Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Induction of apoptosis in 70Z/3 cells treated with 3CPA at 250 μ M and 500 μ M . ( A ) Cytochrome c release into the cytosol after exposure to 3CPA for indicated time periods measured by Western blot analysis. ( B ) Pro-caspase-9 processing determined in cytosolic fractions after exposure to 3CPA. Actin was used as an internal standard of the protein load and the results shown are representatives of two separate experiments. ( C ) Caspase inhibitors reduce 3CPA-induced apoptosis in 70Z/3 cells. Apoptosis, measured as AnnexinV + 7AAD − cells, was determined after exposure to 3CPA at 250 μ M (left) and 500 μ M (right) for 24 h. The pan-caspase inhibitor ZVAD-fmk (50 μ M , n =4), the caspase-9 inhibitor LEHD-fmk (50 μ M , n =3), or the caspase-8 inhibitor IETD-fmk (50 μ M , n =3) were added 1 h before treatment with 3CPA. The level of apoptosis is expressed relative to the level in cell cultures treated with the inhibitor only. Results show mean±s.d., and (*) indicates a statistical difference compared to 3CPA only (paired t -test, P <0.05).

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Over-expression of Bcl-2 in 70Z/3 cells inhibits 3CPA-induced apoptosis. ( A ) FACS-analysis of 70Z/3 (upper panel) and 70Z/3 Bcl-2+ (lower panel) cells after exposure to 3CPA (500 μ M ) for 18 h. 7AAD negative (viable) cells were gated (gate A) and analysed for Annexin V + expression (% of gated cells). ( B ) Levels of Bcl-2 and cytochrome c expression were determined by Western blot analysis in mitochondrial enriched fractions after 18 h incubation with 3CPA at the indicated concentrations (30 μg protein per lane). ( C ) Pro-caspase-9 processing into active caspase-9 was determined in the cytosolic fraction after 18 h incubation with 3CPA at the indicated concentrations. Actin was used as an internal standard to control the amount of cytosolic protein loaded in each lane. Results shown are representatives of two independent experiments.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Over Expression, Expressing, Western Blot, Incubation

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: N-substituted benzamides induce cytochrome c release and activation of caspase-9 in HL60 cells. ( A ) Cytochrome c release induced by MCA (500 μ M ) and 3CPA (250 μ M ) in HL60 cells after 24 and 48 h, measured by Western blot analysis of mitochondria or cytosol fractions (30 μg protein/lane). Cells were incubated with etoposide for 6 and 18 h at 100 μ M as a positive control. ( B ) Activation of caspase-9 after treatment of HL60 cells with 3CPA at 250 and 500 μ M for 12 and 18 h (48 μg protein per lane). One representative experiment out of two is shown.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Activation Assay, Western Blot, Incubation, Positive Control

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Involvement of HMGB1-targeted receptors in the CIPN caused by bortezomib. Bortezomib at 0.4 mg/kg or vehicle was administered i.p. on days 0, 2, 5, 7, 9, and 12. ( A , B ) Preventive ( A ) or therapeutic ( B ) effect of FPS-ZM1, TAK-242, and AMD3100, antagonists of RAGE, TLR4, and CXCR4, respectively. The mice received repeated i.p. administration of FPS-ZM1 at 1 mg/kg, TAK-242 at 3 mg/kg, AMD3100 at 8 mg/kg, or vehicle, 30 min before each dose of bortezomib ( A ), or single i.p. administration of each of them on day 14 ( B ). ( C , D ) Protein levels of RAGE, TLR4, and CXCR4 in the dorsal root ganglion ( C ) and sciatic nerves ( D ) on day 14 after the onset of bortezomib treatment. ( E , F ) Lack of preventive ( E ) and therapeutic ( F ) effects of TH1020, a TLR5 antagonist. The mice received repeated ( E ) or single ( F ) i.p. administration of TH1020 at 1 mg/kg or vehicle, in mice treated with bortezomib according to the above-mentioned schedules. Data show the mean with S.E.M for 5–6 ( A , B , F ), 5–7 ( C ), 6–7 ( D ), or 6 ( E ) mice. V, vehicle; BTZ, bortezomib; DRG, dorsal root ganglion; * p < 0.05, ** p < 0.01 vs. V ( C ) or V in V-treated mice ( A , B , E , F ). † p < 0.05, †† p < 0.01 vs. V in BTZ-treated mice.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques:

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Scheme for bortezomib-induced caspase-dependent HMGB1 release from macrophages and CIPN development, in contrast to caspase-independent mechanisms for paclitaxel. ( A ) Inhibition of proteasome by bortezomib causes caspase-dependent apoptosis of macrophages followed by the release of HMGB1, which in turn causes neuronal sensitization via activation of RAGE and acceleration of CXCL12/CXCR4 signals, leading to CIPN. ( B ) Paclitaxel causes HMGB1 release from macrophages through activation of the ROS/p38MAPK/NF-κB pathway , independently of caspase (see D), and the extracellular HMGB1 develops CIPN in a manner dependent on RAGE and CXCR4 , as shown in the CIPN caused by bortezomib.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques: Inhibition, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and FcγRI are arranged in discrete nanoclusters at macrophage surfaces. (A and B) TIRF and dSTORM images of SIRPα (A) and FcγRI (B) at the surface of human macrophages seeded onto PLL- (nonactivated, top) or hIgG-coated slides (bottom) for 10 min and stained with fluorescently labeled specific antibodies. Bars, 5 µm. Regions delineated by white squares are zoomed-in and shown with corresponding density maps (pseudocolor scale), thresholded binary maps and Ripley’s K analysis of the molecules in the selected regions. Bars, 1 µm. L(r)-r represents the degree of clustering relative to simulated random distributions, indicated by the 99% confidence intervals (CIs); r is the radial scale. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions were calculated by subjecting dSTORM data to spatial point-pattern analysis and thresholding. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F and G) Label-density variation analysis for SIRPα (F) and FcγRI (G) yields characteristic normalized ρ/η curves for clustered proteins. Cells were stained with anti–SIRPα-AF647 (F) or anti–FcγRI-AF488 (G) at different labeling concentrations and imaged by dSTORM. Each data point represents a single cell, color-coded by antibody concentration used for labeling. Red lines indicate reference curves for a random distribution of molecules.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Staining, Labeling, Two Tailed Test, Concentration Assay

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and FcγRI nanoclusters are constitutively associated in nonactivated human macrophages but segregate upon activation with hIgG. (A) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with PLL (nonactivated, top) or hIgG (middle) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right columns) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. As a positive control, macrophages seeded onto PLL-coated slides were stained with anti–FcγRI-AF488 mAb followed by anti–mouse IgG1-AF647 secondary antibody (bottom). (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded onto PLL- (gray) or hIgG-coated (red) slides for 10 min or for positive control data (green). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) Nearest-neighbor (NN) analysis from data shown in (B). Each symbol represents the median NN of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P < 0.0001; two-tailed t test assuming unequal variance. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥ 20,000 clusters from a minimum of 10 cells per condition) from cells seeded onto PLL- (light gray), hIgG-coated (light red) slides, or positive control data (green). Corresponding simulated data are also shown, in which the centroid positions of SIRPα nanoclusters in both nonactivating (dark gray) and hIgG-activating conditions (dark red) were randomized within the cell area.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: SIRPα and the low-affinity Fc receptor, FcγRII, are segregated on a nanometer scale. (A) TIRF and dSTORM images showing FcγRII (green) and SIRPα (red) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with anti–FcγRII-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure is the same as in . Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ***, P < 0.001; one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u. arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Incubation, Staining, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: FcγRs reorganize into concentric rings upon activation. (A) TIRF images of FcγRI (top) and FcγRII (bottom) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with fluorescently labeled specific antibodies. Bars, 10 µm. (B) TIRF and dSTORM images of FcγRI (green) and FcγRII (red) at the surface of macrophages incubated for 10 or 30 min on slides coated with PLL or hIgG and stained with anti–FcγRI-AF488 and anti–FcγRII-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure are the same as in . Data are from a minimum of 10 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Labeling, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Specific activation of FcγRI is required for its reorganization into concentric rings and segregation from SIRPα nanoclusters. (A and B) TIRF (bars, 10 µm) and dSTORM (bars, 5 µm) images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 (A) or 30 min (B) on slides coated with hIgG1 or hIgG2 and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells seeded onto hIgG1- or hIgG2-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Rearrangement of macrophage surface receptors triggered by mobile hIgG. (A) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on SLBs loaded with streptavidin (nonactivating) or with streptavidin-hIgG (activating) and stained with a fluorescently labeled specific antibody. Two example images are shown for each condition. Bars, 10 µm. (B) dSTORM images of FcγRI (green) and SIRPα (red) at the surface of macrophages seeded as in A and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares are shown enlarged with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from two independent experiments. ns, not significant; *, P < 0.05; ****, P < 0.0001; two-tailed t test assuming unequal variance. (F) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded as in A. Data are from a minimum of 30 cells from two independent experiments. Bars represent mean ± SD. (G) NND analysis from data shown in F. Each symbol represents median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P < 0.0001; two-tailed t test assuming unequal variance. (H) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥10,000 clusters from a minimum of 10 cells per condition) from cells seeded onto control nonactivating (light gray) or hIgG-loaded activating (light red) SLBs.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Incubation, Staining, Labeling, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Ligation of SIRPα impairs the reorganization of surface FcγRI. (A) Human macrophages were incubated for 24 h in wells coated with PLL, 20 µg/ml of hCD47, or with increasing concentrations of hCD47 in the presence of 10 µg/ml of hIgG, as indicated. M-CSF release was assessed by ELISA. Bars represent mean ± SD from three donors. Each color represents one individual donor. (B) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on slides coated with hCD47 or hCD47 plus hIgG and stained with fluorescently labeled specific antibody. Bars, 10 µm. (C) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with hCD47 (top) or hCD47 plus hIgG (bottom) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (D and G) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα (D) and for FcγRI and pSHP-1 Y536 (G) in cells seeded onto slides coated with PLL (light gray), hCD47 (light red), hCD47 plus hIgG (dark red), or hIgG (dark gray) for 10 (D) or 5 min (G). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (E and H) NND analysis from data shown in D and G, respectively. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (F and I) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). Graphs compare colocalization between FcγRI and SIRPα (F) and FCγRI and pSHP-1 Y536 (I). a.u., arbitrary units; NN, nearest neighbor; PC, positive control.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Ligation, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Fluorescence, Positive Control

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Segregation and reorganization of FcγRI is dependent on the actin cytoskeleton and formins, but not myosin II. (A) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages pretreated with 1 µM latrunculin A, 0.5 µM jasplakinolide, 10 µM blebbistatin or 10 µM SMIFH2. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min, and stained with anti-FcγRI-AF488 and anti-SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells pretreated with drugs as indicated and seeded onto slides coated with PLL (gray) or hIgG (latrunculin A [Lat A], dark gray; jasplakinolide [Jasp], red; SMIFH2, green; or blebbistatin [Bleb], blue) for 10 min. Data are from a minimum of 30 cells per condition from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P < 0.01; ****, P < 0.0001; two-tailed t test assuming unequal variance. (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition). (E–G) Nanocluster areas (E), density (F), and percentage of localizations in nanoclusters (G) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions after pretreatment of cells with blebbistatin or DMSO control. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from three independent donors. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-tailed t test assuming unequal variance. NN, nearest neighbor.

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Staining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

doi: 10.1083/jcb.201608094

Figure Lengend Snippet: Src-family kinase signaling, but not Syk or PI3K signaling, is indispensable for reorganization of macrophage surfaces. (A) Immunoblots of phosphorylated AKT in nonactivated (PLL) or hIgG-activated human macrophages pretreated with vehicle (DMSO), as a control, 10 µM PP2 (left), 100 µM piceatannol (PCT; middle), or 1 µM wortmannin (Wort; right). Blots represent two independent experiments. (B) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated with vehicle (DMSO), PP2, PCT, or Wort, pretreated as in A. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (C) CBC histograms for FcγRI and SIRPα in cells pretreated as in A and seeded onto slides coated with PLL (gray) or hIgG (DMSO, dark gray; PP2, red; PCT, green; and Wort, blue) for 10 min, as indicated. Data are from a minimum of 30 cells from three independent donors. Bars show mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; ****, P < 0.0001; one-way ANOVA with Tukey’s post-hoc test. (E) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor from the second channel (≥20,000 clusters from a minimum of 10 cells per condition).

Article Snippet: Primary monoclonal antibodies used for microscopy were anti-SIRPα (clone 4C7; AbD Serotec) conjugated in-house with AF647 (Invitrogen), anti–FcγRI-AF488 (clone 10.1; BioLegend), anti-FcγRII (clone FLI8.26; BD) conjugated in-house with Atto488 (Invitrogen) or AF647, and anti–PTPN6(Tyr536)-AF647 (Bioss).

Techniques: Western Blot, Incubation, Staining

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: (A) Cathepsin B and (B) cathepsin L activity in J774A.1 cells (1 × 10 6 cells mL -1 ) was determined after incubation with HOSCN (80–160 μM, white bars) or HOCl (80–160 μM, black bars) for 15 min at 22°C. (C) Cathepsin B and (D) cathepsin L activity in J774A.1 cell lysates (1 × 10 6 cells mL -1 ) after incubation with HOSCN (5–20 μM) for 15 min, followed by further incubation in the absence (white bars) or presence (black bars) of DTT (100 μM) for 15 min. Results are expressed as a percentage of the PBS-treated control cells. * and # represent a significant (p < 0.05) change in cathepsin B/L activity compared with control lysates or the presence / absence of DTT, respectively.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: LDL (1 mg protein mL -1 ) was exposed to 0–500 μM HOSCN (A and B) or HOCl (C and D) for 30 min (white) and 24 h (black) at 22°C and 37°C respectively, prior to addition of each modified LDL (0.1 mg protein mL -1 ) to J774A.1 lysates for 15 min at 22°C, followed by determination of cathepsin B (A, C) or cathepsin L (B, D) activity, which is expressed relative to the no LDL control. Data are means ± SEM for at least 3 independent experiments, with multiple LDL donors. * and # represent a significant (p < 0.05) decrease in cathepsin B or L activity compared to cells exposed to the incubation control LDL or no LDL. “a” represents a significant (p < 0.05) difference between 30 min and 24 h modified LDL.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Modification, Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: LDL (1 mg protein mL -1 ) was exposed to 0–5000 μM KOCN for 24 h at 37°C, prior to addition of each modified LDL (0.1 mg protein mL -1 ) to J774A.1 lysates for 15 min at 22°C, followed by determination of cathepsin B (white bars) or cathepsin L (black bars) activity, which is expressed relative to the no LDL control. * and # represent a significant decrease (p < 0.05) in cathepsin B or L activity compared with cells exposed to the incubation control LDL or no LDL respectively by 1-way ANOVA with Tukey’s post-hoc testing.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Modification, Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: LDL (1 mg protein mL -1 ) was exposed to 0–500 μM HOSCN (A and B) or HOCl (C and D) for 24 h at 37°C respectively, prior to addition of each modified LDL (0.1 mg protein mL -1 ) to J774A.1 cells for 4 (white bars) or 24 h (black bars), and determination of cathepsin B (A, C) or cathepsin L (B, D) activity, which is expressed relative to the no LDL control. * and # represent a significant decrease (p < 0.05) in cathepsin B or L activity compared with cells exposed to the incubation control LDL or no LDL. “a” represents a significant (p < 0.05) difference between cathepsin activity between cells incubated with LDL for 4 or 24 h.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Modification, Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: LDL (1 mg protein mL -1 ) was exposed to 0–500 μM HOSCN (A) or HOCl (B) for 24 h at 37°C, respectively, prior to addition of each modified LDL (0.1 mg protein mL -1 ) to J774A.1 cells, followed by determination of cathepsin B (white) and L (black) protein expression. Cathepsin levels were normalised to β-actin levels, and then calculated as the fold change from the no LDL condition. Representative blots of the cathepsin B band at 25 kDa, the cathepsin L band at 25 kDa, or the β-actin band at 43 kDa, are displayed, of n = 3–4 separate experiments. There was no significant effect of oxidant treatment as determined by 1-way ANOVA on either cathepsin protein levels.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Modification, Expressing