Journal: Molecular cancer research : MCR

Article Title: Centriole Overduplication is the Predominant Mechanism Leading to Centrosome Amplification in Melanoma

doi: 10.1158/1541-7786.MCR-17-0197

Figure Lengend Snippet: (A) Western blot, and (B) qRT-PCR analysis for PLK4 expression in normal adult human epidermal melanocytes (HEMa) and seven human melanoma cell lines (A375, Hs294T, G361, WM35, WM115, 451Lu, and SK-MEL-28). (C) Centrioles were assessed before and after centrinone B treatment by immunofluorescence. Representative images of A375 and Hs294T melanoma cells with or without centrinone B treatment (100 nM for 48 hours). Centrin labels individual centrioles while pericentrin labels the entire centrosome or PCM. Scale = 5 μm. (D) Quantification of centrioles in each cell line before and after treatment with centrinone B. (E) Correlation of centrioles with PLK4 protein expression in the melanoma cell lines. PLK4 expression was normalized to actin. Pearson R = 0.47, P value = 0.24. (F) Correlation of centrioles with PLK4 mRNA expression in the melanoma cell lines. Pearson R = 0.05, P value = 0.90. (G) Quantification of the percent of centrioles with <4 centrioles before and after treatment with centrinone B. (H) The anti-proliferative potential of PLK4 inhibitor centrinone B (treated for 48 hours) was assessed using CytoTox-Glo assay in A375, Hs294T, G361, and SK-MEL-28 melanoma cell lines as well as in normal human melanocytes. All the data are representative of at least three independent experiments. Quantitative results are presented as means ± SEM. Statistical significance are indicated as *p<0.05, **p<0.1, ***p<0.001.

Article Snippet: The human melanoma cell lines A375, Hs294T, G361, WM35, WM115, 451Lu, and SK-MEL-28 and adult human epidermal melanocytes (HEMa) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Glo Assay

Journal: Molecular cancer research : MCR

Article Title: Centriole Overduplication is the Predominant Mechanism Leading to Centrosome Amplification in Melanoma

doi: 10.1158/1541-7786.MCR-17-0197

Figure Lengend Snippet: (A) Melanoma cells were grown to 70% confluency, then treated for 48 hours with 50, 100 nM of centrinone B. The number of apoptotic cells was assessed by flow cytometric analysis of annexinV/propidium iodide (PI) staining. Representative two-dimensional dot plots of annexinV-FITC and PI fluorescence are shown here. (B) Total Annexin V-positive and Annexin V-positive plus propidium iodide-positive (Q2+Q3 quadrants/apoptotic) cells are plotted. Data represent means ± SEM of three replicates. Statistical significance is indicated as *p<0.05, **p<0.1, ***p<0.001, ****p<0.0001. (C) Western blot analysis of PARP cleavage in 25, 50, 100 nM centrinone B-treated A375, Hs294T, and HEMa cells. β-actin was used as a loading control.

Article Snippet: The human melanoma cell lines A375, Hs294T, G361, WM35, WM115, 451Lu, and SK-MEL-28 and adult human epidermal melanocytes (HEMa) cells were obtained from American Type Culture Collection (ATCC).

Techniques: Staining, Fluorescence, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein

doi: 10.1172/JCI150101

Figure Lengend Snippet: ( A ) PKH26- or CFSE-labeled platelets from healthy donors ( n = 5) were mixed at 1:1 and incubated with increasing concentrations of spike protein or S-pseudovirus, and aggregates were detected by flow cytometry. Double-colored events indicated the platelet aggregation, and TRAP was used as the positive control. ( B ) Peripheral blood from healthy donors ( n = 5) was stimulated by spike protein or S-pseudovirus at indicated concentration and analyzed by flow cytometry. Platelet-monocyte aggregation was evaluated using the percentage of CD42b + /CD14 + cells. ( C ) Platelet-monocyte aggregation was visualized by fluorescence microscopy; scale bar: 20 μm. ( D ) Purified platelets from healthy donors ( n = 5) were incubated with spike protein or S-pseudovirus. P selectin, CD40L expression, and fibrinogen binding were shown by histogram and MFI. ( E ) Purified platelets ( n = 4) were pretreated with isotype antibody (indicated as control) or anti-spike RBD before incubation with S-pseudovirus; P selectin and CD40L expression are shown. Mean with SD and P value by paired Student’s t test are displayed. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: In blockade experiments, anti–P selectin (10 μg/mL, R&D Systems, BBA30) and anti-CD40L (10 μg/mL, R&D Systems, MAB3345) were added before spike protein and S-pseudovirus in indicated experiments.

Techniques: Labeling, Incubation, Flow Cytometry, Positive Control, Concentration Assay, Fluorescence, Microscopy, Purification, Expressing, Binding Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein

doi: 10.1172/JCI150101

Figure Lengend Snippet: ( A ) Purified platelets from healthy donors ( n = 3) were incubated with or without spike protein, and Western blot analysis was performed using indicated antibody after IP for spike protein (IP lane). Density of IP lane was analyzed. ( B ) Purified platelets ( n = 3) were pretreated with indicated antibody before incubation with spike protein; spike protein on platelets detected by Western blot. ( C and D ) Purified platelets were pretreated with anti-CD42b or isotype antibody before incubation by spike protein or S-pseudovirus; spike protein binding ( n = 5) and the expressions of P selectin and CD40L ( n = 4) on platelets were examined by flow cytometry. As anti-CD42b control, red controls were indicated as spike protein and isotype antibody; blue controls indicated as S-pseudovirus and isotype antibody. ( E ) Recombinant S-RBD was incubated with ACE-2 or CD42b, and their interaction was measured by co-IP. ( F ) Fixed platelets were incubated with S-RBD, and platelet-vWF complexes were detected by flow cytometry using recombinant vWF and ristocetin. Comparisons were made with paired Student’s t test, except in B , which was assessed by 1-way ANOVA with Dunnett’s multiple-comparison test. Mean with SD and P value are displayed. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: In blockade experiments, anti–P selectin (10 μg/mL, R&D Systems, BBA30) and anti-CD40L (10 μg/mL, R&D Systems, MAB3345) were added before spike protein and S-pseudovirus in indicated experiments.

Techniques: Purification, Incubation, Western Blot, Protein Binding, Flow Cytometry, Control, Recombinant, Co-Immunoprecipitation Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein

doi: 10.1172/JCI150101

Figure Lengend Snippet: ( A ) Purified platelets from healthy donors ( n = 5) were incubated with spike protein or S-pseudovirus. Phosphorylation of PKC-substrates (pPKC-Subs, assessed PKC activation through the resulting phosphorylation of PKC substrates on specific serine residues) and Akt (pAkt) were detected by Western blot. TRAP was used as the positive control. ( B ) Purified platelets ( n = 5) were pretreated with indicated inhibitors before incubation with spike protein. Phosphorylation of pPKC-Subs or Akt shown. ( C ) Purified platelets ( n = 3) were pretreated with isotype or CD42b antibodies before incubation by spike protein. Phosphorylation of pPKC-Subs or Akt shown. ( D ) Purified platelets ( n = 4) were pretreated with Ro31-8220 or LY294002 before incubation with spike protein or S-pseudovirus. P selectin and CD40L expression measured by flow cytometry. Comparisons between groups were measured by paired Student’s t test in A and C , or 1-way ANOVA with Dunnett’s multiple-comparison test in B and D . Mean with SD and P value are displayed. * P < 0.05; ** P < 0.01; *** P < 0.001. **** P < 0.0001.

Article Snippet: In blockade experiments, anti–P selectin (10 μg/mL, R&D Systems, BBA30) and anti-CD40L (10 μg/mL, R&D Systems, MAB3345) were added before spike protein and S-pseudovirus in indicated experiments.

Techniques: Purification, Incubation, Phospho-proteomics, Activation Assay, Western Blot, Positive Control, Expressing, Flow Cytometry, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein

doi: 10.1172/JCI150101

Figure Lengend Snippet: ( A ) Peripheral blood from healthy donors ( n = 3) was stimulated by spike protein or S-pseudovirus in the presence of anti–P selectin or anti–PSGL-1. Platelet-monocyte aggregation was evaluated using the percentage of CD42b + /CD14 + cells. ( B ) Anti–P selectin or anti-CD40L was added into monocyte-platelet cocultures with spike protein or S-pseudovirus. IL-1β expression in monocytes ( n = 4) was measured. ( C ) Purified monocytes ( n = 3) were cultured with/without recombinant P selectin and/or CD40L, and their expression of IL-1β and IL-10 is shown. Mean with SD and P value by paired Student’s t test are displayed. * P < 0.05.

Article Snippet: In blockade experiments, anti–P selectin (10 μg/mL, R&D Systems, BBA30) and anti-CD40L (10 μg/mL, R&D Systems, MAB3345) were added before spike protein and S-pseudovirus in indicated experiments.

Techniques: Expressing, Purification, Cell Culture, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein

doi: 10.1172/JCI150101

Figure Lengend Snippet: SARS-CoV-2 spike protein interacts with CD42b to activate platelets, which express CD40L via PI3K/PKC signaling and P selectin via an unknown signaling pathway. The activated platelets induce inflammatory monocyte differentiation through the engagement of P selectin/PGSL-1 and CD40L/CD40.

Article Snippet: In blockade experiments, anti–P selectin (10 μg/mL, R&D Systems, BBA30) and anti-CD40L (10 μg/mL, R&D Systems, MAB3345) were added before spike protein and S-pseudovirus in indicated experiments.

Techniques:

Journal: Science advances

Article Title: Two-in-one nanoparticle platform induces a strong therapeutic effect of targeted therapies in P-selectin-expressing cancers.

doi: 10.1126/sciadv.adr4762

Figure Lengend Snippet: Fig. 4. Talazoparib increased PD-L1 expression levels and evaluation of P-selectin expression in human and murine melanoma, as well as in BRCA-mutated murine BC. (A and B) dSTORM imaging and analysis showing PD-L1 localizations per square micrometer in EMT6 cells with higher concentrations of talazoparib (Tal) after 48 hours. Blue: TIRF microscopy cell membrane; red: TIRF microscopy PD-L1 and dSTORM microscopy PD-L1 representative images. Data represent mean ± SD (N = 3). Scale bars, 10 μm. (C) Representative images of EMT6 3D spheroids cocultured with activated splenocytes, at a 1:100 ratio, respectively, following treatments with Tal and PD-L1i, separately or combined. Scale bars, 300 μm. (D) Quantification of mCherry % area. Spheroids were either untreated (UT), supplemented with splenocytes (UT + spleen), or treated with 1 μM Tal and 10 μM PD-L1i, sepa- rately or combined. Data represent mean ± SD (N = 3, n = 10), and statistical significance was calculated using two-sided repeated-measures ANOVA (P < 0.05). (E and F) Representative images and quantification of immunofluorescence staining of melanoma (E) Murine D4M.3A. Scale bars, 100 μm. (F) Patient-derived specimens. Scale bars, 10 μm. The nucleus was stained with DAPI (blue), and P-selectin was stained with Cy5-labeled antibody (SELP, cyan). Data represent mean ± SD; at least three fields were imaged from each specimen, N = 3. (G) P-selectin expression in BRCA-mutated EMT6 primary (left) and brain metastases (right). Nuclei are stained with DAPI in blue and P-selectin (SELP) in red. Scale bars, 100 μm.

Article Snippet: Recombinant human P- selectin (rhSELP; Cat. ADP3) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Imaging, Microscopy, Membrane, Immunofluorescence, Staining, Derivative Assay, Labeling

Journal: Science advances

Article Title: Two-in-one nanoparticle platform induces a strong therapeutic effect of targeted therapies in P-selectin-expressing cancers.

doi: 10.1126/sciadv.adr4762

Figure Lengend Snippet: Fig. 5. Sulfate conjugation to PLGA-PEG increased the internalization of NPs into 3D spheroids. (A) Representative images of time course internalization of Cy5-labeled NPs into D4M.3A 3D spheroids. Scale bars, 400 μm. (B) Time course quantification of Cy5 intensity within D4M.3A 3D spheroids. (C) Representative images of Cy5-labeled NPs internalization into D4M.3A 3D spheroids after 22 hours. The spheroids were preincubated with SELPi for 1 hour before NPs addition. Scale bars, 400 μm. (D) Representative Z-stack images of Cy5-labeled PLGA-PEG-GLY-(OSO3Na)2 NPs internalization into the core of D4M.3A 3D spheroids after 24 hours, preincubated in the presence or absence of 10 μM SELPi. Scale bars, 200 μm. (E) Quantification of Cy5 intensity over time of nontargeted NPs and P-selectin–targeted NPs, after preincubation of the D4M.3A 3D spheroids with increasing SELPi concentrations. All quantifications are representative of three independent experiments. The data correspond to the mean ± SD of at least eight 3D spheroids per group, and statistical significance was determined using a two-way ANOVA test. (F and G) P-selectin binding assay of PLGA-PEG-GLY-(OSO3Na)2 NPs and PLGA-PEG NPs. Plates were coated with human recombinant P-selectin (rhSELP) or with skim milk (nonspecific binding control) and then incubated with PLGA-PEG-GLY- (OSO3Na)2 NPs or PLGA-PEG NPs for 15 min. (F) Fluorescence intensity of Cy5-labeled NPs after incubation with rhSELP or skim milk. (G) The fluorescence intensity ratio of rhSELP/skim milk. Bars represent mean ± SD, and a two-way ANOVA was used for statistical analysis.

Article Snippet: Recombinant human P- selectin (rhSELP; Cat. ADP3) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Conjugation Assay, Labeling, Binding Assay, Recombinant, Control, Incubation, Fluorescence

Journal: Nature

Article Title: GLP-1-directed NMDA receptor antagonism for obesity treatment

doi: 10.1038/s41586-024-07419-8

Figure Lengend Snippet: a , Chemical synthesis of GLP-1–MK-801 conjugates with different cysteine homologues. b , In vitro stability assay in human plasma. The assay was performed as biological replicates of GLP-1–MK-801 (Cys-linked) ( n = 3), GLP-1–MK-801 (hCys-linked) ( n = 2), GLP-1–MK-801 (Pen-linked) ( n = 3). c , Change in body weight of DIO mice treated with once-daily s.c. injections of 100 nmol kg −1 MK-801 ( n = 8 mice), 100 nmol kg −1 GLP-1 ( n = 8 mice), 100 nmol kg −1 GLP-1–MK-801 (Cys-linked) ( n = 8 mice) or vehicle (isotonic saline, n = 8 mice) for 5 days. d , Change in body weight of DIO mice treated with once-daily s.c. injections of 100 nmol kg −1 GLP-1 ( n = 6 mice), 100 nmol kg −1 GLP-1–MK-801 (Pen-linked) ( n = 6 mice) or vehicle (isotonic saline, n = 6 mice) for 7 days. e , Degradation assay. GLP-1–MK-801 was incubated in PBS with 200 mM glutathione at pH 7.0. The assay was performed as triplicates ( n = 3). f , Murine GLP-1 receptor activation of GLP-1, GLP-1–MK-801, liraglutide and semaglutide in transiently transfected HEK293 cells. The data represents dose-response at 2 min and is normalized to the maximal GLP-1 response (100%). The assay was performed as duplicates ( n = 2). g , h , Electrophysiological recordings of GLP-1 receptor-positive neurons, which were identified using a tdtomato reporter, being stimulated with NMDA after 30 min of bath application of GLP-1 or GLP-1–MK-801. g , Current responses at a holding potential of −70 mV elicited by NMDA bath application in GLP-1 receptor-positive neurons of the arcuate nucleus (ARC) with bath application of 50 µM GLP-1–MK-801, 50 µM GLP-1, 50 µM MK-801 or artificial cerebrospinal fluid (aCSF). h , Bar graph summarizing the effect of NMDA-induced inward current after control (aCSF, n = 5 neurons), GLP-1 (50 μM, n = 5 neurons), MK-801 (50 μM, n = 5 neurons) or GLP-1–MK-801 (50 μM, n = 5 neurons). Bath application normalized to the first bath application of NMDA (100 μM). i - m , Electrophysiological recordings of POMC neurons stimulated with GLP-1–MK-801. i , Representative trace showing that 50 μM GLP-1–MK-801 acute bath application induces a depolarization of POMC neurons in ARC ( n = 6 of 17 neurons). Bar graphs summarizing the acute effect of 50 μM GLP-1–MK-801 on POMC neurons. j , Resting membrane potential, k , Action potential frequency n . l , Excitatory postsynaptic current (EPSC) frequency. m , EPSC amplitude of POMC neurons that were depolarized in response to GLP-1–MK-801 bath application. n - r , Calcium imaging of ARC brain slices. n , Representative image of Fura-2AM loaded cells in an ARC brain slice. Scalebar is 20 µm. o , Application of GLP-1 and GLP-1–MK-801 induced changes in fluorescence indicative of rises in intracellular calcium (%ΔF/F 0 ) as shown by these representative fluorescent responses of two different Fura 2-AM loaded cells to application of GLP-1 (1 µM) or GLP-1–MK-801 (1 µM). p , The amplitude of the change in fluorescence (%ΔF/F 0 ) elicited by GLP-1 (1 µM, n = 20 neurons) or GLP-1–MK-801 (1 µM, n = 38 neurons). q , Representative traces of intracellular calcium levels in response to application of NMDA (50 µM) following bath application of GLP-1 (1 µM) or GLP-1–MK-801 (1 µM). r , Quantification of NMDA-induced (50 µM) intracellular calcium rises (%ΔF/F 0 ) following bath application of GLP-1 (1 µM, n = 20 neurons) or GLP-1–MK-801 (1 µM, n = 38 neurons). s , t , Treatment of DIO mice with once-daily s.c. injections of 50 nmol kg −1 GLP-1–MK-801 ( n = 6 mice and n = 3 cages), 100 nmol kg −1 GLP-1–MK-801 ( n = 6 mice and n = 3 cages) or vehicle (isotonic saline, n = 6 mice and n = 3 cages) for 6 days. s , Change in body weight. t , Cumulative food intake. u , v , Treatment of C57BL/6 J DIO mice with once-daily s.c. injections of 100 nmol kg −1 GLP-1–MK-801 ( n = 8 mice and n = 4 cages), 300 nmol kg −1 GLP-1–MK-801 ( n = 8 mice and n = 4 cages) or vehicle (isotonic saline, n = 8 mice and n = 4 cages) for 14 days. u , Change in body weight. v , Cumulative food intake. Data analysed by paired two-tailed Student’s t-test ( j - m ), unpaired two-tailed Student’s t-test ( p and r ), one-way ANOVA, multiple comparison, Bonferroni post hoc test ( h ) and two-way repeated measures ANOVA to assess main effects of treatment ( c , d and s - v ). Data represents mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Detailed statistics are in Supplementary Table .

Article Snippet: Drug working concentrations and stock preparation were as follows: GLP-1–MK-801 and MK-801 (both 50 μM, dissolved in aCSF or magnesium-free aCSF), NMDA (100 μM, dissolved in magnesium free aCSF), CNQX (10 μM, dissolved in DMSO, Alomone Labs), picrotoxin (100 μM, dissolved in DMSO).

Techniques: In Vitro, Stability Assay, Clinical Proteomics, Saline, Degradation Assay, Incubation, Activation Assay, Transfection, Control, Membrane, Imaging, Slice Preparation, Fluorescence, Two Tailed Test, Comparison