Journal: Cell reports

Article Title: Alterations of ceramide synthesis induce PD-L1 internalization and signaling to regulate tumor metastasis and immunotherapy response

doi: 10.1016/j.celrep.2024.114532

Figure Lengend Snippet:

Article Snippet: Tris based unmasking solution , VectorLabs , Cat# H-3301-250.

Techniques: Recombinant, Red Blood Cell Lysis, Lysis, Staining, Transfection, In Situ, Proximity Ligation Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Sequencing, shRNA, Software

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet: (A) Flow cytometric analysis of the colon cancer cell lines HCT116 (left panel) and SW480 (right panel) with antibodies directed against CD44 and EpCAM. EpCAM/CD44-positive and -negative regions (gray quadrants) were defined as in Figure supplement 1 using multiple isotype controls and are shown by the quadrants in the plots. Notably, both HCT116 and SW480 revealed a continuum of different EpCAM and CD44 expression levels with a large CD44 high EpCAM high (EpCAM hi ) cluster followed by a tail of gradually decreasing EpCAM and increasing CD44 levels. By applying specific gates, cells were divided in a large EpCAM hi cluster, together with a considerably smaller CD44 high EpCAM low (EpCAM lo ) subpopulation. To ensure good separation from the large EpCAM hi cluster and maximal sorting purity, EpCAM lo cells were gated as CD44 hi events ≤ 60% of the EpCAM fluorescence intensity of the left border of the EpCAM hi gate and sorted from ≤50% of that value. Variable percentages of EpCAM lo cells were found to feature the HCT116 (5.0% ± 2.5%) and SW480 (16.7% ± 13%) cell lines, respectively. For the sake of simplicity, gates are shown in the figure only if they encompass sizeable percentages of cells. Graphs show representative analysis of one experiment. ( B ) Phase-contrast microscopy images of sorted EpCAM hi and EpCAM lo cells from HCT116 (upper images) and SW480 (lower images) cells. While EpCAM hi cells formed compact colonies with characteristic epithelial morphology, EpCAM lo cells showed a more spindle- and mesenchymal-like appearance. Scale bar: 100 µm. ( C ) Intrasplenic injection of bulk, EpCAM hi , and EpCAM lo cells from HCT116 (left panel) and SW480 (right panel). For each transplantation experiment, 2 × 10 4 cells were injected in the spleen of a recipient NSG mouse. 4 (HCT116) and 8 (SW480) weeks after injection, mice were sacrificed and individual tumors counted. Single and double asterisks indicate significant differences (p<0.05 and p<0.01, respectively). HCT116: bulk (n = 8), EpCAM hi (n = 9), and EpCAM lo (n = 7). SW480: bulk (n = 4), EpCAM hi (n = 4), and EpCAM lo (n = 4). ( D ) Images of mouse livers 4 (HCT116) and 8 (SW480) weeks after orthotopic injection with 10 4 cells. Scale bar: 5 mm. Figure 1—source data 1. EpCAM lo cells among colon cancer cell lines. The percentage of CD44 hi /EpCAM lo subpopulation was determined in a panel of commonly employed colon cancer cell lines by flow cytometric analysis. Please note that, within the same cell line, these percentages can vary depending on the passage number and culture conditions. If indicated by Lindeman et al., consensus molecular subtype classification of the cell lines is shown. Figure 1—source data 2. Cell cycle analysis of EpCAM hi and EpCAM lo cells in HCT116 and SW480. Cells fractions were sorted and plated in culture. After 72 hr, cells were fixed and stained with propidium iodide. Cell cycle distribution was assayed by flow cytometry. Tables demonstrate average and standard deviation of three independent experiments. Figure 1—source data 3. Quantification of EpCAM hi/lo percentages of all liver metastases as determined by FACS.

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Expressing, Fluorescence, Microscopy, Injection, Transplantation Assay, Cell Cycle Assay, Staining, Flow Cytometry, Standard Deviation

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet: ( A ) FSC-A/SSC-A, FSC-W/FSC-A, and SSC-W/SSC-A single-cell gates (confirmed by gating on FSC-A/FSC-H). Purity of sorted single cells was confirmed by microscopy. ( B ) Acquisition parameters used for FACS analysis. ( C ) Gating strategy employed to select for live cells by DAPI staining (top: unstained cells; bottom: after DAPI staining). ( D ) Top: unstained and isotype controls, showing absence of relevant unspecific antibody binding. Bottom: fluorescence minus one (FMO) samples, showing the absence of uncompensated spillover between fluorescence channels. For the sake of clarity, a quadrant is placed to show the relative position of unstained and single stains in both cells lines. For the EpCAM-FITC antibody, a mouse IgG1-FITC isotype control S. Cruz sc-2855 was used; for the CD44-APC antibody, a Rat IgG2a-APC isotype control S. Cruz sc-2895 was used. Similar results were obtained with SW480 cells (not shown). ( E ) Full EpCAM/CD44 staining, also showing the rationale behind the definition of high and low referred to EpCAM and CD44 levels. Despite the differences in CD44 levels between EpCAM hi and EpCAM lo cells, for the sake of simplicity the populations were defined ‘low’ for a defined marker if they were mainly covering a region within the 1st log above the negative gate, high if they were above the 1st log. FSC-H: forward-scatterheight; FSC-W: forward-scatter width.

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Microscopy, Staining, Binding Assay, Fluorescence, Control, Marker

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet: ( A ) Top panel: transwell migration assay of EpCAM hi (black bar) and EpCAM lo (gray bar) cells from the HCT116 and SW480 lines. 10 5 cells were plated on TC-coated membrane in triplicate and left overnight before counting the number of migrated cells on the bottom side of the membrane. Each bar represents the mean ± SD of two independent experiments. Asterisks indicate significant differences (p<0.05). Bottom panel: invasion assay of EpCAM hi (black bar) and EpCAM lo (gray bar) cells from the HCT116 and SW480 lines. 10 5 cells were plated in triplicate on top of an extracellular matrix-coated membrane and left overnight before counting the number of cells migrated to other side of the membrane. Each bar represents the mean ± SD of two independent experiments. Asterisks indicate significant differences (p<0.05). ( B ) RT-qPCR expression analysis of epithelial ( EPCAM and CDH1 ) and mesenchymal ( VIM ) markers in sorted EpCAM hi (black bars) and EpCAM lo (gray bars) from the HCT116 and SW480 lines. GAPDH was employed for normalization purposes. Each bar represents the mean ± SD of three independent experiments. Asterisks indicate significant differences (p<0.05). ( C ) RT-qPCR expression analysis of epithelial to mesenchymal transition transcription factors ( ZEB1, ZEB2, TWIST, FOXC2, SLUG, and SNAIL ) in EpCAM hi (black bars) and EpCAM lo (gray bars) cells. Left panel: HCT116. Right panel: SW480. GAPDH was employed for normalization. Each bar represents the mean ± SD of three independent experiments. Asterisks indicate significant differences (p<0.05). ( D ) Immunofluorescence (IF) analysis of EpCAM hi and EpCAM lo cells. Cells were sorted and directed plated on cover slips. After 4 days, cells were fixed with 4% paraformaldehyde and stained with antibodies against EpCAM (green) and ZEB1 (red). Nuclei were visualized by DAPI staining of DNA (blue). Scale bar: 50 µm.

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Transwell Migration Assay, Membrane, Invasion Assay, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet: ( A ). qRT-PCR expression analysis of ZEB1 in HCT116 and SW480 transduced with an inducible control (shCT) or ZEB1-shRNA (shZEB1) construct. shRNA expression was induced with 1 µg/mL of doxycycline. Each bar represents the mean ± SD of three independent experiments. ( B ) Bar graph of flow cytometric analysis (see B ). Each bar represents the relative mean ± SD of three independent experiments. ( C ) Representative analysis of the flow cytometric analysis of the shCT- and shZEB1-transfected HCT116 and SW480 cell lines using antibodies against CD44 and EpCAM. Cells were induced with 1 µg/mL doxycycline for 72 hr before analysis. ( D ) RT-qPCR expression analysis of the members of the miRNA 200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) in EpCAM hi (black bars) and EpCAM lo (gray bars) cells. Upper panel: HCT116. Bottom panel: SW480. U6 was employed for normalization. Each bar represents the mean ± SD of three independent experiments. Single asterisks indicate significant differences of p<0.05, double asterisks of p<0.01, and triple asterisks of p<0.001. ( E ) Cell proliferation assay. Sorted bulk, EpCAM hi and EpCAM lo cells were seeded in triplicate in plates and cultured in conventional medium. HCT116 and SW480 cells were harvested and number of cells was counted at 4 and 11 days, respectively. Each bar represents the mean ± SD three independent experiments. ( F ) Cell cycle analysis of EpCAM hi and EpCAM lo cells in HCT116 (upper panel) and SW480 (lower panel). Cell fractions were sorted and plated in culture. After 72 hr, cells were fixed and stained with propidium iodide. Cell cycle distribution was assayed by flow cytometry. Graphs show representative analysis of one experiment. Tables demonstrate average and standard deviation of three independent experiments. White graph: EpCAM hi ; gray graph: EpCAM lo . Asterisks show the significant (p<0.05) differences between EpCAM hi and EpCAM lo cells in G 1 and G 2 M-phases.

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Expressing, Transduction, Control, shRNA, Construct, Transfection, Proliferation Assay, Cell Culture, Cell Cycle Assay, Staining, Flow Cytometry, Standard Deviation

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet: ( A ) Hematoxylin and eosin (H&E; first two panels) and immunohistochemistry (IHC) with antibody directed against β-catenin (third and fourth panels) in liver metastasis obtained 4 or 8 weeks after intrasplenic injection with HCT116 (upper panels) and SW480 (lower panels) cells, respectively. Second and fourth panels show zoom of the marked area in respectively first and third panels. Scale bar first and third panels: 100 µm. Scale bar second and fourth panels: 50 µm. ( B ) FACS analysis of liver metastases obtained by spleen injection of HCT116 bulk, EpCAM hi , and EpCAM lo cells. Upper panel: representative FACS plots. ( C ) Microscopic (top, left panel) analysis of AKP-Z organoids tagged with GFP and click beetle luciferase. The graph (bottom, left panel) shows the RT-qPCR expression analysis of Zeb1 in AKP (left) and AKP-Z (right) organoids upon in vitro doxycycline treatment for 48 hr. Black bars: no doxycycline treatment; gray bars: 1 μg/mL doxycycline. Each bar represents the mean ± SD of three independent experiments. ( D ) Upon establishment of a primary tumor in the caecum, transplanted mice were administered doxycycline in the drinking water to induce Zeb1 expression. FACS analysis of the primary tumor was performed 1 week after the start of the doxycycline. The panels show representative FACS plots of a control and two dox-treated primary tumors. ( E ) Quantification of the number of lung and liver metastases in uninduced (black; n = 4) and dox-induced (gray; n = 5) AKP-Z transplanted mice. Liver tissue was cut into 500 µm slices, processed for IHC, stained for β-catenin to visualize tumor cells, scanned using a NanoZoomer, and counted using NDP view software. The area of tissue analyzed was used to normalize the data.

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Immunohistochemistry, Injection, Luciferase, Quantitative RT-PCR, Expressing, In Vitro, Control, Staining, Software

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet: ( A ) RT-qPCR expression analysis of EPCAM , CDH1 , VIM , and ZEB1 in ‘early’ (e.g., cells that were used shortly after FACS sorting) and ‘late’ (e.g., cells that were cultured for an extended period of time before performing the experiment) sorted EpCAM hi and EpCAM lo cell from HCT116 and SW480 cell lines. GAPDH was employed for normalization purposes. Each bar represents the mean ± SD of two independent experiments. ( B ) Transwell migration assay of ‘early’ and ‘late’ EpCAM hi (black bar) and EpCAM lo (gray bar) cultures in HCT116. 10 5 cells were plated in triplicate on TC-coated membrane and left overnight before counting the number of migrated cells on the bottom side of the membrane. Each bar represents the mean ± SD of two independent experiments. Asterisks indicate significant differences (p<0.05). ( C ) Analysis of the HCT116 scRNAseq data as a Markov diffusion process. Markov forward (left) and backward (right) diffusion indicating the presence of sink and source points in both EpCAM hi and EpCAM lo populations. ( D ) Partition-based graph abstraction velocity graph mapping out the direction of velocity on a subpopulation level in HCT116.

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Transwell Migration Assay, Membrane, Diffusion-based Assay

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet: ( A ) Multidimensional scaling analysis of RNAseq profiles of EpCAM hi and EpCAM lo cells from the HCT116 and SW480 lines. Red: HCT116, black: SW480, circle: EpCAM hi , triangle: EpCAM lo . ( B ) Ingenuity Pathway Analysis (IPA) of the HCT116 and SW480 expression profiles from the multicell line analysis (p adjusted value <0.01; log 2 fold change <–1.5 and >1.5). Red marked pathways highlight the enhanced involvement of pathways involved in epithelial to mesenchymal transition, Wnt signaling, and the formation of colon cancer metastasis in the EpCAM lo subpopulation compared to EpCAM hi cells. ( C ) TOP-Flash luciferase reporter analysis of Wnt signaling activity in colon cancer cell lines HCT116 and SW480 upon treatment with 4 µM Chiron for 3 days. Each bar represents the mean ± SD of two independent experiments. ( D ) Flow cytometric analysis using antibodies directed against CD44 and EpCAM of control and 4 µM Chiron—figure supplemented HCT116 ( A ) and SW480 ( B ) cultures. Graphs show percentage of cells within the CD44 hi EpCAM hi and CD44 hi EpCAM lo gates relative to the control. Each bar represents the mean ± SD of two independent experiments. ( E ) Immunofluorescence analysis of control and Chiron-treated HCT116 (left panel) and SW480 (right panel) cells. After 3 days of treatment, cells were fixed with 4% paraformaldehyde and stained with antibodies against EpCAM (green) and ZEB1 (red). Nuclei were visualized by DAPI staining of DNA (blue). Scale bar: 100 µm. ( F ) Flow cytometric analysis of three HCT116 cell lines with differential β-catenin mutation status, a parental HCT116 (HCT116-P) cell line harboring one WT and one mutant allele (Ser45 del), and two HCT116-WT and HCT116-MT cell lines harboring one WT or one mutant allele, respectively, generated by disruption of the other allele in the parental cell line . Figure 3—source data 1. List of differentially expressed genes in EpCAM lo vs. EpCAM hi cells in HCT116 and SW480. A total of 152 and 353 differentially regulated genes were identified between the CD44 hi EpCAM hi and CD44 hi EpCAM lo cells in HCT116 and SW480, respectively (p adjusted<0.01, when applying a log 2 fold change of <–1.5 and > 1.5).

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Expressing, Luciferase, Activity Assay, Control, Immunofluorescence, Staining, Mutagenesis, Generated, Disruption

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet: ( A ) tSNE of the SW480 cell line indicating an additional subpopulation in the EpCAM hi population (left panel). Using a signature derived from the bulk RNAseq, this population was identified as the ‘sphere’ population (middle panel) and annotated to be excluded for further analysis (right panel). ( B ) Left panel: the SW480 cell line, after exclusion of the ‘sphere’ population, contains slightly higher variability compared to the HCT116 cell line, as evidenced by the variance of the top 50 principal components. Right panel: while in HCT116 most of the variable expressed genes are differentially expressed between the EpCAM hi and EpCAM lo population, this is not the case in SW480, where most of the highly variable genes do not differ between the two populations. ( C ) Top panels: expression values of VIM , ZEB1, and CD44 on the UMAP embedding of the HCT116 cell line. Lower panels: projection of the RNA velocity direction of the same genes. ( D ) HCT116 UMAP embedding annotated with the eight unsupervised clusters. ( E ) Heatmap of HCT116 with expression values of the epithelial to mesenchymal transition (EMT) signature averaged by the eight clusters. Clusters were ranked according to their EMT score, and genes were clustered in four distinct gene sets using k-means clustering. ( F ) Schematic diagram showing a transcriptional trajectory with distinct gene arrays through which pEMT cells arise.

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Derivative Assay, Expressing

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet:

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Mutagenesis, Expressing, Plasmid Preparation, Sample Prep, Software

Journal: eLife

Article Title: Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

doi: 10.7554/eLife.61461

Figure Lengend Snippet:

Article Snippet: Cells were then exposed overnight at 4°C to primary antibodies against EpCAM (mouse, 1:250; sc-66020; Santa Cruz Biotechnology) and ZEB1 (rabbit, 1:200; sc-25388, Santa Cruz Biotechnology).

Techniques:

Journal: iScience

Article Title: A transfer learning framework to elucidate the clinical relevance of altered proximal tubule cell states in kidney disease

doi: 10.1016/j.isci.2024.109271

Figure Lengend Snippet: Characterization of altered proximal tubule cells (A) UMAP representation of the PT compartment from AKI and control patients. (B) Relative abundance of PT1 and PT2 clusters in AKI and control patients. (C) Expression of known injury marker genes in PT1 and PT2 clusters. (D and E) Gene-weighted density of HAVCR1 and VCAM1. (F) Enrichment score calculated by gene set enrichment analysis using Reactome pathway database (positive enrichment means an enrichment in PT2 cluster). (G) Pathway activity in PT1 and PT2 clusters (inferred from PROGENy). (H) Collagen, extracellular matrix (ecm) proteoglycan (pg) and glycoprotein (gp) scores in PT1 and PT2 clusters. (I) Potential of heat-diffusion for affinity-based trajectory embedding (PHATE) dimension reduction projecting pathway enrichment estimated by GSVA for each cell type. (J and K) Representative immunostainings of HAVCR1 (J) and VCAM-1 (K) proteins in fibrotic area. ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001.

Article Snippet: Immunochemistry staining were performed as followed: after antigen retrieval with pressurized heating chamber in citrate buffer pH7 or tris-EDTA pH9, 5μm tissue sections were incubated with antibodies mouse monoclonal anti-human HAVCR1 (dilution 1:250, clone 219211, RD Systems) and mouse monoclonal anti-human VCAM1 (dilution 1:25, clone 1.4C3, Invitrogen) for 1h at room temperature.

Techniques: Control, Expressing, Marker, Activity Assay, Diffusion-based Assay

Journal: iScience

Article Title: A transfer learning framework to elucidate the clinical relevance of altered proximal tubule cell states in kidney disease

doi: 10.1016/j.isci.2024.109271

Figure Lengend Snippet:

Article Snippet: Immunochemistry staining were performed as followed: after antigen retrieval with pressurized heating chamber in citrate buffer pH7 or tris-EDTA pH9, 5μm tissue sections were incubated with antibodies mouse monoclonal anti-human HAVCR1 (dilution 1:250, clone 219211, RD Systems) and mouse monoclonal anti-human VCAM1 (dilution 1:25, clone 1.4C3, Invitrogen) for 1h at room temperature.

Techniques: Control, Software

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet: Neogenin forms nanoclusters with the WRC on dendritic spines (A) Schematic outlines the experimental workflow. (B and C) Endogenous Neogenin colocalizes with the WRC subunits Cyfip1 (B) and WAVE1 (C) on spines of hippocampal neurons expressing GFP. (D) Super-resolution confocal microscopy (90 nm resolution) shows that Neogenin and Cyfip1 colocalize with the PSD protein PSD-95. (E) STORM imaging (20 nm resolution): Neogenin is clustered within Cyfip1 nanodomains and also forms nanoclusters independently of Cyfip1. (F) Percentage of Neogenin associated with Cyfip1 nanodomains ( N = 53 spines) and percentage of Cyfip1 nanoclusters associated with Neogenin ( N = 55 spines). (G) Quantification of Neogenin/Cyfip1 and Neogenin-only nanocluster diameters (unpaired Student’s t test, p < 0.0001, F(46, 46) = 1.516; p = 0.1620, N = 47 spines, mean ± SEM, ∗∗∗∗ p < 0.0001). (H) Line-scan analysis of fluorescence intensities shows that Neogenin is surrounded by a ring of Cyfip1. (I and J) Percentage of Neogenin ( N = 61 spines) or Cyfip1 nanoclusters ( N = 55 spines) per spine. (K) Pearson correlation analysis of Neogenin nanoclusters/spine (red, R 2 = 0.55, slope = 2.38 ± 0.30, F(1, 50) = 61.11, p < 0.0001) and Cyfip1 nanoclusters/spine (green, R 2 = 0.12, slope = 0.25 ± 0.09, F(1, 50) = 6.484, p = 0.0141) correlates with increasing spine size ( N = 52 spines). (L) Pearson correlation analysis of Neogenin cluster size with increasing spine size (R 2 = 0.28, slope = 0.17 ± 0.03, F(1, 65) = 25.34, p < 0.0001, N = 67 spines). Also see Figures S1 and .

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: Expressing, Confocal Microscopy, Imaging, Fluorescence

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet: Neogenin mobility is confined within spines compared to shafts (A) Schematic outlines the experimental workflow. (B) Representative image of GFP epifluorescence in hippocampal neurons cotransfected with Neo shRNA and Neo-mEos. (C–E) (C) Neo-mEos localization intensity map (blue, higher localization intensity), (D) map of individual trajectories, and (E) diffusion coefficient map (blue, higher mobility). (F) Average mean squared displacement as a function of time for Neo-mEos trajectories calculated for (i) spines (red = mean) or (ii) shafts (blue = mean) and (iii) mean squared displacement for shafts versus spines. (G) Neo-mEos mobility (Area Under the Curve, AUC) is reduced for spines compared to shafts. (H) Frequency distribution curves for Neo-mEos diffusion coefficients [D] calculated for (i) spines (mean = red) or (ii) shafts (mean = blue) and (iii) mean diffusion coefficient frequency distribution for shafts versus spines. Dotted line: threshold value (−1.45 μm 2 /s) segregating immobile and mobile fractions. (I) Neo-mEos immobile fraction is increased in spines compared to shafts. (F and H) Gray lines represent single neurons. Unpaired Student’s t test; (G) p < 0.0001; F(15, 15) = 2.683, p = 0.0652, (I) p = 0.0031; F(15, 15) = 1.547, p = 0.407. (G and I) N = 16 neurons, mean ± SEM, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (J) Neo-mEos trajectories on the spine membrane (red) are more confined compared to the shaft (blue). Also see Figure S3 .

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: shRNA, Diffusion-based Assay, Membrane

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet: BDNF increases Neogenin/Cyfip1 association with the postsynaptic density (A) Immunolabelling was performed in hippocampal neurons expressing GFP before and after BDNF treatment. Images were captured using the super-resolution Airyscan 2 microscope. (B and C) Representative images of GFP (gray), Cyfip1 (green), Neogenin (yellow) and PSD-95 (magenta) in spines in BDNF untreated (B) and treated (C) neurons. (D and E) Quantification of (D) Neogenin clusters and (E) Cyfip1 clusters associated with PSD-95 +/− BDNF. Unpaired Student’s t test: (D) Neo only, p < 0.0001; F(80, 74) = 1.817, p = 0.0099; Neo+PSD-95, p < 0.0001; F(74, 80) = 1.968, p = 0.0032, (E) Cyfip1 only, p < 0.0001; F(80, 74) = 1.817, p = 0.0099; Cyfip1+PSD-95, p < 0.0001; F(74, 80) = 1.968, p = 0.0032. N = 75–81 spines, 11 neurons/condition, mean ± SEM, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: Expressing, Microscopy

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet: BDNF induces WRC-dependent diffusion trapping of Neogenin on spines (A) sptPALM was performed on hippocampal spines after shRNA depletion of Neogenin and rescue with either Neo-mEos or NeoΔWIRS-mEos. (B–E) Neo-mEos (B and D) and NeoΔWIRS-mEos (C and E) trajectory maps, localization intensity maps (blue, higher localization intensity) and diffusion coefficient maps (blue, higher mobility) in untreated (B and C) or BDNF-treated (D and E) cultures. (F–K) Quantification of the average mean squared displacement as a function of time (F and I), mobility (area under the curve) (G and J) and immobile fraction (H and K) for spines (F–H) and shafts (I–K). Two-way ANOVA, Tukey’s post hoc test: (G) F1(17, 36) = 1.077, p = 0.4098; F2(3, 26) = 11.00, p < 0.0001, (J) F1(19, 34) = 1.064, p = 0.4247; F2(3, 34) = 1.345, p = 0.2761, (H) F1(17, 36) = 1.231, p = 0.2910; F2(3, 26) = 10.38, p < 0.0001, (K) F1(18, 35) = 1.711, p = 0.0851; F2(3, 35) = 3.075, p = 0.0402. (F–K) N = 13–16 neurons, mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: Diffusion-based Assay, shRNA

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet: BDNF induces WRC-dependent Neogenin nanoclustering on spines (A) NASTIC analysis of hippocampal spines after shRNA depletion of Neogenin and rescue with either Neo-mEos or NeoΔWIRS-mEos. (B) Representative images of the distribution of Neo and NeoΔWIRS trajectories and nanoclusters (outlined in color) in BDNF untreated and treated neurons. (C–G) BDNF induces nanoclustering in spines (C–F) but not shafts (G). Quantification of cluster density (C and G), cluster membership (D), cluster lifetime (E), and cluster radius (F). Two-way ANOVA, Tukey’s post hoc test: (C) F1(18, 34) = 1.546, p = 0.1336; F2(3, 34) = 8.752, p = 0.0002, (G) F1(17, 27) = 0.9043, p = 0.5765; F2(3, 27) = 0.2020, p = 0.8941, (D) F1(367, 379) = 1.189, p = 0.0478; F2(3, 379) = 5.564, p = 0.0010, (E) F1(359, 384) = 1.130, p = 0.1198; F2(3, 384) = 4.498, p = 0.0041, (F) F1(371, 388) = 1.169, p = 0.0638; F2(3, 388) = 1.065, p = 0.3640. (C and G) N = 13–14 neurons. (D–F) Neo ( N = 6,947 trajectories, 286 clusters), NeoΔWIRS ( N = 3,383 trajectories, 99 clusters), Neo + BDNF ( N = 6,840 trajectories, 255 clusters) and NeoΔWIRS + BDNF ( N = 4,709 trajectories, 114 clusters). (C–G) Mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: shRNA

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet: WRC diffusion trapping is dependent on Neogenin in spines (A) sptPALM was performed on hippocampal spines after cotransfection with Cyfip1 shRNA, Cyfip1-mEos, and Neo (shNeo) or control shRNA (shCtl). (B–E) (B and D) shCtl and (C and E) shNeo trajectory maps, localization intensity maps (blue, higher localization intensity) and diffusion coefficient maps (blue, higher mobility) in untreated (B and C) or BDNF-treated (D and E) neurons. (F–K) Quantification of the average mean squared displacement as a function of time (F and I), mobility (area under the curve) (G and J), and immobile fraction (H and K) for spines (F–H) and shafts (I–K). Two-way ANOVA, Tukey’s post hoc test: (G) F1(18, 28) = 0.9875, p = 0.4992; F2(3, 28) = 6.017, p = 0.0027, (J) F1(15, 31) = 1.980, p = 0.0528; F2(3, 31) = 4.538, p = 0.0095, (H) F1(18, 28) = 0.6421, p = 0.8351; F2(3, 28) = 2.882, p = 0.0535, (K) F1(15, 31) = 1.989, p = 0.0518; F2(3, 31) = 3.765, p = 0.0205. (F–K) N = 11–14 neurons, mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01. Also see Figures S4–S6 .

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: Diffusion-based Assay, Cotransfection, shRNA, Control

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet: Neogenin promotes nanoclustering of the WRC in the presence of BDNF (A) NASTIC analysis of hippocampal spines after cotransfection with Cyfip1 shRNA, Cyfip1-mEos, and Neo (shNeo) or control shRNA (shCtl). (B) Representative images of the distribution of Cyfip1-mEos trajectories and nanoclusters (outlined in color) +/− BDNF. (C–G) BDNF increases Cyfip1-mEos nanocluster density in spines (C) but not shafts (G). Quantification of cluster density (C and G), cluster membership (D), cluster lifetime (E), and cluster radius (F). Two-way ANOVA, Tukey’s post hoc test: (C) F1(13, 31) = 1.332, p = 0.2479; F2(3, 31) = 13.19, p < 0.0001, (G) F1(15, 31) = 0.4047, p = 0.9668; F2(3, 31) = 1.357, p = 0.2740, (D) F1(279, 596) = 0.8803, p = 0.8886; F2(3, 596) = 2.281, p = 0.0782, (E) F1(279, 603) = 0.9491, p = 0.6898; F2(3, 603) = 4.410, p = 0.0044, (F) F1(279, 603) = 0.9545, p = 0.6704; F2(3, 603) = 3.880, p = 0.0091. (C and G) N = 11–13 neurons. (D–F) shCtl ( N = 4,325 trajectories, 256 clusters), shNeo ( N = 2,667 trajectories, 172 clusters), shCtl + BDNF ( N = 6,425 trajectories, 334 clusters) and shNeo + BDNF ( N = 2,390 trajectories, 178 clusters). Mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: Cotransfection, shRNA, Control

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet: Neogenin-WRC interactions are required for BDNF-induced actin polymerization and calcium signaling (A) FRAP analysis of LifeAct-RFP-transfected hippocampal spines after shRNA depletion of Neogenin and rescue with either Neo-mEos or NeoΔWIRS-mEos. (B) Relative intensity curve showing F-actin recovery in shCtl spines before and after BDNF treatment. (C) Quantification of the stable F-actin fraction (unpaired Welch’s t test, p = 0.0448; F(14, 7) = 3.996, p = 0.0726, N = 8–15 neurons). (D) Relative intensity curve after BDNF addition showing F-actin recovery in the presence of Neo-mEos but not NeoΔWIRS-mEos. (E) Quantification of the stable F-actin fraction. (F) Representative FRAP images for (D). (G) Relative intensity curve showing F-actin recovery in the presence of Neo-mEos or NeoΔWIRS-mEos without BDNF. (H) Quantification of the stable F-actin fraction. (I) Schematic outlines the experimental workflow in J and K. (J and K) Relative fluorescence intensities (ΔF/F0) of calcium rises in response to BDNF. One-way ANOVA, Tukey’s post hoc test: (E) F = 6.385, p = 0.0008; F(3, 59) = 3.802, p = 0.0147, N = 15–17 neurons, (H) F = 0.6156, p = 0.6103; F = 6.385, F(3, 30) = 0.5987, p = 0.6208, N = 8–9 neurons), (K) F = 15.39, p < 0.0001; F(3, 46) = 1.176, p = 0.3290, N = 10–15 neurons. Mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01. Also see Figure S7 .

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: Transfection, shRNA, Fluorescence

Journal: iScience

Article Title: BDNF-dependent nano-organization of Neogenin and the WAVE regulatory complex promotes actin remodeling in dendritic spines

doi: 10.1016/j.isci.2024.110621

Figure Lengend Snippet:

Article Snippet: Immunostaining was performed as described above using goat anti-C-terminal Neogenin (1:250; C-20, Santa Cruz) and rabbit anti-Cyfip1 (1:400; Upstate).

Techniques: Recombinant, Derivative Assay, Protease Inhibitor, In Situ, Membrane, Sequencing, shRNA, Software, Imaging, Microscopy