recombinant s100a8 a9 protein  (Thermo Fisher)

Journal: iScience

Article Title: Whitening of brown adipose tissue inhibits osteogenic differentiation via secretion of S100A8/A9

doi: 10.1016/j.isci.2024.108857

Figure Lengend Snippet: Loss of Rheb in BAT induces transcription and secretion of S100A8/A9 Interscapular BAT from either control or Rheb AD KO (A), or from control or Rheb BAD KO (B) mice, were used for transcriptome analysis by RNA-sequencing. The significantly upregulated genes were further subjected to gene ontology (GO) analysis. Detailed biological processes of the significantly upregulated genes in the Rheb AD KO mice are shown as a CNET plot (C). (D) Significantly up- or downregulated genes (at least two-fold, p < 0.01) in the BAT of control or Rheb BAD KO mice are shown as a volcano plot, with S100A8 and S100A9 labeled. S100A8 and S100A9 expression in the BAT of control or Rheb AD KO (or control or Rheb BAD KO) mice was analyzed by RT-qPCR (E) or western blotting (F). (G) Serum S100A8 and S100A9 levels of these mice were determined by ELISA or western blotting. Interscapular BAT (H) or serum (I) of the 3-month and 20-month C57BL/6J mice were analyzed for S100A8 expression by western blotting. (J) Conditional medium (CM) collected from interscapular BAT or bone marrow cells from these mice were analyzed for S100A8 by western blotting. Silver staining was used to assess equal loading. Data are shown as box-and-whisker plots (with median and interquartile ranges) from max to min, with all data points shown. Analyses were performed as two-tailed unpaired t-test.

Article Snippet: BMSC and C3H/10T1/2 were treated with 200, 500, and 1000 ng/mL of endotoxin-free S100A8/A9 RP (8916-S8-050, R&D, USA) for 7 days in osteogenic medium.

Techniques: RNA Sequencing Assay, Labeling, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Silver Staining, Whisker Assay, Two Tailed Test

Journal: iScience

Article Title: Whitening of brown adipose tissue inhibits osteogenic differentiation via secretion of S100A8/A9

doi: 10.1016/j.isci.2024.108857

Figure Lengend Snippet: Brown adipocytic S100A8/A9 mediates the effect of BAT Rheb loss on osteogenesis (A) Interscapular BAT from either control or Rheb AD KO, or from control or Rheb BAD KO mice, were analyzed for S100A8 or S100A9 expression by immunohistochemistry. The conditional medium (CM) was collected from both interscapular BADs and epididymal WADs of the control and Rheb AD KO mice (B), or from interscapular BADs of the control and Rheb BAD KO mice (C). The cells were then lysed, followed by both the whole-cell lysates and CM analyzed for S100A8 and S100A9 expression by WB. Primary brown adipocyte progenitors (BPADs) from wild-type C57BL/6J mice were transfected with lentivirus carrying the control or Rheb shRNA and induced into mature BADs, followed by analysis of whole-cell or secreted S100A8 level via RT-qPCR (D) or WB (E), respectively. BPADs were induced into BADs and treated with various concentrations of rapamycin for 12 h, then analyzed for S100A8 expression via RT-qPCR (F) and WB (G). The efficiency of rapamycin treatment was assessed by monitoring phosphorylated S6 (S235/236) (G). S100A8/A9 neutralizing antibody was injected into the tibial medullary cavity of Rheb BAD KO mice (10-month) every 3 days for 2 months, and the effects on bone formation were analyzed by 3D-microCT (H) or HE staining (I). Bone histomorphometric parameters of the tibiae were shown in the lower panel of (H). Data are shown as box-and-whisker plots (with median and interquartile ranges) from max to min, with all data points shown. Two-tailed unpaired t-test was used for two-group comparison; for comparison between multiple groups, one-way analysis of variance with multiple comparisons were used, followed by the Bonferroni post-hoc test for significance.

Article Snippet: BMSC and C3H/10T1/2 were treated with 200, 500, and 1000 ng/mL of endotoxin-free S100A8/A9 RP (8916-S8-050, R&D, USA) for 7 days in osteogenic medium.

Techniques: Expressing, Immunohistochemistry, Transfection, shRNA, Quantitative RT-PCR, Injection, Staining, Whisker Assay, Two Tailed Test, Comparison

Journal: iScience

Article Title: Whitening of brown adipose tissue inhibits osteogenic differentiation via secretion of S100A8/A9

doi: 10.1016/j.isci.2024.108857

Figure Lengend Snippet: S100A8/A9 inhibits OB differentiation of BMSCs through targeting toll-like receptor 4 (TLR4) (A) Expression of the S100A8/A9 receptor TLR4 and RAGE in primary BMSCs was analyzed by RT-qPCR. C2C12 stromal cells (B) and C3H10T1/2 MSCs (C) were treated with recombinant S100A8/A9 and analyzed for Myd88 expression and localization by confocal microscopy. Original magnification, ×200 or ×600; scale bar was shown as indicated. (D) C3H10T1/2 cells were treated with S100A8/A9 and TLR4 inhibitor TAK242 as indicated, followed by assay for OB differentiation by ALP staining (upper panel) and WB (lower panel). For comparisons between 2 groups, two-tailed unpaired t-tests were used. For comparisons between multiple groups, one-way analysis of variance with multiple comparisons were used, followed by the Bonferroni post-hoc test for significance. Data are shown as box-and-whisker plots (with median and interquartile ranges) from max to min, with all data points shown. Two-tailed unpaired t-test was used for two-group comparison; for comparison between multiple groups, one-way analysis of variance with multiple comparisons were used, followed by the Bonferroni post-hoc test for significance.

Article Snippet: BMSC and C3H/10T1/2 were treated with 200, 500, and 1000 ng/mL of endotoxin-free S100A8/A9 RP (8916-S8-050, R&D, USA) for 7 days in osteogenic medium.

Techniques: Expressing, Quantitative RT-PCR, Recombinant, Confocal Microscopy, Staining, Two Tailed Test, Whisker Assay, Comparison

Journal: iScience

Article Title: Whitening of brown adipose tissue inhibits osteogenic differentiation via secretion of S100A8/A9

doi: 10.1016/j.isci.2024.108857

Figure Lengend Snippet:

Article Snippet: BMSC and C3H/10T1/2 were treated with 200, 500, and 1000 ng/mL of endotoxin-free S100A8/A9 RP (8916-S8-050, R&D, USA) for 7 days in osteogenic medium.

Techniques: Virus, Negative Control, Recombinant, Staining, Silver Staining, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Real-time Polymerase Chain Reaction, Software

Journal: Oncology Research

Article Title: β-1,3-Galactosyl- O -Glycosyl-Glycoprotein β-1,6- N -Acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility

doi: 10.3727/096504017X15031557924123

Figure Lengend Snippet: Effects of glycosyltransferases on S100A8/A9-induced migration and invasion. Migration (left) and invasion (right) of WM-115 cells transfected with indicated genes [green fluorescence protein (GFP) as a control] were assessed by the Boyden chamber method. The transfected WM-115 cells were placed in the insert chamber, and purified recombinant S100A8/A9 (final concentration of 100 ng/ml) was added to the bottom well. The quantified results were viewed from the upper surface of the insert chamber membrane, and representative images of migrating or invading cells were obtained using crystal violet staining at the lower side. Data are shown as means ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: In brief, human S100A8/A9 recombinant protein was expressed using the FreeStyle 293 Expression System (Invitrogen), which enabled the production of a large amount of secreted S100A8/A9 in the culture medium.

Techniques: Migration, Transfection, Fluorescence, Purification, Recombinant, Concentration Assay, Staining

Journal: Oncology Research

Article Title: β-1,3-Galactosyl- O -Glycosyl-Glycoprotein β-1,6- N -Acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility

doi: 10.3727/096504017X15031557924123

Figure Lengend Snippet: Effects of GCNT3 (β-1,3-galactosyl- O -glycosyl-glycoprotein β-1,6- N -acetylglucosaminyltransferase 3) combined with each S100 soil sensor receptors (SSSR) on S100A8/A9-induced migration and invasion. Migration (A) and invasion (B) of WM-115 cells transfected with indicated combinations of the genes [empty vector (EV) and GFP used as controls] were assessed by the Boyden chamber method. The transfected WM-115 cells in the top chamber were stimulated or not stimulated with the purified recombinant S100A8/A9 (final concentration of 100 ng/ml). Data are shown as means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: In brief, human S100A8/A9 recombinant protein was expressed using the FreeStyle 293 Expression System (Invitrogen), which enabled the production of a large amount of secreted S100A8/A9 in the culture medium.

Techniques: Migration, Transfection, Plasmid Preparation, Purification, Recombinant, Concentration Assay

Journal: Oncology Research

Article Title: β-1,3-Galactosyl- O -Glycosyl-Glycoprotein β-1,6- N -Acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility

doi: 10.3727/096504017X15031557924123

Figure Lengend Snippet: Effect of inhibition of intrinsic GCNT3 on S100A8/A9-induced migration and invasion. (A) Protein levels of MCAM and GCNT3 were compared between WM-115 and WM-266-4 cells by Western blotting. Protein levels of MCAM were determine by Western blotting after treatment of WM-266-4 cells with the indicated siRNAs (final concentration of 20 nM) (B) and with talniflumate (Tal) (final concentration of 100 μM) (C). After treatment of WM-266-4 cells with the indicated siRNAs (final concentration of 20 nM) (D) or Tal (final concentration of 100 μM) (E), migration of the treated cells was assessed by the Boyden chamber method. The treated cells in the top chamber were stimulated or not stimulated with purified recombinant S100A8/A9 (final concentration of 100 ng/ml). The quantified results are displayed on the left, and representative images of migrating cells detected by crystal violet staining are shown on the right. Data are shown as means ± SD. * p < 0.05, *** p < 0.001.

Article Snippet: In brief, human S100A8/A9 recombinant protein was expressed using the FreeStyle 293 Expression System (Invitrogen), which enabled the production of a large amount of secreted S100A8/A9 in the culture medium.

Techniques: Inhibition, Migration, Western Blot, Concentration Assay, Purification, Recombinant, Staining

Journal: Oncology Research

Article Title: β-1,3-Galactosyl- O -Glycosyl-Glycoprotein β-1,6- N -Acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility

doi: 10.3727/096504017X15031557924123

Figure Lengend Snippet: Effect of aberrant overexpression of GCNT3 on S100A8/A9-induced migration and invasion. (A) Protein levels of MCAM expression were examined by Western blotting in GCNT3-stable clones established from WM-115 cells. (B) mRNA levels of MCAM expression were examined by Northern blotting. (−), nontransfection; GFP and GCNT3-Myc, transgene expression with the indicated genes; parental, equal to (−); GCNT3#1, 2, 4, stable clones for GCNT3 overexpression. All specimens were prepared from WM-115 cells. Migration (C) and invasion (D) of WM-115-derived GCNT3 stable clones were assessed by the Boyden chamber method. The cells in the top chamber were stimulated or not stimulated with the purified recombinant S100A8/A9 (final concentration of 100 ng/ml). Data are shown as means ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: In brief, human S100A8/A9 recombinant protein was expressed using the FreeStyle 293 Expression System (Invitrogen), which enabled the production of a large amount of secreted S100A8/A9 in the culture medium.

Techniques: Over Expression, Migration, Expressing, Western Blot, Clone Assay, Northern Blot, Derivative Assay, Purification, Recombinant, Concentration Assay

Journal: Oncology Research

Article Title: β-1,3-Galactosyl- O -Glycosyl-Glycoprotein β-1,6- N -Acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility

doi: 10.3727/096504017X15031557924123

Figure Lengend Snippet: Effect of catalytic dead GCNT3 (mutGCNT3) on MCAM stability and on S100A8/A9-induced migration. (A) Schematic of protein compositions of wtGCNT3 and mutGCNT3. The mutGCNT3 was designed to produce C-terminal deletion mutant of GCNT3, which lacks the catalytic domain in part but does not lose the endoplasmic reticulum (ER) localization. (B) WM-115 cells were transiently expressed with aberrant wtGCNT3, mutGCNT3, or GFP as a control for 48 h. The expressed foreign GCNT3 (wt and mut) was detected by myc antibody. After immunoprecipitation of the confirmed extracts, we used 400 mM NaCl to wash out the precipitate in the hope of removing proteins that interact with MCAM and also with the beads. The endogenous MCAM proteins and their GlcNAc levels from the prepared cell samples were determined by immunoprecipitation and following detection with either MCAM antibody or HRP-conjugated WGA. (C) The wtGCNT3-overexpressed WM-115 clone #4 with stable mode was transiently transfected with either GFP or mutGCNT3 for 48 h. The foreign expression of the GCNT3 with stable wild and temporal mutant was detected simultaneously by myc antibody, and the following immunoprecipitation and detection of the intrinsic MCAM and its GlcNAc level were carried out by a similar method as shown in (B). (D) Migration activity of WM-266-4 cells was assessed by the Boyden chamber method. The transfected cells (GFP, wtGCNT3, and mutGCNT3) in the top chamber were stimulated or not stimulated with purified recombinant S100A8/A9 (final concentration of 100 ng/ml). The quantified results are displayed. Data are shown as means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: In brief, human S100A8/A9 recombinant protein was expressed using the FreeStyle 293 Expression System (Invitrogen), which enabled the production of a large amount of secreted S100A8/A9 in the culture medium.

Techniques: Migration, Mutagenesis, Immunoprecipitation, Transfection, Expressing, Activity Assay, Purification, Recombinant, Concentration Assay