Journal: Journal of Cell Science

Article Title: Syntaxin-2 balances phagocytic uptake and phagolysosomal clearance in macrophages

doi: 10.1242/jcs.263855

Figure Lengend Snippet: Stx2 exhibits multiple subcellular distributions in macrophages, and inhibits engagement and uptake of IgG-opsonized particles. (A–F) DIC and confocal images of endogenous Stx2 with endogenous (A) β-actin (ACTB), (B) RAB5A, (C) Rab11, (D) VAMP4, (E) VAMP7 and (F) LAMP1 at steady-state. (G) DIC and confocal images of RAW 264.7 macrophages fed with OPZ (white asterisks) for 1h and probed for endogenous Stx2 and LAMP1. Boxed areas enlarged in the insets. ‘ r ’ values show degree of colocalization. Scale bars: 10 μm (main images) and 5 μm (insets). DAPI denotes the nucleus. All images representative of three independent repeats. (H) Western blot of Stx2 and β-actin (ACTB, loading control) from RAW 264.7 macrophages (WT) and macrophages transduced with scrambled (sc) shRNA (control) or shRNAs targeted to Stx2 (Stx2-KD). (I) Quantified Stx2 band density normalized to ACTB (loading control). N =3 independent experiments. Results are mean±s.e.m. ns, not significant; * P <0.05. (J) DIC and stacked (three consecutive optical sections) confocal images from 15, 30 and 60 min OPZR-incubated macrophages also probed for Stx2. DAPI marks the nucleus. Scale bars: 10 μm. (K) Quantification of engaged OPZR per macrophage (15 min: control, n =61; Stx2-KD, n =82; 30 min: control, n =56; Stx2-KD, n =109; 60 min: control, n =111; Stx2-KD, n =86). N =3 independent experiments. Results are mean±s.e.m. **** P <0.0001. (L) Maximum intensity projection confocal images of inside-outside stained macrophages incubated with OPZR for 15, 30 and 60 min. Scale bars: 10 µm. (M,N) Quantification of (M) phagocytic index and (N) phagocytic rate for OPZR analyzed at 15 min (control, n =75; Stx2-KD, n =68), 30 min (control, n =273; Stx2-KD, n =344) and 60 min (control, n =165; Stx2-KD, n =190). N =3 independent experiments. M shown as violin plots with median and quartiles marked. For N, results are mean±s.e.m. ** P <0.01, **** P <0.0001. All statistical tests were two-tailed unpaired Student's t -tests. See also Fig. S1 .

Article Snippet: Cells intended for surface staining were skipped for permeabilization and directly used for blocking after fixation and PBS wash. Primary antibodies diluted in blocking buffer were used for 1 h to immunostain cells for Stx2 (Synaptic Systems, catalog #110123, 1:200 dilution; R&D Systems, catalog #AF2568, 1:200 dilution), Rab5A (Cell Signaling Technology, catalog #46449, 1:100 dilution), Rab11 (Cell Signaling Technology, catalog #46449, 1:200 dilution), VAMP4 (Thermo Fisher Scientific, catalog #PA1-768, 1:100 dilution), VAMP7 (Thermo Fisher Scientific, catalog #PA5-116892, 1:100 dilution), LAMP1 (Thermo Fisher Scientific, catalog #14-1071-81, 1:500 dilution), Fcγ (Thermo Fisher Scientific, catalog #14-0161-82, 1: 100 dilution), TfR (Abcam, catalog #ab214039, 1:200 dilution), Cat L (R&D Systems, catalog #AF1515, 1:100 dilution), Cat B (Cell Signaling Technology, catalog #31718, 1:100 dilution), Cat D (Cloud-Clone Corporation, catalog #MAB280Hu22, 1:100 dilution) and Phalloidin647 (Thermo Fisher Scientific, catalog # A22287, 1:500 dilution).

Techniques: Western Blot, Control, Transduction, shRNA, Incubation, Staining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

doi: 10.1083/jcb.202401057

Figure Lengend Snippet: Distinct N-cad localization and dynamics in leader and follower cells. (A) Localization of N-cad in PBT-05 cells migrating on laminin. Maximum intensity projections. Insets show enlarged orthogonal views of follower (r1) and leader (r2) cells. Arrowheads indicate cell-cell junctions and arrows indicate vesicles. Scale bars, 10 or 7 μm (inset). (B) Intracellular N-cad or transferrin receptor-positive vesicles per cell were counted in leader and follower cells. N = 3 experiments. Unpaired t test. ***P < 0.0001. ns, not significant. (C) Representative images showing colocalization between N-cad and endosomal markers. Arrowheads indicate N-cad-positive endocytic vesicles colocalized with Rab5 or Rab11. Scale bars, 10 or 2 μm (inset). (D) Mander’s colocalization coefficients. N = 7–12 cells, 2–3 spheroids for each marker protein analyzed. (E and F) N-cad antibody uptake assay. Surface N-cad was labeled with N-cad ECD antibodies on ice before warming for various times, fixing, and staining for surface and intracellular antibody as described in . (E) Representative images. Scale bars, 10 μm. (F) Quantification. a.u., arbitrary units. N = 7–24 cells, 2–4 spheroids, 2 experiments. Two-way ANOVA Tukey’s multiple comparisons test between leader and follower cells at the same time point. ****P < 0.0001. (G) Recycling of intracellular N-cad Abs to the surface in leader and follower cells, measured as described in . N = 4 experiments each representing the average of 10 cells in each of 3–5 spheroids. Paired t test. *P < 0.05. (H) Quantification of total N-cad fluorescence intensity in leader and follower cells. Migrating cells were fixed, permeabilized, and stained with N-cad Ab. Fluorescence intensity was integrated for multiple leader and follower cells across the z stack. Paired t test. ***P < 0.001. N = 8 spheroids, five experiments. Error bars show mean ± SEM.

Article Snippet: Mouse anti-Rab11 , BD Biosciences , 610656 , IF (1:500).

Techniques: Marker, Labeling, Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

doi: 10.1083/jcb.202401057

Figure Lengend Snippet: N-cad endocytosis, recycling, and surface levels in leader and follower cells. (A) Schematic diagram of the N-cad antibody (Ab) internalization assay. Cells were labeled with N-cad ECD Ab in the cold, warmed for various times to allow internalization, fixed, and surface and internalized Ab detected with different fluorescent secondaries. (B) Immunofluorescence detection of internalized N-cad Ab with EEA1, Rab5, or Rab4 in leader cells after 40 min internalization. Arrowheads indicate colocalization. Scale bars, 10 or 2 μm (inset). (C) Object-based colocalization quantification of internalized N-cad Ab with EEA1, Rab5, Rab4, Rab11, LAMP1, or GM130. N = 5–10 spheroids, three experiments. (D) Surface N-cad Ab increases between 40 and 60 min; a.u., arbitrary units. Two-way ANOVA Šídák’s multiple comparisons test multiple comparisons test. *P < 0.05, ***P < 0.001. N = 3–4 spheroids, three experiments. (E and F) Schematic and representative images of N-cad recycling assay. Cells were incubated with N-cad ECD antibody in the cold and warmed for 40 min to allow internalization. N-cad Ab remaining on the surface was blocked in the cold with excess F(ab’) 2 . Cells were then warmed to allow recycling before fixation and detection of surface and internalized Ab with different fluorescent secondaries. Scale bars, 10 μm. (G and H) Schematic diagram and representative images showing photoconversion of histone H2B-Dendra2 in leader and follower cells. (I–K) Flow cytometry. (I) Definition of leader, follower and spheroid cells according to extent of photoconversion. (J) N-cad surface intensity histograms. (K) Normalized median N-cad fluorescent intensity. Paired t test. *P = 0.0374. N = 3 experiments. Error bars show mean ± SEM.

Article Snippet: Mouse anti-Rab11 , BD Biosciences , 610656 , IF (1:500).

Techniques: Labeling, Immunofluorescence, Incubation, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

doi: 10.1083/jcb.202401057

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Mouse anti-Rab11 , BD Biosciences , 610656 , IF (1:500).

Techniques:

Journal: eLife

Article Title: ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

doi: 10.7554/eLife.89261

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Rab11 (mouse monoclonal) , Thermo Fisher Scientific , 71-5300; RRID: AB_2533987 , WB 1/1000.

Techniques: Isolation, Transfection, Construct, Plasmid Preparation, CRISPR, Purification, Sequencing, Recombinant, Immunohistochemistry, Immunofluorescence, Cryo-EM Sample Prep, Tomography, Bioprocessing, Immunoprecipitation