Journal: BMC Cell Biology

Article Title: Functional blockade of α 5 β 1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

doi: 10.1186/1471-2121-11-81

Figure Lengend Snippet: Β1-integrin expression and adhesive properties of MLP29 and Hep16 cells . (A) Quantitative analysis by flow cytometry revealed that most (90%) of cells were positive for β 1 staining and that the intensity levels (Geo Mean fluorescence) of β 1 expression was higher in MLP29 than in Hep16 cells. The experiment was performed three times with similar results. (B) MTT assay was used to assess MLP29 and Hep16 cell adhesion capacity to different components of the extracellular matrix, including FN, VN, LMN and COL I. To prevent nonspecific cell adhesion, plates were blocked with BSA. Cell adhesion to the different substrates was estimated by measuring the optic density at A570 nm. The data presented summarize the mean (±SD) of three independent experiments, each performed in triplicate.

Article Snippet: Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α 5 β 1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1.

Techniques: Expressing, Adhesive, Flow Cytometry, Staining, Fluorescence, MTT Assay

Journal: BMC Cell Biology

Article Title: Functional blockade of α 5 β 1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

doi: 10.1186/1471-2121-11-81

Figure Lengend Snippet: α 5 β 1 integrin functional block induced spreading and scattering of MLP29 hepatic progenitor cells . (A) Immunofluorescent detection of F-actin in MLP29 cells showed that the cells grow in packed islands, while after the treatment with a specific α 5 β 1 -functional blocking antibody the cells undergo spreading and scattering. Higher magnification (252×) of untreated and treated MLP29 cells shows the characteristic actin microfilaments reorientation associated with cytoskeleton re-organization. Immunofluorescent and immunoblot detection of E-cadherin in MLP29 cells following α 5 β 1 functional blockade showed that the expression levels of E-cadherin decreased at the cell-cell junctions in cells treated with the specific functional blocking antibody against α 5 β 1 , which was corroborated by immunoblot analysis. (B) Representative images of the phospho-MAPKs arrays. Activation of the different members of the MAPKs family was identified by means of a key provided with the kit. MLP29 cells were treated with the α 5 β 1 functional blocking antibody or HGF/SF1, in the presence or absence of the specific MEK inhibitor U0126. Cells were lysed and total protein (20 μg) was resolved by SDS-PAGE and analyzed by immunoblot for ERK1/ERK2 MAPK and phospho-ERK1/ERK2 MAPKs. Blots were then reprobed with an antibody for β-actin as a control for protein loading. Results are representative of at least three independent experiments.

Article Snippet: Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α 5 β 1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1.

Techniques: Functional Assay, Blocking Assay, Western Blot, Expressing, Activation Assay, SDS Page, Control

Journal: BMC Cell Biology

Article Title: Functional blockade of α 5 β 1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

doi: 10.1186/1471-2121-11-81

Figure Lengend Snippet: Most significant up-regulations in MLP29 cells upon α 5 β 1 integrin functional blockade

Article Snippet: Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α 5 β 1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1.

Techniques: Functional Assay

Journal: BMC Cell Biology

Article Title: Functional blockade of α 5 β 1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

doi: 10.1186/1471-2121-11-81

Figure Lengend Snippet: Most significantly up-regulated genes (p < 0.0005) in MLP29 cells upon α 5 β 1 integrin functional blockade

Article Snippet: Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α 5 β 1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1.

Techniques: Functional Assay

Journal: BMC Cell Biology

Article Title: Functional blockade of α 5 β 1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

doi: 10.1186/1471-2121-11-81

Figure Lengend Snippet: Gene expression profiling of MLP29 hepatic progenitor cells after α 5 β 1 integrin blockade and HGF/SF stimulation . (A) (B) Plots showing the changes in the expression of gene sets involved in cell adhesion and migration, respectively. Data from three independent experiments were pooled and subjected to permutation analyses to assess the expression level changes of gene sets obtained from the Molecular Signatures Database. The resulting data are presented as color-encoded plots in which a p-value close to 1 indicates significant up-regulation, and a p-value close to 0 indicates a significant down-regulation.

Article Snippet: Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α 5 β 1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1.

Techniques: Gene Expression, Expressing, Migration

Journal: BMC Cell Biology

Article Title: Functional blockade of α 5 β 1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

doi: 10.1186/1471-2121-11-81

Figure Lengend Snippet: Changes in the expression level of genes encoding chromatin remodeling and transcription factors in MLP29 cells during migration . (A) Changes in the expression level of genes encoding chromatin remodeling factors were plotted according to their level of significance as a color-encoded map, in which a p-value close to 1 indicates significant up-regulation, and a p-value close to 0 indicates a significant down-regulation. (B) Changes in the expression level of transcription factors following the treatment with α 5 β 1 integrin blocking antibody or with HGF/SF1 for 24 hours.

Article Snippet: Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α 5 β 1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1.

Techniques: Expressing, Migration, Blocking Assay

Journal: BMC Cell Biology

Article Title: Functional blockade of α 5 β 1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

doi: 10.1186/1471-2121-11-81

Figure Lengend Snippet: Radial position of integrin genes during migration of MLP29 cells . Three-dimensional FISH analysis was performed with BAC clones for the Itgb1 or Itgb3 loci and the position of fluorescent signals was determined using computational programs for image analysis. Left panels, 3D reconstruction of MLP29 cell nuclei subjected to FISH analysis with a probe for the Itgb1 and Itgb3 genes. Right panel, absolute radial position of these genes in control and treated cells. Scale bar = 3 μm.

Article Snippet: Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α 5 β 1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1.

Techniques: Migration, Clone Assay, Control

Journal: The Journal of Neuroscience

Article Title: Deleted in Colorectal Carcinoma and Differentially Expressed Integrins Mediate the Directional Migration of Neural Precursors in the Rostral Migratory Stream

doi: 10.1523/JNEUROSCI.22-09-03568.2002

Figure Lengend Snippet: Six integrins are differentially expressed in the rostral migratory stream. The α1 (A) and β8 (B) integrin subunits are expressed (arrows) from the anterior horn of the subventricular zone to the center of the olfactory bulb from P0 mice.C, The β1-integrin subunit is expressed in the RMS (arrows), blood vessels (BV), and the choroid plexus (CPX) of the lateral ventricle (LV) of a P2 mouse. D, The αv-integrin subunit is found in the RMS (arrows) from a P30 mouse. E, The β6-integrin subunit is expressed in the RMS (arrows) of a P15 mouse. F, The β3-integrin subunit is observed in P30 rat RMS (arrows). Scale bar, 1 mm. AOB, Accessory olfactory bulb; CC, corpus callosum; CX, cerebral cortex.

Article Snippet: The function-blocking anti-β1-integrin rat monoclonal antibody (clone 9EG7) was obtained from PharMingen (San Diego, CA); its specificity has been reported previously ( Lenter et al., 1993 ; Lenter and Vestweber, 1994 ).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Deleted in Colorectal Carcinoma and Differentially Expressed Integrins Mediate the Directional Migration of Neural Precursors in the Rostral Migratory Stream

doi: 10.1523/JNEUROSCI.22-09-03568.2002

Figure Lengend Snippet: Summary of the stage-specific expression of integrin and laminin subunits in the RMS. Tenascin-C is expressed along the sides of the RMS but not in the RMS itself.

Article Snippet: The function-blocking anti-β1-integrin rat monoclonal antibody (clone 9EG7) was obtained from PharMingen (San Diego, CA); its specificity has been reported previously ( Lenter et al., 1993 ; Lenter and Vestweber, 1994 ).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Deleted in Colorectal Carcinoma and Differentially Expressed Integrins Mediate the Directional Migration of Neural Precursors in the Rostral Migratory Stream

doi: 10.1523/JNEUROSCI.22-09-03568.2002

Figure Lengend Snippet: Integrins mediate migration of neural precursors in the RMS. Brain slices from a P12 mouse were labeled with DiI and cultured in CCM1 medium with 5% horse serum for 5 hr in the presence of either control or anti-integrin antibodies. Three hours after addition of DiI, the migration was confirmed by fluorescence time lapse, and then a control or function-blocking anti-αv-integrin antibody was added for (Figure legend continued.) 5 hr, after which migration was recorded over the next 3 hr. A, Time-lapse sequence of three cells (a–c) in a slice migrating from the SVZ (bottom) toward the olfactory bulb (top) in the presence of a control antibody. Thearrow pointing to each cell shows the leading process, and the line shows the cell body. The interval between each image is 5 min. See Movie 1 (available at www.jneurosci.org).B, Graphical representation of the migration of the three cells (a–c) in A. Eachpoint represents the position of the cell body at 5 min time points. Note the unidirectional pathway and the bursts of rapid migration followed by slower meandering. C, Time-lapse sequence of images of seven cells (d–j) in a slice migrating from the SVZ (left) to the olfactory bulb (right) in the presence of an anti-αv-integrin antibody. Seven cells (d–j) are marked for reference. See Movie 2 (available at www.jneurosci.org). D, Graphical representation of the migration of the seven cells as described in C. The olfactory bulb is at theright. Note the inhibited migration. The interval between each image is 5 min. Scale bars, 50 μm.

Article Snippet: The function-blocking anti-β1-integrin rat monoclonal antibody (clone 9EG7) was obtained from PharMingen (San Diego, CA); its specificity has been reported previously ( Lenter et al., 1993 ; Lenter and Vestweber, 1994 ).

Techniques: Migration, Labeling, Cell Culture, Fluorescence, Blocking Assay, Sequencing

Journal: The Journal of Neuroscience

Article Title: Deleted in Colorectal Carcinoma and Differentially Expressed Integrins Mediate the Directional Migration of Neural Precursors in the Rostral Migratory Stream

doi: 10.1523/JNEUROSCI.22-09-03568.2002

Figure Lengend Snippet: Inhibited migration of RMS cells by function-blocking anti-integrin antibodies. Living brain slices were prepared from P3, P5, and P12 mice, and the cells were labeled with a small crystal of DiI placed on the center of the RMS. The slices were cultured in CCM1 medium supplemented with HEPES and 5% horse serum. The slices were preincubated with anti-integrin antibodies for 5 hr, and the migrating cells were traced by time-lapse recording for 3 hr. At P3, when α1- and β1-integrins are expressed, corresponding blocking antibodies reduced the migration speed. Anti-β3 antibody did not inhibit the migration significantly. At P5, when αv- and β1-integrins are expressed, antibodies against these integrins inhibited the migration speed. At P12, when αv-integrin is expressed, anti-αv-integrin antibody inhibited the speed as well; however, anti-α1 and -β3 antibodies did not inhibit the migration speed significantly, and they are not expressed at this stage. Each value represents the mean ± SD. Statistical analysis was performed by one-way ANOVA with Scheffé's multiple comparison procedure (significance of p < 0.01). The groups withasterisks do not differ from each other, nor do the nonmarked groups, but in all other comparisons, the differences are significant.

Article Snippet: The function-blocking anti-β1-integrin rat monoclonal antibody (clone 9EG7) was obtained from PharMingen (San Diego, CA); its specificity has been reported previously ( Lenter et al., 1993 ; Lenter and Vestweber, 1994 ).

Techniques: Migration, Blocking Assay, Labeling, Cell Culture

Journal: The Journal of Neuroscience

Article Title: Deleted in Colorectal Carcinoma and Differentially Expressed Integrins Mediate the Directional Migration of Neural Precursors in the Rostral Migratory Stream

doi: 10.1523/JNEUROSCI.22-09-03568.2002

Figure Lengend Snippet: Expression of DCC, neogenin, and netrin-1 and function of DCC in RMS migrations. A, Neogenin, a netrin-1 receptor, immunoreactivity coincides with the contour of RMS beginning at the anterior horn of the lateral ventricle (LV) and ending at the center of the olfactory bulb in a P0 mouse. B, The rostral part of the RMS strongly expresses the DCC protein, which is also present in the lateral olfactory tract (LOT) in P2 rats.AOB, Accessory olfactory bulb. C, Netrin-1 protein is expressed in the basal portion of olfactory mitral cells in the mitral cell layer (MCL) from an embryonic day 18 mouse. EPL, External plexiform layer;GCL, granule cell layer; GL, glomerular layer; ONL, olfactory nerve layer. (Figure legend continued.) D, Preabsorbed netrin-1 antibody (control) was prepared by coincubation of antibody and antigen peptide. The absorbed antibody did not show immunoreactivity. E, Sequence of 21 time-lapse images of a living slice from a P3 mouse treated with anti-DCC antibody as described in the legend to Figure ​Figure5.5. The olfactory bulb is located at the top. Three migrating cells (a–c) are indicated. Arrows indicate retracting leading process, and crossed arrows indicate processes pointing toward the anterior region of the subventricular zone (bottom). Note that the migration is no longer unidirectional, and that the processes form and retract frequently, which also distinguishes these migrations from those of normal cells shown in Figure ​Figure5.5. The interval between each image is 10 min. See Movie 3 (available atwww.jneurosci.org). F, Graphical representation of three migrating cells (a–c) shown in E. Cell bodies are tracked as described in Figure ​Figure5,5, and the time interval is 5 min. S, Start point; E, end point for cell b. See Movie 3 (available at www.jneurosci.org).G, Inhibited migration of cells by anti-DCC function-blocking antibodies. Living brain slices were prepared from P3 mice, and then the cells were labeled with DiI placed at the center or end of the RMS. The slices were cultured in CCM1 medium with HEPES and 5% horse serum with or without anti-DCC antibody for 5 hr, and the migration was observed by time-lapse recording. Each value represents the mean ± 1 SD. Statistical analysis was performed by one-way ANOVA with Scheffé's multiple comparison procedure (significance of p < 0.01). The control groups without anti-DCC antibody differ from every other group with anti-DCC. The groups withasterisks do not differ from each other, nor do the nonmarked groups, but in all other comparisons, the differences are significant. In contrast to cell treated with anti-integrin antibodies (Fig. ​(Fig.6),6), these cells show a larger net translocation. Scale bars:A, B, 1 mm; C, D, 100 μm;E, 50 μm.

Article Snippet: The function-blocking anti-β1-integrin rat monoclonal antibody (clone 9EG7) was obtained from PharMingen (San Diego, CA); its specificity has been reported previously ( Lenter et al., 1993 ; Lenter and Vestweber, 1994 ).

Techniques: Expressing, Sequencing, Migration, Blocking Assay, Labeling, Cell Culture, Translocation Assay

Journal: The Journal of Neuroscience

Article Title: Deleted in Colorectal Carcinoma and Differentially Expressed Integrins Mediate the Directional Migration of Neural Precursors in the Rostral Migratory Stream

doi: 10.1523/JNEUROSCI.22-09-03568.2002

Figure Lengend Snippet: Diagram depicting a working hypothesis for the roles of DCC and netrin and integrin in the migration of neural precursors from the SVZ to the center of the olfactory bulb. Netrin-1 secreted from mitral cells attracts DCC- or neogenin-expressing migrating cells, or both, to the olfactory bulb. Slit proteins from the septum inhibit migration out of the RMS into the septum or surrounding tissues by their repulsive activity. Integrins and laminins provide the traction for the motive force, and PSA-N-CAM provides the cellular milieu where the cells can move easily and maintenance of chains. Theshort arrow indicates the differentiation of neural precursors to granule cells; the long arrow marks the periglomerular cells. EPL, External plexiform layer;GCL, granule cell layer; GL, glomerular layer; MCL, mitral cell layer; ONL, olfactory nerve layer.

Article Snippet: The function-blocking anti-β1-integrin rat monoclonal antibody (clone 9EG7) was obtained from PharMingen (San Diego, CA); its specificity has been reported previously ( Lenter et al., 1993 ; Lenter and Vestweber, 1994 ).

Techniques: Migration, Expressing, Activity Assay

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 8. β1-integrin is an SNX17 cargo in neurons and plays a role in dendritic spine density. (A) DIV11 rat cortical neurons were infected with len- tiviruses carrying scrambled or SNX17 shRNAs, and the surface levels of β1-integrin were determined at DIV17 using a surface biotinylation assay. SNX17 knockdown was validated by Western blot of the lysate and GAPDH was used as a loading control. (B) The levels of surface β1-integrin protein were quantified and normalized to total β1-integrin levels (lysate). Data are expressed as a percentage of ctrl-shRNA (ctrl-shRNA: 100%, SNX17-shRNA: 57.630 ± 3.058%). N = 4 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. (C) Rep- resentative confocal images of surface β1-integrin levels of DIV17 hippocampal neurons that were infected at DIV11 with lentiviruses carrying either ctrl-shRNA or SNX17-shRNA. Neurons were treated in the presence or absence of cLTP and live labeled with an anti-surface ß1-integrin antibody for 15 min, followed by fixation and immunostaining for MAP2. Scale bar, 5 µm. (D) The intensity of ß1-integrin in the first 50 µm of secondary dendrites was quantified and values were normalized to crtl-shRNA. ctrl-shRNA: 1.000 ± 0.038, N = 32 neurons; ctrl-shRNA cLTP: 1.139 ± 0.039, N = 29 neurons; SNX17-shRNA: 0.839 ± 0.040, N = 28 neurons; SNX17-shRNA cLTP: 0.786 ± 0.035, N = 28 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, *P < 0.05. Error bars are SEM. (E) Validation of an shRNA clone (V2LMM_39157, Horizon Discovery) to knockdown rat ITGB1. pGIPZ scrambled non- target (RHS4346, Horizon Discovery) was used as a control. HEK293 cells stably expressing the tet repressor (TR-HEK293) were either transfected with control-shRNA or ITGB1-shRNA in the absence or presence of eGFP or ITGB1-GFP, as indicated. 5 d post-infection, cells were treated with 1 μg/ml of dox- ycycline to promote the expression of eGFP or ITGB1-GFP. 24 h later, extracts were generated and analyzed by Western blot. (F) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or ITGB1-shRNA. Scale bar, 5 µm. Treated with either β1-integrin blocking or isotype control antibodies 24 h before fixation. Scale bar, 5 µm. (G) The numbers of dendritic spines in the first 30 μm of secondary dendrites were quantified. ctrl-shRNA: 0.705 ± 0.044, N = 31 neurons; ITGB1-shRNA: 0.505 ± 0.043, N = 33 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, **P < 0.01. Error bars are SEM. (H) Hippocampal neurons were transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or SNX17-shRNA. Neurons were treated with either β1-integrin blocking or isotype control antibodies 24 h before fixation at DIV16. The number of dendritic spines in the first 30 μm of secondary dendrites was quantified. ctrl-shRNA + isotype ctrl: 0.689 ± 0.030, N = 26 neurons; ctrl-

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Infection, Surface Biotinylation Assay, Knockdown, Western Blot, Control, shRNA, Two Tailed Test, Labeling, Immunostaining, Biomarker Discovery, Stable Transfection, Expressing, Transfection, Generated, Blocking Assay

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 9. β1-integrin has roles in structural and functional plasticity during cLTP. (A) Diagram of experiment. DIV16-18 hippocampal neurons were treated with either β1-integrin blocking or isotype control antibodies for 30 min, followed by a 5 min cLTP stimulus. mEPSCs were recorded during the first 30 min after cLTP (cLTP < 30) or from 30 to 90 min after cLTP (cLTP > 30). (B) Examples of mEPSC recordings of neurons that were treated with isotype ctrl or β1-integrin blocking antibodies. Recordings were performed in the absence of cLTP (baseline), during the first 30 min after cLTP, or from 30 to 90 min after cLTP. (C) Quantification of mEPSC amplitude. Isotype ctrl baseline: 12.240 ± 0.570, N = 19 neurons; isotype ctrl cLTP < 30 min: 16.790 ± 1.106, N = 9 neurons; isotype ctrl cLTP > 30 min: 16.900 ± 0.954, N = 10 neurons; β1-integrin blocking baseline: 11.570 ± 0.373, N = 9 neurons; β1-integrin blocking cLTP < 30 min: 18.110 ± 1.247, N = 6 neurons; β1-integrin blocking neurons cLTP > 30 min: 13.170 ± 0.458, N = 13 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (D) Quantification of mEPSC frequency. Isotype ctrl baseline: 1.191 ± 0.227, N = 19 neurons; isotype ctrl cLTP < 30 min: 3.333 ± 0.800, N = 9 neurons; isotype ctrl cLTP > 30 min: 3.110 ± 0.740, N = 10 neurons; β1-integrin blocking baseline: 1.167 ± 0.296, N = 9 neurons; β1-integrin blocking cLTP < 30 min: 3.952 ± 1.214, N = 6 neurons; β1-integrin blocking cLTP > 30 min: 1.096 ± 0.302, N = 13 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, **P < 0.01. Error bars are SEM. (E) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected with eGFP (filler) at DIV12. Neurons were treated with β1- integrin blocking or isotype control antibodies for 30 min, followed by a 5-min cLTP stimulus in the presence of antibodies where indicated. Neurons were further incubated in the presence of antibodies for 50 min before fixation. Scale bar, 5 µm. (F) The maximum width for each spine was quantified, and the average size of the dendritic spines in the first 30 μm of secondary dendrites was calculated. Isotype ctrl: 0.626 ± 0.016, N = 28 neurons; isotype ctrl with cLTP: 0.717 ± 0.018, N = 25 neurons; β1-integrin blocking: 0.621 ± 0.015, N = 31 neurons; β1-integrin blocking with cLTP: 0.628 ± 0.010, N = 32 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ***P < 0.005. Error bars are SEM. (G) Quantification of dendritic spine density (spines/μm). Isotype ctrl: 1.044 ± 0.048, N = 28 neurons; isotype ctrl with cLTP: 0.977 ± 0.050, N = 25 neurons; β1-integrin blocking: 0.931 ± 0.054, N = 31 neurons; β1-integrin blocking with cLTP: 0.931 ± 0.039, N = 32 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Functional Assay, Blocking Assay, Control, Transfection, Incubation

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 10. Model of SNX17-mediated modulation of synaptic structure and function. The SNX17-Retriever pathway is required for dendritic spine maintenance and the cLTP-dependent increase in dendritic spine size. Glycine-mediated cLTP (1) stimulates calcium entry through the NMDA receptor, which activates the CaMKII pathway (2). CaMKII activation is necessary and sufficient to promote the recruitment of SNX17 and the Retriever complex to dendritic spines (3), and activates the recycling of β1-integrin from endosomes to the plasma membrane (4). The surface levels of β1-integrin increase during cLTP and promote dendritic spine growth (5). Endosomal PI(3)P increases upon cLTP and may help with the recruitment of SNX17 to synapses. Created with BioRender. com.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1,000, HPA043867; Atlas Antibodies), VPS35L rabbit pAb (1:1,000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1,000, 11938-1-AP; Proteintech), GFP rabbit mAb (1:1,000, Ab32146; Abcam), β1-integrin goat pAb (1: 1,000, AF2405; R&D Systems), and GAPDH rabbit mAb (1:2,000, 2118; Cell Signaling).

Techniques: Activation Assay, Clinical Proteomics, Membrane

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 8. β1-integrin is an SNX17 cargo in neurons and plays a role in dendritic spine density. (A) DIV11 rat cortical neurons were infected with len- tiviruses carrying scrambled or SNX17 shRNAs, and the surface levels of β1-integrin were determined at DIV17 using a surface biotinylation assay. SNX17 knockdown was validated by Western blot of the lysate and GAPDH was used as a loading control. (B) The levels of surface β1-integrin protein were quantified and normalized to total β1-integrin levels (lysate). Data are expressed as a percentage of ctrl-shRNA (ctrl-shRNA: 100%, SNX17-shRNA: 57.630 ± 3.058%). N = 4 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, ****P < 0.001. Error bars are SEM. (C) Rep- resentative confocal images of surface β1-integrin levels of DIV17 hippocampal neurons that were infected at DIV11 with lentiviruses carrying either ctrl-shRNA or SNX17-shRNA. Neurons were treated in the presence or absence of cLTP and live labeled with an anti-surface ß1-integrin antibody for 15 min, followed by fixation and immunostaining for MAP2. Scale bar, 5 µm. (D) The intensity of ß1-integrin in the first 50 µm of secondary dendrites was quantified and values were normalized to crtl-shRNA. ctrl-shRNA: 1.000 ± 0.038, N = 32 neurons; ctrl-shRNA cLTP: 1.139 ± 0.039, N = 29 neurons; SNX17-shRNA: 0.839 ± 0.040, N = 28 neurons; SNX17-shRNA cLTP: 0.786 ± 0.035, N = 28 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, *P < 0.05. Error bars are SEM. (E) Validation of an shRNA clone (V2LMM_39157, Horizon Discovery) to knockdown rat ITGB1. pGIPZ scrambled non- target (RHS4346, Horizon Discovery) was used as a control. HEK293 cells stably expressing the tet repressor (TR-HEK293) were either transfected with control-shRNA or ITGB1-shRNA in the absence or presence of eGFP or ITGB1-GFP, as indicated. 5 d post-infection, cells were treated with 1 μg/ml of dox- ycycline to promote the expression of eGFP or ITGB1-GFP. 24 h later, extracts were generated and analyzed by Western blot. (F) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or ITGB1-shRNA. Scale bar, 5 µm. Treated with either β1-integrin blocking or isotype control antibodies 24 h before fixation. Scale bar, 5 µm. (G) The numbers of dendritic spines in the first 30 μm of secondary dendrites were quantified. ctrl-shRNA: 0.705 ± 0.044, N = 31 neurons; ITGB1-shRNA: 0.505 ± 0.043, N = 33 neurons. Statistical significance was determined using unpaired two-tailed Student’s t test, **P < 0.01. Error bars are SEM. (H) Hippocampal neurons were transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or SNX17-shRNA. Neurons were treated with either β1-integrin blocking or isotype control antibodies 24 h before fixation at DIV16. The number of dendritic spines in the first 30 μm of secondary dendrites was quantified. ctrl-shRNA + isotype ctrl: 0.689 ± 0.030, N = 26 neurons; ctrl-

Article Snippet: Immunofluorescence and labeling of surface exposed β1-integrin Primary antibodies used were SNX17 rabbit polyclonal antibody (pAb; 1:200, HPA043867; Atlas Antibodies), SNX17 mouse monoclonal antibody (mAb; 1:50, sc-166957; Santa Cruz Biotechnology), VPS35L rabbit pAb (1:200; Daniel D. Billadeau, previously described in Phillips-Krawczak et al. (2015), COMMD1 rabbit pAb (1:200, 11938-1-AP; Proteintech), VPS35 goat pAb (1:200, ab10099; Abcam), PSD-95 mouse mAb (1:200, MAB1596; Millipore Sigma), EEA1 mouse mAb (1:200, 48453; Cell Signaling), vGLUT1 guinea pig pAb (1:2,000, AB5905;

Techniques: Infection, Surface Biotinylation Assay, Knockdown, Western Blot, Control, shRNA, Two Tailed Test, Labeling, Immunostaining, Biomarker Discovery, Stable Transfection, Expressing, Transfection, Generated, Blocking Assay

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 9. β1-integrin has roles in structural and functional plasticity during cLTP. (A) Diagram of experiment. DIV16-18 hippocampal neurons were treated with either β1-integrin blocking or isotype control antibodies for 30 min, followed by a 5 min cLTP stimulus. mEPSCs were recorded during the first 30 min after cLTP (cLTP < 30) or from 30 to 90 min after cLTP (cLTP > 30). (B) Examples of mEPSC recordings of neurons that were treated with isotype ctrl or β1-integrin blocking antibodies. Recordings were performed in the absence of cLTP (baseline), during the first 30 min after cLTP, or from 30 to 90 min after cLTP. (C) Quantification of mEPSC amplitude. Isotype ctrl baseline: 12.240 ± 0.570, N = 19 neurons; isotype ctrl cLTP < 30 min: 16.790 ± 1.106, N = 9 neurons; isotype ctrl cLTP > 30 min: 16.900 ± 0.954, N = 10 neurons; β1-integrin blocking baseline: 11.570 ± 0.373, N = 9 neurons; β1-integrin blocking cLTP < 30 min: 18.110 ± 1.247, N = 6 neurons; β1-integrin blocking neurons cLTP > 30 min: 13.170 ± 0.458, N = 13 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Error bars are SEM. (D) Quantification of mEPSC frequency. Isotype ctrl baseline: 1.191 ± 0.227, N = 19 neurons; isotype ctrl cLTP < 30 min: 3.333 ± 0.800, N = 9 neurons; isotype ctrl cLTP > 30 min: 3.110 ± 0.740, N = 10 neurons; β1-integrin blocking baseline: 1.167 ± 0.296, N = 9 neurons; β1-integrin blocking cLTP < 30 min: 3.952 ± 1.214, N = 6 neurons; β1-integrin blocking cLTP > 30 min: 1.096 ± 0.302, N = 13 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, **P < 0.01. Error bars are SEM. (E) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected with eGFP (filler) at DIV12. Neurons were treated with β1- integrin blocking or isotype control antibodies for 30 min, followed by a 5-min cLTP stimulus in the presence of antibodies where indicated. Neurons were further incubated in the presence of antibodies for 50 min before fixation. Scale bar, 5 µm. (F) The maximum width for each spine was quantified, and the average size of the dendritic spines in the first 30 μm of secondary dendrites was calculated. Isotype ctrl: 0.626 ± 0.016, N = 28 neurons; isotype ctrl with cLTP: 0.717 ± 0.018, N = 25 neurons; β1-integrin blocking: 0.621 ± 0.015, N = 31 neurons; β1-integrin blocking with cLTP: 0.628 ± 0.010, N = 32 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ***P < 0.005. Error bars are SEM. (G) Quantification of dendritic spine density (spines/μm). Isotype ctrl: 1.044 ± 0.048, N = 28 neurons; isotype ctrl with cLTP: 0.977 ± 0.050, N = 25 neurons; β1-integrin blocking: 0.931 ± 0.054, N = 31 neurons; β1-integrin blocking with cLTP: 0.931 ± 0.039, N = 32 neurons. Three independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM.

Article Snippet: Immunofluorescence and labeling of surface exposed β1-integrin Primary antibodies used were SNX17 rabbit polyclonal antibody (pAb; 1:200, HPA043867; Atlas Antibodies), SNX17 mouse monoclonal antibody (mAb; 1:50, sc-166957; Santa Cruz Biotechnology), VPS35L rabbit pAb (1:200; Daniel D. Billadeau, previously described in Phillips-Krawczak et al. (2015), COMMD1 rabbit pAb (1:200, 11938-1-AP; Proteintech), VPS35 goat pAb (1:200, ab10099; Abcam), PSD-95 mouse mAb (1:200, MAB1596; Millipore Sigma), EEA1 mouse mAb (1:200, 48453; Cell Signaling), vGLUT1 guinea pig pAb (1:2,000, AB5905;

Techniques: Functional Assay, Blocking Assay, Control, Transfection, Incubation

Journal: The Journal of cell biology

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

doi: 10.1083/jcb.202207025

Figure Lengend Snippet: Figure 10. Model of SNX17-mediated modulation of synaptic structure and function. The SNX17-Retriever pathway is required for dendritic spine maintenance and the cLTP-dependent increase in dendritic spine size. Glycine-mediated cLTP (1) stimulates calcium entry through the NMDA receptor, which activates the CaMKII pathway (2). CaMKII activation is necessary and sufficient to promote the recruitment of SNX17 and the Retriever complex to dendritic spines (3), and activates the recycling of β1-integrin from endosomes to the plasma membrane (4). The surface levels of β1-integrin increase during cLTP and promote dendritic spine growth (5). Endosomal PI(3)P increases upon cLTP and may help with the recruitment of SNX17 to synapses. Created with BioRender. com.

Article Snippet: Immunofluorescence and labeling of surface exposed β1-integrin Primary antibodies used were SNX17 rabbit polyclonal antibody (pAb; 1:200, HPA043867; Atlas Antibodies), SNX17 mouse monoclonal antibody (mAb; 1:50, sc-166957; Santa Cruz Biotechnology), VPS35L rabbit pAb (1:200; Daniel D. Billadeau, previously described in Phillips-Krawczak et al. (2015), COMMD1 rabbit pAb (1:200, 11938-1-AP; Proteintech), VPS35 goat pAb (1:200, ab10099; Abcam), PSD-95 mouse mAb (1:200, MAB1596; Millipore Sigma), EEA1 mouse mAb (1:200, 48453; Cell Signaling), vGLUT1 guinea pig pAb (1:2,000, AB5905;

Techniques: Activation Assay, Clinical Proteomics, Membrane