Journal: EFSA Journal

Article Title: Re‐evaluation of neohesperidine dihydrochalcone (E 959) as a food additive

doi: 10.2903/j.efsa.2022.7595

Figure Lengend Snippet:

Article Snippet: Statistical methods , All experimental values are presented as means 6 standard deviations (SD). OriginLab software (Origin 2017) and GraphPad Prism software (Graph‐Pad 7.0) were used for data analysis and graphical illustrations. All data between different groups were statistically analysed by the Mann–Whitney U test or the Kruskal–Wallis analysis of variance (ANOVA) test. p Values of 0.05 were considered significant..

Techniques: Animal Model, Concentration Assay, Histopathology, Software, Mann-Whitney U-Test

Journal: EFSA Journal

Article Title: Re‐evaluation of neohesperidine dihydrochalcone (E 959) as a food additive

doi: 10.2903/j.efsa.2022.7595

Figure Lengend Snippet:

Article Snippet: Statistical methods , All experimental values are presented as means 6 standard deviations (SD). OriginLab software (Origin 2017) and GraphPad Prism software (Graph‐Pad 7.0) were used for data analysis and graphical illustrations. All data between different groups were statistically analysed by the Mann–Whitney U test or the Kruskal–Wallis analysis of variance (ANOVA) test. p Values of 0.05 were considered significant..

Techniques: Animal Model, Concentration Assay, Mann-Whitney U-Test

Journal: Developmental cell

Article Title: T cell cytoskeletal forces shape synapse topography for targeted lysis via membrane curvature bias of perforin.

doi: 10.1016/j.devcel.2022.08.012

Figure Lengend Snippet: Figure 3. The CTL synapse exhibits negative membrane curvatures at regions cleared of F-actin (A) Timelapse confocal images of live CTL F-actin distribution at the synapse during cognate tumor interaction in the DPA assay. Top: maximum intensity projection. Middle/bottom: SynapseMech analysis of CTL F-actin distribution and membrane curvature within the synapse. Displayed F-actin signal restricted to synapse region and intensity-thresholded. Scale bar, 5 mm. (B) Dynamics of area cleared from F-actin (gray shaded region) and average mean curvature of complete (dark gray) and F-actin-cleared (brown) synapse area for data shown in (A). (C) Time-averaged mean curvature of complete, F-actin-cleared, and F-actin-rich (outside of cleared space) synapse area. Boxplot shows median, IQR, and range for n = 6 cells. p values by matched one-way ANOVA test with prior Kolmogorov-Smirnov test for normality. (D) Surface tension of resting CTLs, tumor cells and tumor cytoplasts. Boxplots show median, IQR and range for n = 24 (CTL), n = 36 (tumor cells), and n = 19 (tumor cytoplasts). p values by Mann-Whitney test. (E) Timelapse confocal images of a live CTL interacting with a cognate tumor cytoplast in the DPA assay. t = 0 s: time of contact. Scale bar, 10 mm. (F) Main: dynamics of average mean synapse curvature of CTL interacting with cognate tumor cell or tumor cytoplast. Data are mean ± SEM (shaded area) for n = 7 (tumor cell) and n = 8 (tumor cytoplast). Inset: time-averaged mean synapse curvature of CTLs interacting with cognate tumor cells or tumor cytoplasts. p value by Mann-Whitney test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Experimental models: Cell lines EL4 mouse lymphoma cells ATCC Cat. #: TIB-39 WM266-4 (Elkin1 KO) human melanoma cells Patkunarajah et al. (2020) N/A Experimental models: Organisms/strains Mouse: C57BL/6 OT-1 Gift from E. Deenick (Garvan Institute of Medical Research) N/A Mouse: C57BL/6 OT-1 x Lifeact-EGFP Galeano Niño et al. (2020) N/A Mouse: C57BL/6 OT-1 x TdTomato Galeano Niño et al. (2020); Muzumdar et al. (2007) N/A Human: primary CTLs transduced with 1G4 TCR Gift from J. Goyette (University of New South Wales) N/A Recombinant DNA Myosin IIA-GFP plasmid Addgene RRID: Addgene_38297 Software and algorithms SynapseMech This paper [Github]: https://github.com/BiroLab/ SynapseMech; https://doi.org/10.5281/ zenodo.7014229 FIJI LimeSeg plugin Machado et al. (2019) https://imagej.net/plugins/limeseg Imaris v8.4.1 Bitplane https://imaris.oxinst.com Prism v8 Graphpad https://graphpad.com Matlab R2021a Mathworks https://mathworks.com Other AccuCount Blank Particles Spherotech Cat. #: ACBP-70-10 Sensoplate Microplate, 96 well, PS, F-bottom Greiner Cat. #: 655892 35 mm Dish | No. 1.5 Coverslip | 14 mm Glass Diameter | Uncoated Mattek Cat. #: P35G-1.5-14-C

Techniques: Membrane, MANN-WHITNEY

Journal: Cell

Article Title: Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

doi: 10.1016/j.cell.2024.03.015

Figure Lengend Snippet: Figure 3. Tau propagation in 4R-P301S neurons is associated with endolysosomal dysfunction (A) Representative TEM images from soma of D35 4R neurons, D40 4R-P301S neurons, and 1.5 mg/mL K18-seeded D40 (7 + 33) 4R-P301S neurons. Scale bar, 1 mm. (B) Quantification of #MLB/neuron from D35 4R and 4R-P301S, D40 4R and 4R-P301S, and D43 (7 + 36) 4R-P301S + K18 neurons (3 mg/mL) from TEM images. n = 16 from two/ three independent experiments with eight neurons each. ***p < 0.001, one-way ANOVA, Tukey post-hoc test. (C) Representative TEM images from soma of 3 mg/mL K18-seeded D43 (7 + 36) 4R-P301S neurons at different magnifications. Scale bars left to right: 2 mm, 500 nm, 200 nm. (D) Representative TEM images from neuronal processes of 3 mg/mL K18-seeded D35 (7 + 28) 4R and 4R-P301S neurons. Scale bars left to right: 500 nm, 500 nm, 200 nm, and 100 nm. (E) VAMP7 dominant-negative (VAMP7DN) functional knockdown mechanism. TM, transmembrane. (F) Strategy for VAMP7 functional knockdown and analysis of 4R-P301S neurons. (G) Quantification of total Tau secreted from 4R and 4R-P301S neurons ±3 mg/mL K18 and overexpressing GFP or GFP-VAMP7DN incubated with 50 mM KCl. n = 7–8 from two independent experiments. **p < 0.01, one-way ANOVA, Tukey’s post-hoc test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human Tau (Total) ELISA Kit Thermo Fisher Scientific Cat#KHB0042 NucleoBond Xtra Maxi EF Kit Macherey-Nagel Cat#740424.5 Laminin coated coverslips Neuvitro Corporation Cat#GG-12-15-Laminin ToxinSensor Chromogenic LAL Endotoxin Assay Kit GenScript Cat#L00350C VECTASHIELD Antifade Mounting Medium without DAPI Vector Laboratories Cat#H-1000-10 VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories Cat#H-1200-10 NucleoSpin Blood L kit Macherey Nagel Cat#740954.20 MOM Immunodetection Kit Vector Laboratories Cat#BMK-2202 Deposited data Raw bulk RNA-seq data This paper GEO: GSE255902 Raw single-cell RNA-seq data This paper GEO: GSE255904 Experimental models: Cell lines Human: HEK293T line ATCC Cat#CRL-11268 Human: i3N iPSC line Wang et al.15 N/A Experimental models: Organisms/strains Mouse: Human Tau P301S (PS19) transgenic mice Jackson Laboratory Cat#008169 Oligonucleotides Primers for sequencing; see Table S1 This paper N/A Primers for qPCR; see Table S6 This paper N/A Recombinant DNA pCDH-EF1-FLPe Kazuhiro Oka Addgene #72262 psPAX Didier Trono Addgene #12260 pMD2.G Didier Trono Addgene #12259 pMDLg/pRRE Didier Trono Addgene #12251 pRSV-REV Didier Trono Addgene #12253 pC13N-CLYBL-CAG-dCas9-BFP-KRAB Martin Kampmann Addgene #127968 pMK1334 Martin Kampmann Addgene #127965 pFUGW-hSyn-jGCaMP8f This paper N/A PFUGW-hSyn-hM4D(Gi)-mCherry This paper N/A pFUGW-eGFP Manu Sharma Gift pFUGW-GFP-VAMP72-120 (VAMPDN) Manu Sharma Gift Software and algorithms R 4.2.2 The R project https://www.r-project.org/ RStudio 2022.07.2 RStudio: Integrated Development for R. RStudio https://rstudio.com Gene set enrichment analysis (GSEA) Subramanian et al.70 https://www.gsea-msigdb.org/gsea/ msigdb/index.jsp ClueGO Bindea et al.71; Shannon et al.72 https://apps.cytoscape.org/apps/cluego Adobe Illustrator Illustrator v26.5.2 https://www.adobe.com/products/ illustrator.html GraphPad Prism 6 Prism v9.2 https://www.graphpad.com FlowJo FlowJo v10 https://www.flowjo.com/ Cell Ranger- 6.1.2 10x Genomics https://support.10xgenomics.com/ single-cell-gene-expression/software/ pipelines/latest/what-is-cell-ranger (Continued on next page) ll e3 Cell 187, 1–19.e1–e13, May 9, 2024 Please cite this article in press as: Parra Bravo et al., Human iPSC 4R tauopathy model uncovers modifiers of tau propagation, Cell (2024), https://doi.org/10.1016/j.cell.2024.03.015 Article

Techniques: Dominant Negative Mutation, Functional Assay, Knockdown, Incubation

Journal: Cell

Article Title: Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

doi: 10.1016/j.cell.2024.03.015

Figure Lengend Snippet: Figure 4. Tau inclusions impair spontaneous and evoked neuronal activity (A) Strategy for targeting HaloTag at the 50 end of the MAPT locus in the 4R-P301S iPSC line. (B) Representative fluorescence image of 3 mg/mL K18-seeded 4R-P301S-HaloTag neurons (D24) harboring Tau inclusions incubated with JFX549-HaloTag ligand. Scale bar, 50 mm. (C) Representative fluorescence images expressing GCaMP8f, HaloTag, and overlay in D24 K18-seeded 4R-P301S-HaloTag spontaneous activity. Scale bar, 50 mm. (D) Quantification of spike amplitude, firing rate, and synchrony index from calcium imaging of K18-seeded 4R-P301S-HaloTag neurons (D24–D30). n = inclusion/+inclusion for spike amplitude (74/121), firing rate (76/122), and network (14/16) from 3 biological replicates. *p < 0.05, unpaired t test. (E) Representative fluorescence images of GCaMP8f, HaloTag, and overlay in D30 K18-seeded 4R-P301S-HaloTag treated with 50 mM KCl. Scale bar, 50 mm. (F) Representative averaged calcium traces from one KCl stimulation experiment in 4R-P301S-HaloTag neurons (D30) with and without Tau inclusions. Recording is 400 s. n = 11 inclusion and 8 +inclusion. Mean ± SEM. (G) Quantification of peak amplitude from KCl-stimulation-induced neuronal responses. n = 33 inclusion and 20 +inclusion from 3 biological replicates. *p < 0.05, unpaired t test. (H) Strategy for sustained neuronal activity repression and analysis of 4R-P301S-HaloTag neurons. (I) Representative spontaneous calcium traces (right) of each cell from D36 4R-P301S expressing hM4Di (left) upon 10 mM CNO addition at 60 s. Scale bar, 50 mm. (J) Quantification of MC1 area/DAPI+ nuclei from immunofluorescence images of chronically silenced D29 K18-seeded 4R-P301S neurons. n = 50 CNO and 30 +CNO from 10 images; 5 CNO biological replicates and 3 +CNO biological replicates. **p < 0.01, unpaired t test. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human Tau (Total) ELISA Kit Thermo Fisher Scientific Cat#KHB0042 NucleoBond Xtra Maxi EF Kit Macherey-Nagel Cat#740424.5 Laminin coated coverslips Neuvitro Corporation Cat#GG-12-15-Laminin ToxinSensor Chromogenic LAL Endotoxin Assay Kit GenScript Cat#L00350C VECTASHIELD Antifade Mounting Medium without DAPI Vector Laboratories Cat#H-1000-10 VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories Cat#H-1200-10 NucleoSpin Blood L kit Macherey Nagel Cat#740954.20 MOM Immunodetection Kit Vector Laboratories Cat#BMK-2202 Deposited data Raw bulk RNA-seq data This paper GEO: GSE255902 Raw single-cell RNA-seq data This paper GEO: GSE255904 Experimental models: Cell lines Human: HEK293T line ATCC Cat#CRL-11268 Human: i3N iPSC line Wang et al.15 N/A Experimental models: Organisms/strains Mouse: Human Tau P301S (PS19) transgenic mice Jackson Laboratory Cat#008169 Oligonucleotides Primers for sequencing; see Table S1 This paper N/A Primers for qPCR; see Table S6 This paper N/A Recombinant DNA pCDH-EF1-FLPe Kazuhiro Oka Addgene #72262 psPAX Didier Trono Addgene #12260 pMD2.G Didier Trono Addgene #12259 pMDLg/pRRE Didier Trono Addgene #12251 pRSV-REV Didier Trono Addgene #12253 pC13N-CLYBL-CAG-dCas9-BFP-KRAB Martin Kampmann Addgene #127968 pMK1334 Martin Kampmann Addgene #127965 pFUGW-hSyn-jGCaMP8f This paper N/A PFUGW-hSyn-hM4D(Gi)-mCherry This paper N/A pFUGW-eGFP Manu Sharma Gift pFUGW-GFP-VAMP72-120 (VAMPDN) Manu Sharma Gift Software and algorithms R 4.2.2 The R project https://www.r-project.org/ RStudio 2022.07.2 RStudio: Integrated Development for R. RStudio https://rstudio.com Gene set enrichment analysis (GSEA) Subramanian et al.70 https://www.gsea-msigdb.org/gsea/ msigdb/index.jsp ClueGO Bindea et al.71; Shannon et al.72 https://apps.cytoscape.org/apps/cluego Adobe Illustrator Illustrator v26.5.2 https://www.adobe.com/products/ illustrator.html GraphPad Prism 6 Prism v9.2 https://www.graphpad.com FlowJo FlowJo v10 https://www.flowjo.com/ Cell Ranger- 6.1.2 10x Genomics https://support.10xgenomics.com/ single-cell-gene-expression/software/ pipelines/latest/what-is-cell-ranger (Continued on next page) ll e3 Cell 187, 1–19.e1–e13, May 9, 2024 Please cite this article in press as: Parra Bravo et al., Human iPSC 4R tauopathy model uncovers modifiers of tau propagation, Cell (2024), https://doi.org/10.1016/j.cell.2024.03.015 Article

Techniques: Activity Assay, Incubation, Expressing, Imaging

Journal: Cell

Article Title: Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

doi: 10.1016/j.cell.2024.03.015

Figure Lengend Snippet: Figure 5. Identification of Tau inclusion modifiers by CRISPRi screening (A) Strategy for CRISPRi screening based on Tau inclusions in 4R-P301S-dCas9 neurons. (B) Volcano plot summarizing knockdown phenotypes and statistical significance (Mann-Whitney U test) for genes targeted in the pooled screen. Dashed lines are cutoff for hit genes (FDR = 0.05). (C) Subcellular classification of gene hits (product above 2.5 and below 2.5; see STAR Methods). (D and E) Representative immunofluorescence images (D) and quantification (E) of the percentage of DAPI+/MC1+ cells in D21 (7 + 14) 1.5 mg/mL K18-seeded 4R- P301S;VPS29/ neurons from clone #1 and #2 compared to the parent line. n = 4 replicates from one independent experiment per clone. ****p < 0.0001, one-way ANOVA, Dunnett’s multiple comparisons test. Scale bar, 50 mm. (F and G) Representative flow cytometry analysis (F) and quantification (G) of the percentage of MC1+ cells in D21 (7 + 14) 1.5 mg/mL K18-seeded 4R- P301S;VPS29/ neurons from clone #1 and clone #2 compared to the parent line. N = 6 replicates from one independent experiment per clone. ****p < 0.0001, one-way ANOVA, Dunnett’s multiple comparisons test. See also Figures S5 and S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human Tau (Total) ELISA Kit Thermo Fisher Scientific Cat#KHB0042 NucleoBond Xtra Maxi EF Kit Macherey-Nagel Cat#740424.5 Laminin coated coverslips Neuvitro Corporation Cat#GG-12-15-Laminin ToxinSensor Chromogenic LAL Endotoxin Assay Kit GenScript Cat#L00350C VECTASHIELD Antifade Mounting Medium without DAPI Vector Laboratories Cat#H-1000-10 VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories Cat#H-1200-10 NucleoSpin Blood L kit Macherey Nagel Cat#740954.20 MOM Immunodetection Kit Vector Laboratories Cat#BMK-2202 Deposited data Raw bulk RNA-seq data This paper GEO: GSE255902 Raw single-cell RNA-seq data This paper GEO: GSE255904 Experimental models: Cell lines Human: HEK293T line ATCC Cat#CRL-11268 Human: i3N iPSC line Wang et al.15 N/A Experimental models: Organisms/strains Mouse: Human Tau P301S (PS19) transgenic mice Jackson Laboratory Cat#008169 Oligonucleotides Primers for sequencing; see Table S1 This paper N/A Primers for qPCR; see Table S6 This paper N/A Recombinant DNA pCDH-EF1-FLPe Kazuhiro Oka Addgene #72262 psPAX Didier Trono Addgene #12260 pMD2.G Didier Trono Addgene #12259 pMDLg/pRRE Didier Trono Addgene #12251 pRSV-REV Didier Trono Addgene #12253 pC13N-CLYBL-CAG-dCas9-BFP-KRAB Martin Kampmann Addgene #127968 pMK1334 Martin Kampmann Addgene #127965 pFUGW-hSyn-jGCaMP8f This paper N/A PFUGW-hSyn-hM4D(Gi)-mCherry This paper N/A pFUGW-eGFP Manu Sharma Gift pFUGW-GFP-VAMP72-120 (VAMPDN) Manu Sharma Gift Software and algorithms R 4.2.2 The R project https://www.r-project.org/ RStudio 2022.07.2 RStudio: Integrated Development for R. RStudio https://rstudio.com Gene set enrichment analysis (GSEA) Subramanian et al.70 https://www.gsea-msigdb.org/gsea/ msigdb/index.jsp ClueGO Bindea et al.71; Shannon et al.72 https://apps.cytoscape.org/apps/cluego Adobe Illustrator Illustrator v26.5.2 https://www.adobe.com/products/ illustrator.html GraphPad Prism 6 Prism v9.2 https://www.graphpad.com FlowJo FlowJo v10 https://www.flowjo.com/ Cell Ranger- 6.1.2 10x Genomics https://support.10xgenomics.com/ single-cell-gene-expression/software/ pipelines/latest/what-is-cell-ranger (Continued on next page) ll e3 Cell 187, 1–19.e1–e13, May 9, 2024 Please cite this article in press as: Parra Bravo et al., Human iPSC 4R tauopathy model uncovers modifiers of tau propagation, Cell (2024), https://doi.org/10.1016/j.cell.2024.03.015 Article

Techniques: Knockdown, MANN-WHITNEY, Cytometry