Reporting Checklist for Nature Neuroscience

Transcription

1 Corresponing Author: Manuscript Number: Manuscript Type: Albert La Spaa NNA4471A Article Reporting Checklist for Nature Neuroscience # Main Figures: 8 # Supplementary Figures: 9 # Supplementary Tables: 0 # Supplementary Vieos: 0 This checklist is use to ensure goo reporting stanars an to improve the reproucibility of publishe results. For more information, please rea Reporting Life Sciences Research. Please note that in the event of publication, it is manatory that authors inclue all relevant methoological an statistical information in the manuscript. Statistics reporting, by figure example example Please specify the following information for each panel reporting quantitative ata, an where each item is reporte (section, e.g. Results, & paragraph number). Each figure legen shoul ieally contain an exact sample size (n) for each experimental group/conition, where n is an exact number an not a range, a clear efinition of how n is efine (for example x cells from x slices from x animals from x litters, collecte over x ays), a escription of the statistical use, the results of the s, any escriptive statistics an clearly efine error bars if applicable. For any experiments using custom statistics, please inicate the use an stats obtaine for each experiment. Each figure legen shoul inclue a statement of how many times the experiment shown was replicate in the lab; the etails of sample collection shoul be sufficiently clear so that the replicability of the experiment is obvious to the reaer. For experiments reporte in the text but not in the figures, please use the paragraph number instea of the figure number. Note: Mean an stanar eviation are not appropriate on small samples, an plotting inepenent ata points is usually more informative. When technical replicates are reporte, error an significance measures reflect the experimental variability an not the variability of the biological process; it is misleaing not to state this clearly. FIGURE NUMBER 1a results, TEST USED WHICH TEST? oneway unpaire t legen Results EXACT VALUE 9, 9, 10, 15 n DEFINED? mice from at least litters/group 15 slices from 10 mice Methos para 8 Results DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? mean / SEM mean / SEM legen Results P VALUE EXACT VALUE p = p = legen Results DEGREES OF FREEDOM & F/t/z/R/ETC VALUE VALUE F(, ) = 2.97 t(28) = legen Results Nature Neuroscience: oi:10.108/nn.787 1

2 FIGURE NUMBER 1a 1 1f a b c TEST USED WHICH TEST? oneway posthoc Tukey oneway posthoc Tukey oneway posthoc Tukey One way (Tukey's post ) One way (Tukey's post ) One way (Tukey's post ) EXACT VALUE > n DEFINED? cells counte per conition inepenent western blot runs of entire set inepenent western blot runs of entire set motor neurons/ mouse group motor neurons/ mouse group autophagosomes/ autolysosomes per group Methos Methos Methos para 11 Methos para 11 Methos para 11 DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? mean SEM mean SEM mean SEM mean SEM mean SEM mean SEM P VALUE EXACT VALUE Autolysosome s: p=0.01 Post t: Wt vs AR24Q p=0.72 Wt vs AR5Q p= AR24Q vs AR5Q p=0.01 Autophagoso mes: p=.9e08 Post t: Wt vs AR24Q p=0.02 Wt vs AR5Q p= 1.9E07 AR24Q vs AR5Q p= p=0.05 p=0.028 Post t: Wt vs AR24Q p=.50 Wt vs AR5Q p= AR24Q vs AR5Q p=0.1 M: p=0.5 14M: p=0.015 M: p= M: p=0.012 M: p= M p=0.002 DEGREES OF FREEDOM & F/t/z/R/ETC VALUE VALUE Autolysosomes: F(2,105)=.5 Autophagosome: F(2,105)=20.1 F= F(2,9)=5.45 F(2,147)=0. F(2,175)=4.2 F(2,148)=4.18 F(2,157)=4.5 F(2,)= F(2,)=82.82 Nature Neuroscience: oi:10.108/nn.787 2

3 4b 4c 4 5a 5b 5 Untreate: oneway posthoc Tukey Untreate vs. Sucrose: unpaire t oneway posthoc Tukey oneway posthoc Tukey Unpaire T (Tukey's post ) Oneway posthoc Tukey 18 Inepenent complete sets of luciferase runs in triplicate Inepenent sets of RNA lysates/cell line Inepenent sets of RNA lysates of E1 motor neurons/genotype Inepenent complete sets of luciferase runs in triplicate Inepenent complete sets of luciferase runs in triplicate 24 cells per conition Methos mean SEM mean SEM mean SEM mean SEM mean SEM mean SEM Untreate **p<0.01 t: Untreate vs Sucrose: Wt/ AR24Q: **p<0.01 AR5Q: p=0.5 Sucrose ***p<.001 p< (* p<0.5, **p<0.01) CTSF p=0.027 p=0.014 ATP1V1H p=0.018 GLA p=0.021 Wt: p< AR24Q: p< AR5Q: P= p< (***p<0.001) Autolysosome s: p=0.242 Post s: n/a Autophagoso mes: p=1.01e0 Post s: Untransfecte vs BFPempty p=0.08 Untransfecte vs BFPTFEB p=2.17e09 BFPempty vs BFPTFEB p= Autophagy inex: p=0.001 Untreate: F(2,15)=.48 Untreate vs Sucrose: Wt: t(10)=8.1 AR24Q t(10)=.11 AR5Q t(10)=0.44 Sucrose F(2,15)= 2.49 LAMP1 F(2,)=7.51 Atp1VH1: F(2,)=57.2 F(2,)=251.0 GLA F(2,)=75.51 CTSF F(2,15)=4. F(2,15)=.4 ATP1V1H F(2,15)=5. GLA F(2,15)=5.5 Wt: t(4)=48.79 AR24Q: t(4)=22.91 AR5Q: t(4)=14.02 Untr:F(2,):182.7 STV:F(2,)=.4 NH4Cl: F(2,)=291.9 Rapa F(2,)=1.28 Autolysosomes: F(2,77)=1.44 Autophagosomes F(2,77)=1.59 Autophagy inex: F(2,)=24.4 Nature Neuroscience: oi:10.108/nn.787

4 e f g h 7a 7b 7 7e oneway posthoc Tukey Unpaire T Unpaire T Unpaire T for each gene Unpaire T Unpaire T Unpaire T (# of vesicles/cell) Unpaire T Unpaire T for each iniviual gene > cells/conition in triplicate Inepenent complete sets of luciferase runs in triplicate Inepenent complete sets of luciferase runs in triplicate Inepenent knockown experiments ifferent control samples an ifferent SBMA NPCs ifferent control samples an ifferent SBMA NPCs about 15 cells were counte per clone; at least clones for each of the SBMANPCs an of the control samples (SBMA n=15 an control n=15) ifferent control samples an ifferent SBMA NPCs Methos Methos para 2 Methos mean SEM mean SEM mean SEM mean SEM mean SEM Tukey box whisker plot mean SEM mean SEM Untreate p=0.57 Post t: n/a Testosterone treate: p=.0089 Post ts: Wt vs AR24Q p= WT vs AR5Q p=0.4 AR24Q vs AR5Q p=0.009 Control p= NH4Cl p=0.02 Rapamycin p=0.0 Untreate: p=0.52 Rapamycin p=0.004 Scr control vs ARKD GLA p=0.02 ATP1 p=0.0 p=0.00 CTSD p=0.015 LAMP1 p=0.014 p= p=0.014 (# of vesicles/ cell) autophagoso mes p= autolysosome p=0.142 CYCS p=0.047 p=0.015 GLA p=0.07 ADCAM p=0.011 CDXA1 p=0.005 TFEB p= Untreate F(2,1)=0.5 Testosterone treate F(2,1)=5.51 Control: t()=15.7 NH4Cl: t(4)=4.108 Rapa: t(4)=4.58 Ctl t(4)=0.51 Rapa: t(4)=7.15 GLA t(4)=4.77 ATPV t(4)=.75 t(4)= 1.74 CTSD t(4)=7.91 LAMP1 t(4)=.4 t(4)=4.02 t(4)=.98 (# of vesicles/ cell) autophagosomes t(18)=2.799 autolysosomes t(17)=1.522 YCS t(4)=2.844 t(4)=.24 GLA t(4)=.174 ADCAM t(4)=4.405 CDXA1 t(4)=5.584 TFEB t(4)=0.594 Nature Neuroscience: oi:10.108/nn.787 4

5 8b 8 S1 S Unpaire T Unpaire T oneway posthoc Tukey oneway posthoc Tukey 2 9 ifferent control samples an ifferent SBMA NPCs about 15 cells were counte from the selecte affecte clone with more pronounce phenotype; comparison was performe in the context of treate an untreate (with TFEB) samples/ conition 9 mine/group Methos Tukey box whisker plot mean SEM mean SEM mean SEM p= (# of vesicles/ cell) autophagoso mes p= autolysosome s p= (Autophagy Inex) p=0.012 Posthoc s: MN1 WT vs. MN1 AR5Q p<.05 Lamp1 p=0.005 Posts AR20 p= AR100 p= AR20 vs AR100 p= Atp1V1H p= e0 Posts AR20 p= AR100 p= AR20 vs AR100 p=5.2745e0 5 GLA p= Posts AR20 p= AR100 p= AR20 vs AR100 p= CTSD p=4.7082e0 5 Posts t()=.217 (# of vesicles/ cell) autophagosomes t(0)=4.744 autolysosomes t(2)=1.525 (Autophagy Inex) F(2,)=10.12 Lamp1 F(2,12)=8.15 Atp1V1G F(2,12)=49.95 GLA F(2,12)=8. CTSD F(2,12)=25.57 F(2,12)= Nature Neuroscience: oi:10.108/nn.787

6 S4 S8 1b Unpaire T Unpaire T oneway posthoc Tukey Fig legen 2 at least 15 BFP cells were counte from MN1 Wt type cells (total n=0) at least 15 cells were counte for each control iniviual. Total of controls were analyze (Total n= 45 per conition) >5 cells per conition Methos Methos Methos mean SEM mean SEM mean SEM legen Autolysosome s: p=.0089 Post t: Untransfecte vs BFPempty p=0.118 Untransfecte vs BFPTFEB p=0.04 BFPempty vs BFPTFEB p= Autophagoso mes: p=0.90 Post ts: n/a autophagoso mes p= autolysosome s p= Autolysosome s: p=0.014 Post t: Mock vs AR25Q p= Mock vs AR125Q p=0.015 AR25Q vs AR125Q p=0.01 Autophagoso mes: p=7.81e08 Post t: Mock vs AR25Q p= Mock vs AR125Q p=7.18e11 AR25Q vs AR125Q p=1.01e0 Figure Autolysosomes: F(2,87)=4.97 Autophagosome: (F2,87)=0.04 autophagosomes t(4)= autolysosomes t(4)= Autolysosomes F(2,7)=4.49 Autophagosomes F(2,7)=20.5 Fig legen Nature Neuroscience: oi:10.108/nn.787

7 Representative figures 1. Are any representative images shown (incluing Western blots an immunohistochemistry/staining) in the paper? If so, what figure(s)? 2. For each representative image, is there a clear statement of how many times this experiment was successfully repeate an a iscussion of any limitations in repeatability? If so, where is this reporte (section, paragraph #)? Statistics an general methos 1. Is there a justification of the sample size? If so, how was it justifie? Even if no sample size calculation was performe, authors shoul report why the sample size is aequate to measure their effect size. 2. Are statistical s justifie as appropriate for every figure? a. If there is a section summarizing the statistical methos in the methos, is the statistical for each experiment clearly efine? b. Do the ata meet the assumptions of the specific statistical you chose (e.g. normality for a parametric )? Where is this escribe (section, paragraph #)? c. Is there any estimate of variance within each group of ata? Is the variance similar between groups that are being statistically compare? Where is this escribe (section, paragraph #)? Yes. Figures: 1, 2, 4, 5,, 7 an 8. Supplementary Figures: S2, S an S7 Yes. In the corresponing Figure legen. Yes. In the corresponing Methos paragraph 1. For cell lines, each experiment was repeate in triplicate or quaruplicate an results average for final ata presentation. For experiments involving mice, groups of n=4/genotype (non transgenic, YAC AR20 an YAC AR100) were use. This is the stanar for cellular an molecular phenotype characterization in mice samples. For the stem cell experiments we use clones per line, an lines per group (SBMA an control). Total of 18 samples in accorance with the highest stanar of ips cell research for statistical purposes for the autophagy flux experiments. For other assay ranom clones were selecte, at least 1 clone por line with a total of ifferent lines per group (SBMA an control) Statistical s were selecte base upon number of samples, an all statistical s are stanar parametric s. Yes. We escribe our statistical analysis in the Methos section on page 22. We always efine each statistical performe in the corresponing Figure legen. Yes, we are only analyzing sample sets that conform to the requirements of stanar parametric ing. Yes, we apply one way to compare variance within groups for sample sizes of n>2, an we use the t for analysis of inepenent samples of n = 2. The analysis an t are escribe in the corresponing Figure legens.. Are s specifie as one or twosie? Yes, we perform oneway an onetaile ts. Nature Neuroscience: oi:10.108/nn.787 7

8 e. Are there ajustments for multiple comparisons? Yes, we perform a posthoc Tukey for multiple comparisons when we perform.. Are criteria for excluing ata points reporte? Was this criterion establishe prior to ata collection? Where is this escribe (section, paragraph #)? 4. Define the metho of ranomization use to assign subjects (or samples) to the experimental groups an to collect an process ata. If no ranomization was use, state so. Where oes this appear (section, paragraph #)? 5. Is a statement of the extent to which investigator knew the group allocation uring the experiment an in assessing outcome inclue? If no blining was one, state so.. For experiments in live vertebrates, is a statement of compliance with ethical guielines/regulations inclue? 7. Is the species of the animals use reporte? 8. Is the strain of the animals (incluing backgroun strains of KO/ transgenic animals use) reporte? 9. Is the sex of the animals/subjects use reporte? 10. Is the age of the animals/subjects reporte? 11. For animals house in a vivarium, is the light/ark cycle reporte? 12. For animals house in a vivarium, is the housing group (i.e. number of animals per cage) reporte? No ata was exclue from the presente analysis. No group ranomization was use in our stuy. Yes, bline s were performe an a statement escribing this is inclue in the applicable methos section. Methos paragraph 1. Yes, see Methos paragraph 7. Yes, see Methos paragraph 7. Yes, see Methos paragraph 7. Yes, see Methos paragraph 7. Yes, see Methos paragraph 7. Yes, see Methos paragraph 7. Yes, see Methos paragraph For behavioral experiments, is the time of ay reporte (e.g. light or ark cycle)? Nature Neuroscience: oi:10.108/nn.787 No behavioral analysis were performe in this stuy 8

9 14. Is the previous history of the animals/subjects (e.g. prior rug aministration, surgery, behavioral ing) reporte? a. If multiple behavioral s were conucte in the same group of animals, is this reporte? 15. If any animals/subjects were exclue from analysis, is this reporte? Reagents a. How were the criteria for exclusion efine? Where is this escribe (section, paragraph #)? b. Specify reasons for any iscrepancy between the number of animals at the beginning an en of the stuy. Where is this escribe (section, paragraph #)? 1. Have antiboies been valiate for use in the system uner stuy (assay an species)? a. Is antiboy catalog number given? Where oes this appear (section, paragraph #)? b. Where were the valiation ata reporte (citation, supplementary information, Antiboypeia)? Where oes this appear (section, paragraph #)? 2. If cell lines were use to reflect the properties of a particular tissue or isease state, is their source ientifie? a. Were they recently authenticate? Where is this information reporte (section, paragraph #)? No iniviual animals were exclue from final ata representation Yes. Yes, see Methos paragraph 5 an. Yes, see Methos paragraph 5 an. Yes, see Methos paragraph 2. SBMA IPSc/NPCs were generate an authenticate in this stuy (see Supplementary Figures S5 an S). Nature Neuroscience: oi:10.108/nn.787 9

10 Data eposition Data eposition in a public repository is manatory for: a. Protein, DNA an RNA sequences b. Macromolecular structures c. Crystallographic ata for small molecules. Microarray ata Deposition is strongly recommene for many other atasets for which structure public repositories exist; more etails on our ata policy are available here. We encourage the provision of other source ata in supplementary information or in unstructure repositories such as Figshare an Drya. 1. Are accession coes for eposit ates provie? Computer coe/software Any custom algorithm/software that is central to the methos must be supplie by the authors in a usable an reaable form for reaers at the time of publication. However, referees may ask for this information at any time uring the review process. 1. Ientify all custom software or scripts that were require to conuct the stuy an where in the proceures each was use. 2. Is computer source coe/software provie with the paper or eposite in a public repository? Inicate in what form this is provie or how it can be obtaine. Human subjects 1. Which IRB approve the protocol? Where is this state (section, paragraph #)? 2. Is emographic information on all subjects provie?. Is the number of human subjects, their age an sex clearly efine? 4. Are the inclusion an exclusion criteria (if any) clearly specifie? 5. How well were the groups matche? Where is this information escribe (section, paragraph #)? Yes. IRB #107ZF. See Methos paragraph. Yes. See Methos paragraph. Yes. See Methos paragraph. The controls were matche to the SBMA patients by age an gener as escribe. See Methos paragraph. Nature Neuroscience: oi:10.108/nn

11 . Is a statement inclue confirming that informe consent was obtaine from all subjects? 7. For publication of patient photos, is a statement inclue confirming that consent to publish was obtaine? fmri stuies Yes. See Methos paragraph. For papers reporting functional imaging (fmri) results please ensure that these minimal reporting guielines are met an that all this information is clearly provie in the methos: 1. Were any subjects scanne but then rejecte for the analysis after the ata was collecte? a. If yes, is the number rejecte an reasons for rejection escribe? 2. Is the number of blocks, trials or experimental units per session an/ or subjects specifie?. Is the length of each trial an interval between trials specifie? 4. Is a blocke, eventrelate, or mixe esign being use? If applicable, please specify the block length or how the eventrelate or mixe esign was optimize. 5. Is the task esign clearly escribe?. How was behavioral performance measure? 7. Is an or factorial esign being use? 8. For ata acquisition, is a whole brain scan use? If not, state area of acquisition. a. How was this region etermine? Nature Neuroscience: oi:10.108/nn

12 9. Is the fiel strength (in Tesla) of the MRI system state? a. Is the pulse sequence type (graient/spin echo, EPI/spiral) state? b. Are the fielofview, matrix size, slice thickness, an TE/TR/ flip angle clearly state? 10. Are the software an specific parameters (moel/functions, smoothing kernel size if applicable, etc.) use for ata processing an preprocessing clearly state? 11. Is the coorinate space for the anatomical/functional imaging ata clearly efine as subject/native space or stanarize stereotaxic space, e.g., original Talairach, MNI05, ICBM152, etc? Where (section, paragraph #)? 12. If there was ata normalization/stanarization to a specific space template, are the type of transformation (linear vs. nonlinear) use an image types being transforme clearly escribe? Where (section, paragraph #)? 1. How were anatomical locations etermine, e.g., via an automate labeling algorithm (AAL), stanarize coorinate atabase (Talairach aemon), probabilistic atlases, etc.? 14. Were any aitional regressors (behavioral covariates, motion etc) use? 15. Is the contrast construction clearly efine? 1. Is a mixe/ranom effects or fixe inference use? a. If fixe effects inference use, is this justifie? 17. Were repeate measures use (multiple measurements per subject)? a. If so, are the metho to account for within subject correlation an the assumptions mae about variance clearly state? 18. If the threshol use for inference an visualization in figures varies, is this clearly state? 19. Are statistical inferences correcte for multiple comparisons? a. If not, is this labele as uncorrecte? Nature Neuroscience: oi:10.108/nn

13 20. Are the results base on an ROI (region of interest) analysis? a. If so, is the rationale clearly escribe? b. How were the ROI s efine (functional vs anatomical localization)? 21. Is there correction for multiple comparisons within each voxel? 22. For clusterwise significance, is the clusterefining threshol an the correcte significance level efine? Aitional comments Aitional Comments Nature Neuroscience: oi:10.108/nn.787 1