5-HT agonists). Photostimulation of the motor cortex induced a significant increase of the step height that scaled up with laser intensity, as shown in the bar plots ( n = 4 mice per group; statistics indicate Tukey HSD tests following one-way ANOVA, P = 0.0003, P = 0.025, respectively). e , Leg movements and muscle activity during overground walking without any intervention or support recorded at four weeks after a contusion SCI in a mouse that did not undergo EES REHAB and in a mouse that underwent EES REHAB . Locomotor performance was quantified using principal component analysis applied to 80 gait parameters calculated from kinematic recordings (Supplementary Table ). In this denoised space, each dot represents a gait cycle ( n = 20 per mouse, n = 9 mice per group). Larger dots represent the mean of each experimental group. The first principal component (PC1) distinguished gaits from mice with SCI that did not undergo EES REHAB from mice that underwent EES REHAB . Locomotor performances were thus quantified as the scores on PC1 (reported in Fig. ). Analysis of factor loadings on PC1 revealed that the percentage of paw dragging, the extent of whole-limb oscillation (virtual limb connecting the hip to the toe) and step height were the parameters that showed the highest correlation with PC1. Bars report the mean values of these gait parameters ( n = 9 mice per group; statistics indicate Tukey HSD tests following oneway ANOVA, P = 2.1 × 10 –9 , P = 7.4 × 10 –6 , P = 0.0055, respectively). Data from mice that underwent EES REHAB are shown during EES OFF and EES ON . f , Photographs show whole mouse spinal cords before and after processing with iDISCO+ , , during which the spinal cords underwent immunolabeling of cFos followed by clearing. The spinal cords were imaged with the mesoSPIM lightsheet microscope . Representative microscopy images show a raw coronal optical slice of the cFos labelling in the spinal cord and after application of automated 3D nuclear spot detection. Images were then reconstructed to visualize the entire lumbar spinal cord. g , 3D cFos quantifications were confirmed using immunohistochemistry and labelling for cFos on coronal sections of the lumbar spinal cord, as illustrated in the representative photographs of spinal cord sections from mice with SCI and mice that followed EES REHAB . The bar plot reports the mean number of cFos labeled cells per section in the spinal grey matter across the whole section, in Lamina I–III (dorsal), in Lamina IV–VI (intermediate), in Lamina VII–IX (ventral) and in Lamina X (central canal) ( n = 4 mice per group; independent samples two-tailed t-test, t = –2.7, P = 0.042; t = 0.60, P = 0.57; t = 3.7, P = 0.010; t = 1.2, P = 0.27; t = 1.0, P = 0.35, respectively). Bar plots show the mean with individual data points overlaid. Error bars show the standard error of the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The following AAV plasmids were used and detailed sequence information is available as detailed or upon request: AAVDj-hSyn-flex-mGFP-2A-synaptophysin-mRuby (Stanford Vector Core Facility, reference AAV DJ GVVC-AAV-100), AAV5-CAG-DIO-COMET-tdTomato and AAV5CAG-COMET-GFP (a gift from M. Tuszynski), AAV5-Syn-flex-ChrimsonR-tdT (Addgene 62723), AAV5-CAG-flex-Jaws-KGC-GFP-ER2 (Addgene 84445), AAV5-hSyn-DIO-hm4D (Gi)-mCherry (Addgene 44362), AAV5-hSyn-DIO-hm3D (Gq)-mCherry (Addgene 44361), AAV5-CAG-flex-tdTomato (a gift from S. Arber), AAV5CAG-flex-human diphtheria toxin receptor (DTR) (a gift from S. Arber), AAV5-DIO-TC66T-2A-eGFP-2A-oG (GT3) (Salk Institute) and AAV5-hSyn-DIO-TVAP2A-EGFP-2A-oG (a gift from T. Karayannis).

Techniques: Immunolabeling, Two Tailed Test, Labeling, Preserving, Activity Assay, Activation Assay, Optogenetics, Microscopy, Immunohistochemistry

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , UMAP visualization of 20,990 neurons, coloured by Augur cell type prioritization, identifying perturbation-responsive subpopulations in a representative experimental comparison. b , Identification of perturbation-responsive subpopulations across experimental comparisons with Augur. Top, clustering tree of neuronal subpopulations. Middle, heat map showing scaled areas under the curve (AUCs) for individual comparisons: (1) SCI versus SCI →EES::walking ; (2) SCI versus EES REHAB ; (3) EES REHAB versus EES REHAB →cortex ; (4) EES REHAB versus EES REHAB →EES ; (5) EES REHAB →EES::walking versus SCI →EES::walking ; (6) EES REHAB versus EES REHAB →EES::walking . Bottom, distribution of AUCs across all comparisons. c , Spatial visualization of 22,127 barcodes in the common coordinate space coloured by Magellan spatial prioritization for the same representative experimental comparison as in a . d , Synaptic inputs and outputs from SC Vsx2::Hoxa10 neurons. Inputs: SC Vsx2::Hoxa10 neurons and their projections. Projections from vGi neurons (AAV5-CAG-COMET-GFP) and large-diameter afferent neurons (PV cre::Advillin:tdTomato ) onto SC Vsx2::Hoxa10 neurons. Outputs: synaptic appositions from SC Vsx2::Hoxa10 neurons (AAV-Dj-hSyn-flex-mGFP-2A-synaptophysin-mRuby) onto glutamatergic, GABAergic and motor neurons. C, caudal; R, rostral; PV, parvalbumin; syn, synaptophysin. e , Single-unit recordings of optotagged SC Vsx2::Hoxa10 neurons. Responses of an optotagged single unit to optogenetic stimulations. Right, the number of units responding to each type of stimulation. f , Number of vGluT1 synapses from large-diameter afferents apposing SC Vsx2::Hoxa10 neurons ( n = 6 out of 10 mice per group; independent samples two-tailed t -test: t = 4.9, P = 0.002). g , Number of reticulospinal neurons projecting to SC Vsx2::Hoxa10 neurons ( n = 4 mice per group; independent samples two-tailed t -test: t = 4.8, P = 0.0029). h , Percentage of motor neurons, glutamatergic and GABAergic neurons receiving at least one synapse from SC Vsx2::Hoxa10 neurons ( n = 6; one-way analysis of variance (ANOVA); choline acetyltransferase (ChAT): F = 0.45, P = 0.39; glutamatergic: F = 0.52, P = 0.76; GABAergic: statistics indicate Tukey’s honest significant difference tests: uninjured versus SCI: P = 5.2 × 10 −6; SCI versus EES REHAB : P = 1.8 × 10 −5 ). i , Fluorescent intensity of cFos in Vsx2 cre neurons following walking with EES ON ( n = 5 out of 3 mice per group; independent samples two-tailed t -test: t = 5.7, P = 0.0013). f – i , Bars show mean ± s.e.m. with individual points overlaid. AU, arbitrary units.

Article Snippet: The following AAV plasmids were used and detailed sequence information is available as detailed or upon request: AAVDj-hSyn-flex-mGFP-2A-synaptophysin-mRuby (Stanford Vector Core Facility, reference AAV DJ GVVC-AAV-100), AAV5-CAG-DIO-COMET-tdTomato and AAV5CAG-COMET-GFP (a gift from M. Tuszynski), AAV5-Syn-flex-ChrimsonR-tdT (Addgene 62723), AAV5-CAG-flex-Jaws-KGC-GFP-ER2 (Addgene 84445), AAV5-hSyn-DIO-hm4D (Gi)-mCherry (Addgene 44362), AAV5-hSyn-DIO-hm3D (Gq)-mCherry (Addgene 44361), AAV5-CAG-flex-tdTomato (a gift from S. Arber), AAV5CAG-flex-human diphtheria toxin receptor (DTR) (a gift from S. Arber), AAV5-DIO-TC66T-2A-eGFP-2A-oG (GT3) (Salk Institute) and AAV5-hSyn-DIO-TVAP2A-EGFP-2A-oG (a gift from T. Karayannis).

Techniques: Two Tailed Test

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Implantable optoelectronic device to deliver EES and photostimulation. After EES REHAB , chronophotography of walking during EES ON in a representative mouse. Red-shifted light is delivered for a few seconds to silence SC Vsx2::Hoxa10 neurons (AAV5-Syn-flex-ChrimsonR-tdTomato; n = 4; Tukey’s honest significant difference tests following repeated measures one-way analysis of variance (ANOVA): P = 0.0023). b , Chronophotography of walking after EES REHAB in a representative mouse with EES ON before and after chemogenetic silencing of SC Vsx2::Hoxa10 neurons (AAV5-hSyn-DIO-hm4D-(Gi)-mCherry; n = 4; paired samples two-tailed t -test; t = −21.3, P = 0.0002). c , Chronophotography of walking in representative mice with no EES REHAB , with EES OFF before and after chemogenetic activation of SC Vsx2::Hoxa10 neurons (AAV5-hSyn-DIO-hm3D-(Gq)-mCherry; n = 4; paired samples two-tailed t -test; t = 5.3, P = 0.0013). d , After EES REHAB , chronophotography of walking in representative mice with EES ON . SC Vsx2::Hoxa10 neurons were silenced during the entire period of EES REHAB ( n = 5; independent samples two-tailed t -test: t = −3.5, P = 0.008). e , Chronophotography of representative mice walking without EES after recovery from a lateral hemisection SCI. Kinematic limb reconstruction is overlaid. Right, same condition, but SC Vsx2::Hoxa10 neurons located in lumbar segments were ablated before the SCI (AAV5-CAG-flex-DTR; n = 5 per group; independent samples two-tailed t -test: t = 5.9, P = 0.0004). a – e , Bars show mean ± s.e.m. with individual points overlaid.

Article Snippet: The following AAV plasmids were used and detailed sequence information is available as detailed or upon request: AAVDj-hSyn-flex-mGFP-2A-synaptophysin-mRuby (Stanford Vector Core Facility, reference AAV DJ GVVC-AAV-100), AAV5-CAG-DIO-COMET-tdTomato and AAV5CAG-COMET-GFP (a gift from M. Tuszynski), AAV5-Syn-flex-ChrimsonR-tdT (Addgene 62723), AAV5-CAG-flex-Jaws-KGC-GFP-ER2 (Addgene 84445), AAV5-hSyn-DIO-hm4D (Gi)-mCherry (Addgene 44362), AAV5-hSyn-DIO-hm3D (Gq)-mCherry (Addgene 44361), AAV5-CAG-flex-tdTomato (a gift from S. Arber), AAV5CAG-flex-human diphtheria toxin receptor (DTR) (a gift from S. Arber), AAV5-DIO-TC66T-2A-eGFP-2A-oG (GT3) (Salk Institute) and AAV5-hSyn-DIO-TVAP2A-EGFP-2A-oG (a gift from T. Karayannis).

Techniques: Two Tailed Test, Activation Assay