Journal: International Journal of Molecular Sciences

Article Title: Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells

doi: 10.3390/ijms231810300

Figure Lengend Snippet: Identification of HRG as a binding protein to S100A8/A9. ( A ) Recombinant proteins (GST, GST-S100A8, GST-S100A9) were prepared from the E. coli expression system. Ten micrograms of each individual single protein or a preincubated mixture of GST-S100A8 and GST-S100A9 (GST-S100A8/A9) was incubated with human plasma (1 mL) collected from a healthy volunteer donor. After incubation for 30 min at room temperature under gentle shaking, the GST proteins were all pulled down by the addition of glutathione-conjugated Sepharose beads. After the beads were washed extensively, the bound proteins were eluted with glutathione and subjected to SDS-PAGE for analysis. The Coomassie brilliant blue (CBB)-stained gel displayed three clear bands (BP1, 2, and 3) that co-shed with GST-S100A9 and GST-S100A8/A9. ( B ) Interaction of S100A8/A9 with HRG was confirmed by ELISA. A 96-well plate coated with the highly purified human recombinant HRG protein (100 µg/mL) was incubated with the indicated concentrations of biotinylated human recombinant S100A8/A9 protein after blocking with a chemical-based reagent, Blockmaster DB1130, that is very good for quenching the nonspecific binding of S100A8/A9. The binding of S100A8/A9 to HRG was detected by treatment with HRP-conjugated streptavidin and the chemical reaction between the HRP and the substrate used. The background values of S100A8/A9 binding from the wells without HRG coating were deducted. Data are expressed as optical density (O.D.) means ± SD. *** p < 0.001 by Student’s t -test. ( C ) Schematic drawing of the method used to detect the interaction between S100 family proteins and HRG on the HEKT293 cell surface (left). To effectively increase the probability of interaction between S100 proteins and HRG in the extracellular space, all proteins were modified to express in a membrane-anchored form, so that their density was much greater on the narrow cell surface than in the vast extracellular space. HEK293T cells were co-transfected with myc-tagged modified HRG and HA-tagged S100 family-expressing vectors. After the immunoprecipitation of the expressed cell-surface HRG with myc antibody-conjugated beads, the interacting cell surface S100 proteins were detected by the HA antibody (right).

Article Snippet: The antibodies used were as follows: rabbit anti-human S100A8 (Calgranulin A) polyclonal antibody (#sc-20174; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-human S100A9 (Calgranulin B) polyclonal antibody (#sc-20173; Santa Cruz Biotechnology), rabbit anti-human HRG antibody (#PA5-97051; Thermo Fisher Scientific) that cross-reacts with mouse HRG, and mouse anti-tubulin monoclonal antibody (#T5168, clone B-5-1-2; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Binding Assay, Recombinant, Expressing, Incubation, Clinical Proteomics, Gentle, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay, Modification, Membrane, Transfection, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells

doi: 10.3390/ijms231810300

Figure Lengend Snippet: Prevention of the S100A8/A9-mediated migration and invasion of melanoma cells by extracellular HRG. ( A , B ), B16-BL6 cells ( A ) and A375 cells ( B ) were treated with S100A8/A9 (0.1 µg/mL) in the presence or absence of HRG (1 µg/mL) for 1 h. The treated cells were washed with PBS and collected as cell pellets. The lysed cell pellets were then subjected to SDS-PAGE followed by Western blotting for the detection of cell-bound ectopic S100A8/A9 protein. The Western blotting was repeated three times for the distinct samples prepared from the independent experiments, and the results were shown as the representative images (( A , B ), left) and the quantified data (( A , B ), right). For the quantification, the band intensities were all measured using ImageJ software ( https://imagej.nih.gov/ij/ , accessed on 9 August 2022). The intensities of the individual target bands (S100A8 and S100A9) were then calibrated to those of the corresponding tubulin bands (internal control) and were presented as the fold change compared with those of the indicated setting at the single treatment with S100A8/A9, whose values were set as 1.0. Data are means ± SD, *** p < 0.001 by Student’s t -test ( n = 3), ND: not detected. ( C ) Experimental settings similar to those described in ( B ) were applied to evaluate HRG’s effects on S100A8/A9-mediated cancerous events in culture. Migration ( top ) and invasion ( bottom ) were evaluated using a Boyden chamber set with unburied and buried cell transmembrane with Matrigel, respectively. Cells were placed in the top chamber, and S100A8/A9 (1 µg/mL) with HRG (100 µg/mL) or BSA (a negative control, 100 µg/mL) was added to the bottom well. Eighteen hours later, migrating ( top ) and invaded ( bottom ) cells were detected with H&E staining (rightmost side, representative images) and then counted (left side, quantified data). Data are means ± SD. * p < 0.05 and ** p < 0.01 by Student’s t -test.

Article Snippet: The antibodies used were as follows: rabbit anti-human S100A8 (Calgranulin A) polyclonal antibody (#sc-20174; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-human S100A9 (Calgranulin B) polyclonal antibody (#sc-20173; Santa Cruz Biotechnology), rabbit anti-human HRG antibody (#PA5-97051; Thermo Fisher Scientific) that cross-reacts with mouse HRG, and mouse anti-tubulin monoclonal antibody (#T5168, clone B-5-1-2; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Migration, SDS Page, Western Blot, Software, Control, Negative Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells

doi: 10.3390/ijms231810300

Figure Lengend Snippet: RT-qPCR Primer Sequences.

Article Snippet: The antibodies used were as follows: rabbit anti-human S100A8 (Calgranulin A) polyclonal antibody (#sc-20174; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-human S100A9 (Calgranulin B) polyclonal antibody (#sc-20173; Santa Cruz Biotechnology), rabbit anti-human HRG antibody (#PA5-97051; Thermo Fisher Scientific) that cross-reacts with mouse HRG, and mouse anti-tubulin monoclonal antibody (#T5168, clone B-5-1-2; Sigma-Aldrich, St. Louis, MO, USA).

Techniques:

Journal: The European journal of neuroscience

Article Title: Microstimulation of primate neocortex targeting striosomes induces negative decision-making

doi: 10.1111/ejn.14555

Figure Lengend Snippet: pACC projections to striosomes. (a) Ipsilateral pACC projections in monkey S. pACC projection (hrGFP, green, left), KChIP1-positive striosomes (red, middle) and merged images (right), with enlarged views of the anterior striatum (framed by dashed line) at bottom. Asterisks indicate the same positions. AP positions in mm are based on the brain atlas for rhesus monkeys by Paxinos et al. (2000). Left is lateral, and right is medial. (b) Quantification of pACC projections to striatum calculated from the images of monkeys S, A, Y and N. Tracer densities (crosses) are represented by normalized gray value of each striatal region (Materials and Methods). The normalized gray values were calculated separately for striosomes and matrix, and for anterior (n = 7, 8, 10 and 8 for ipsilateral, respectively, in monkeys S, Y, A and N; n = 10, 9 and 11 for contralateral, respectively, in monkeys Y, A and N), central (n = 9, 9, 9 and 10 for ipsilateral, respectively, in monkeys S, Y, A and N; n = 9, 9 and 9 for contralateral, respectively, in monkeys Y, A and N) and posterior (n = 9, 9, 8 and 11 for ipsilateral, respectively, in monkeys S, Y, A and N; n = 8, 9 and 9 for contralateral, respectively, in monkeys Y, A and N) regions. Figure S7 shows the details for each monkey. Red bars, means. Pink bars, SEM. Tracer densities were significantly greater in striosomes than in matrix in both hemispheres (paired t-test, *P < 0.05). (c) Effective microstimulation and viral injection sites (stars) in monkeys S (red), P (purple) and Y (blue), and stimulation site (square) in monkey N during fMRI (orange), mapped onto a sagittal drawing of a monkey’s left side brain. Colored regions show the extent of viral injection sites (S, red; Y, blue; A, green; N, yellow).

Article Snippet: The striatal sections were incubated with primary antibody solutions containing rabbit anti-hrGFP (for hrGFP; 240141, Agilent Technologies) or rabbit anti-RFP (for mCherry; Rockland, 600-401-379) and mouse anti-KChIP1 (UC Davis/NIH NeuroMab Facility, #75-003) in TSA-block for 24 hrs at 4°C.

Techniques: Injection

Journal: The Journal of Cell Biology

Article Title: c-erbB-3

doi: 10.1083/jcb.200109033

Figure Lengend Snippet: HRGβ1 affects subcellular distribution of c-erbB-3. MTSV1-7 were examined by confocal immunofluorescent microscopy (A,B, and D) and immuno EM (C) for c-erbB-3 (A–C) or HRG (D). (A) Cells on filters pretreated with neutralizing anti-HRG (10 μg/ml, 48 h) were exposed for various times to 1 nM HRGβ1. Note mitotic cells were strongly positive. (B) Endogenous HRG was not blocked by anti-HRG (0 min) before exogenous HRGβ1 (1 nM, 48 h). (C) 0 min, after blocking of endogenous HRG, c-erbB-3 (arrows) was found in the granular component (gc) of the nucleolus (nc). np, nucleoplasm. At 24 h, added HRGβ1 (1 nM) caused relocation of c-erbB-3 into the cytoplasm (cp) and to the cell membrane. (D) Cells were treated as in B but immunostained with anti-pan HRG. Bars: (A, B, and D) 40 μm; (C) 0.1 μm.

Article Snippet: 10 μl Laemmli buffer were added to 10 μl of the concentrates and boiled (5 min), separated by 10% SDS-PAGE, blotted, and immunostained with 1 μg/ml rabbit anti-HRG (Ab-2; Neomarkers) and donkey anti–rabbit Ig F(ab') 2 -HRP conjugate (1:10,000; Amersham Pharmacia Biotech) as above.

Techniques: Microscopy, Blocking Assay