polyclonal rabbit anti-human cd14  (ABclonal Biotechnology)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: S100A9 + CD14 + monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function

doi: 10.1186/s13046-024-02985-1

Figure Lengend Snippet: Monocyte-related gene signature enriched in the PBMC of non-responders. a Schematic diagram of the study design and workflow. b UMAP visualization of 30,667 single-cell transcriptomes of immune cells identifying 23 populations and colored by cell type assignment. c Stacked histograms indicating the proportion of immune subsets in each patient. d Volcano plot depicting DEGs between NR and R. Four top genes enriched in the NR group are boxed with red dashed lines, and their expression is shown as UMAP feature plots ( f ). e GO pathway enrichment analysis of DEGs from PBMCs between NR and R. g UMAP visualization of monocytes. Left: Monocytes stratified by NR group (blue) and R group (yellow). Right: Monocytes stratified into four populations. h Proportions of monocyte subsets in each group. The numbers indicate the percentage of S100A9 + CD14 + monocytes

Article Snippet: Staining was carried out using a 1:200 dilution of rabbit polyclonal antibody for human S100A9 (Proteintech Cat# 26,992–1-AP; RRID: AB_2880716) and a 1:2000 dilution of rabbit polyclonal antibody against human CD14 (Proteintech Cat# 17,000–1-AP; RRID: AB_2074048), followed by incubation with secondary antibodies (Biolynx Cat# I20012C).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: S100A9 + CD14 + monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function

doi: 10.1186/s13046-024-02985-1

Figure Lengend Snippet: Circulating S100A9 + CD14 + monocyte predicts unfavorable responses to anti-PD-1 immunotherapy. a UMAP visualization of immune cells between NR and R. S100A9 + CD14 + monocytes are boxed with red dashed lines. b Percentage of each immune subset between the NR group (blue) and R group (yellow), represented as box-whisker plots. Significance was evaluated using 2-way ANOVA. c Violin plots representing the percentage of S100A9 + CD14 + monocytes in each patient. d Survival curves showing overall survival stratified by the percentage of S100A9 + CD14 + monocyte. e Left: Representative image of dual immunofluorescence demonstrating S100A9 (red) and CD14 (green) positive cells and S100A9 + CD14 + monocytes (white triangles) in HCC tumors of a responder and a non-responder. Right: Number of S100A9 + CD14 + monocytes quantified in five randomly selected fields per patient ( n = 4 in NR; n = 4 in R). Scale bar, 50 μm. f Levels of plasma S100A9 in patients among different ICB best efficacy. g ROC curves and the area under the curve (AUC) of the plasma S100A9 level (red) or NLR (dashed blue) for discrimination between responders and non-responders. The cut-off value for S100A9 level was 308.110 ng/ml. h Best efficacy of patients stratified by plasma S100A9 levels. i Survival curves showing progress-free survival stratified by the plasma S100A9 level. P value in ( c and e ) were calculated using unpaired Student’s t-test. P values in ( d and i ) were calculated using the log-rank test

Article Snippet: Staining was carried out using a 1:200 dilution of rabbit polyclonal antibody for human S100A9 (Proteintech Cat# 26,992–1-AP; RRID: AB_2880716) and a 1:2000 dilution of rabbit polyclonal antibody against human CD14 (Proteintech Cat# 17,000–1-AP; RRID: AB_2074048), followed by incubation with secondary antibodies (Biolynx Cat# I20012C).

Techniques: Whisker Assay, Immunofluorescence, Clinical Proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: S100A9 + CD14 + monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function

doi: 10.1186/s13046-024-02985-1

Figure Lengend Snippet: Higher Mono_S100A9 signature score predicts worse ICB response and T cell dysfunction in cancer patients. a Genes in Mono_S100A9 signature. b Mono_S100A9 signature scores in patients with melanoma between NR and R. c Association of Mono_S100A9-signature score with overall survival within LIHC dataset obtained from TCGA. LIHC: Liver hepatocellular carcinoma. d Volcano plot showing DEGs of T cells between NR and R. e-g GSEA plots of the indicated signature genes from T cells between NR and R. h UMAP visualization of tumor infiltrating leukocytes (TILs) in HCC cohort SRP318499 or in HCC cohort CNP0000650 ( j ). i Heatmap plot showing the expression level of T-cell cytotoxicity genes in tumor-infiltrating T cells from HCC cohort SRP318499 and CNP0000650 ( k ). Patients were divided into two groups (Mono_CD14_S100A9 high (blue) and low (yellow)) based on their infiltration level of S100A9 + CD14 + monocytes

Article Snippet: Staining was carried out using a 1:200 dilution of rabbit polyclonal antibody for human S100A9 (Proteintech Cat# 26,992–1-AP; RRID: AB_2880716) and a 1:2000 dilution of rabbit polyclonal antibody against human CD14 (Proteintech Cat# 17,000–1-AP; RRID: AB_2074048), followed by incubation with secondary antibodies (Biolynx Cat# I20012C).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: S100A9 + CD14 + monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function

doi: 10.1186/s13046-024-02985-1

Figure Lengend Snippet: Endogenous S100A9 enhances PD-L1 expression in monocytes to inhibit T-cell cytotoxicity. a KEGG pathway enrichment analysis of DEGs between S100A9 + CD14 + monocytes and other circulating cells. b Percentage of S100A9 + cells (left) and PD-L1 + cells (right) in PBMC from healthy donors (HD, n = 5) and patients with hepatocellular carcinoma (HCC, n = 7), colorectal cancer (CRC, n = 5), and biliary tract cancer (BTC, n = 7). c-e PD-L1 levels in S100A9 high or S100A9 low cells in CD14 + monocytes from patients with HCC, CRC, and BTC. f-h Correlation analysis between PD-L1 and S100A9 levels in CD14 + monocytes from patients with HCC, CRC, and BTC. i S100A9 and CD274 ( j ) mRNA levels of S100A9 -knockdown THP-1 cells transfected with si-S100A9#1, si- S100A9 #2, and si- S100A9 #3. k Representative flow cytometric plot (left) and quantification (right) of S100A9 levels in S100A9 -knockdown THP-1 cells transfected with sh- S100A9 #1 and sh- S100A9 #2. l Representative flow cytometric plot (left) and quantification (right) of PD-L1 levels in S100A9 -knockdown THP-1 cells transfected with sh- S100A9 #1 and sh- S100A9 #2. m the percentage of highly proliferated CFSE low of T cells co-cultured with S100A9 -knockdown THP-1 cells at 96 h ( n = 3). n TBX21 , PRF1 , IL2 , and GZMB mRNA levels of T cells after co-cultured with S100A9 -knockdown THP-1 cells for 24 h. P value in ( b ) was evaluated using the Mann–whitney U-test. P values in c-e were determined by two-tailed paired sample t-test. P values in i-m were determined by one-way ANOVA. P value in ( n ) was evaluated using 2-way ANOVA. Correlations were analyzed using the Spearman rank correlation test. * P < 0.05, ** P < 0·01, *** P < 0.001 and **** P < 0.0001

Article Snippet: Staining was carried out using a 1:200 dilution of rabbit polyclonal antibody for human S100A9 (Proteintech Cat# 26,992–1-AP; RRID: AB_2880716) and a 1:2000 dilution of rabbit polyclonal antibody against human CD14 (Proteintech Cat# 17,000–1-AP; RRID: AB_2074048), followed by incubation with secondary antibodies (Biolynx Cat# I20012C).

Techniques: Expressing, Knockdown, Transfection, Cell Culture, MANN-WHITNEY, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: S100A9 + CD14 + monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function

doi: 10.1186/s13046-024-02985-1

Figure Lengend Snippet: Exogenous S100A9 enhances PD-L1 expression in monocytes to inhibit T-cell proliferation and cytotoxicity. a Representative flow cytometric plot (left) and quantification (right) of PD-L1 levels in primary human CD14 + monocytes ( n = 3) treated with PBS or rS100A9 for 8 h. b Similar to ( a ), PD-L1 levels in THP-1 cells treated with PBS ( n = 3), rS100A9 ( n = 3), or pre-incubated tasquinimod plus rS100A9 ( n = 3) for 24 h. c Schematic representation of the co-culture system. d Representative CFSE dilution profiles of T cells (left) and the percentage of highly proliferated CFSE low T cells at 96 h (right, n = 3). The peak of the CFSE-labelled unstimulated cells (gray, filled) is also shown. e The percentage of highly proliferated CFSE low T cells at 96 h in four groups: pre-incubated with IgG, pre-incubated with IgG and co-cultured with S100A9-treated THP-1, pre-incubated with αPD-1 antibody, pre-incubated with αPD-1 antibody and co-cultured with S100A9-treated THP-1. ( n = 3). f The RUNX3 and TBX21 mRNA levels in T cells co-cultured with THP-1 cells treated with PBS or rS100A9 for 24 h. g Schematic showing S100A9 binding sites on CD274 promoter (left). Luciferase activities relative to the control were shown on the right ( n = 3). MFI, mean of fluorescence intensity. CFSE: carboxyfluorescein succinimidyl ester. Unsti: unstimulated. Data are represented as mean ± S.E.M. P value in ( a ) was determined by two-tailed paired sample t-test. P value in ( d ) was determined using two-tailed unpaired Student’s t-tests. P value in ( b ) was determined by one-way ANOVA. P values in ( e-g) were evaluated using 2-way ANOVA. * P < 0.05, ** P < 0·01, *** P < 0.001 and **** P < 0.0001

Article Snippet: Staining was carried out using a 1:200 dilution of rabbit polyclonal antibody for human S100A9 (Proteintech Cat# 26,992–1-AP; RRID: AB_2880716) and a 1:2000 dilution of rabbit polyclonal antibody against human CD14 (Proteintech Cat# 17,000–1-AP; RRID: AB_2074048), followed by incubation with secondary antibodies (Biolynx Cat# I20012C).

Techniques: Expressing, Incubation, Co-Culture Assay, Cell Culture, Binding Assay, Luciferase, Control, Fluorescence, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: S100A9 + CD14 + monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function

doi: 10.1186/s13046-024-02985-1

Figure Lengend Snippet: Working model for the effect of S100A9 + CD14 + monocytes contribute to anti-PD-1 immunotherapy resistance. Abundant blood S100A9 + CD14 + monocytes and high concentrate plasma S100A9 linked to poor HCC response to anti-PD-1. Mono_S100A9 signature inversely associated with survival of cancer patients. S100A9 enhanced PD-L1 expression on monocytes to inhibit T-cell proliferation and cytotoxicity. Blockage of S100A9 synergizes with anti-PD-1 drug to enhance HCC eradication

Article Snippet: Staining was carried out using a 1:200 dilution of rabbit polyclonal antibody for human S100A9 (Proteintech Cat# 26,992–1-AP; RRID: AB_2880716) and a 1:2000 dilution of rabbit polyclonal antibody against human CD14 (Proteintech Cat# 17,000–1-AP; RRID: AB_2074048), followed by incubation with secondary antibodies (Biolynx Cat# I20012C).

Techniques: Clinical Proteomics, Expressing

Journal: Kidney international

Article Title: Renal CD14 expression correlates with the progression of cystic kidney disease

doi: 10.1038/ki.2010.175

Figure Lengend Snippet: CD14 is a high affinity lipopolysacharide (LPS) receptor that, in conjunction with Toll-like family receptors (e.g., TLR4), activates LPS-induced immune responses. CD14 is anchored to cell surfaces by a linkage to glycosylphosphatidylinositol (GPI). However, activated cells may release CD14 by proteinase-dependent (48 kDa) or proteinase-independent shedding (55 kDa protein).

Article Snippet: These were xylene-deparafinized, rehydrated, and stained using rabbit polyclonal anti-human CD14 (M-305) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and species-specific secondary antibody conjugated with biotin (Molecular Probes, Eugene, OR).

Techniques:

Journal: Kidney international

Article Title: Renal CD14 expression correlates with the progression of cystic kidney disease

doi: 10.1038/ki.2010.175

Figure Lengend Snippet: Panel a shows a strong correlation of Cd14 expression with kidney volumes in 10-d old mice generated in (C57BL/6J-cpk × CAST/Ei)F1 intercrosses (r=0.94, p<0.001). Black symbols designate affected cpk mice, white symbols correspond to unaffected littermates, diamonds represent males and circles females. This panel also demonstrates that Cd14 expression is significantly increased even in the most mildly affected cpk kidneys vs. unaffected littermate kidneys (p<0.05; bracketed data points). Panel b demonstrates the outcomes of similar analyses for Ccl2, a gene encoding a surrogate PKD progression marker MCP-1. The correlation of Ccl2 expression and kidney volumes was moderately strong (r=0.79, p<0.001). However, the difference in Ccl2 expression levels between unaffected and mildly affected cpk kidneys was not significant. Panels c and d show relative renal Cd14 and Ccl2 expression in early postnatal period. Black diamonds represent mean gene expression values for specific time-points in affected cpk mice, white circles correspond to mean gene expression in unaffected littermates, bars indicate 1SD. Asterisks mark statistically significant differences between data for kidneys from cpk mice and unaffected littermates (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: These were xylene-deparafinized, rehydrated, and stained using rabbit polyclonal anti-human CD14 (M-305) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and species-specific secondary antibody conjugated with biotin (Molecular Probes, Eugene, OR).

Techniques: Expressing, Generated, Marker, Gene Expression

Journal: Kidney international

Article Title: Renal CD14 expression correlates with the progression of cystic kidney disease

doi: 10.1038/ki.2010.175

Figure Lengend Snippet: Panel a shows a representative micrograph of an anti-CD14 antibody stained section of wild type (+/+) 10-d old kidney showing few strongly positive CD14 cells in interstitium (presumably mature monocytes and macrophages; marked by arrows) and weaker staining in tubular cells. In 10-d old cpk kidneys there is a similar weak staining of both non-cystic and cystic renal epithelial cells as well as strong staining of CD14-positive interstitial cells. Panel b shows that the content of strongly stained CD14-positive mononuclear cells in interstitium is not significantly increased in cpk kidneys. Panel c demonstrates the CD14 producing capacity of principal cells from inner medullary collecting duct cell line mIMCD-K2 (IMCD); RT-PCR with Cd14-derived primers produced a cDNA-specific 258 bp product (asterisk), anti-CD14 immunoblotting of IMCD lysates showed a 55 kDa protein that is present in non-activated CD14-producing cells.

Article Snippet: These were xylene-deparafinized, rehydrated, and stained using rabbit polyclonal anti-human CD14 (M-305) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and species-specific secondary antibody conjugated with biotin (Molecular Probes, Eugene, OR).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Produced, Western Blot

Journal: Kidney international

Article Title: Renal CD14 expression correlates with the progression of cystic kidney disease

doi: 10.1038/ki.2010.175

Figure Lengend Snippet: A representative immunoblot of kidney lysates from 10-d old cpk (cpk/cpk) and wild type (+/+) mice demonstrates that while the total CD14 content is reduced in the cpk kidneys, these kidneys contain increased amounts of the 48 kDa CD14 fragment that is generated by shedding of membrane-bound CD14 from activated cells.

Article Snippet: These were xylene-deparafinized, rehydrated, and stained using rabbit polyclonal anti-human CD14 (M-305) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and species-specific secondary antibody conjugated with biotin (Molecular Probes, Eugene, OR).

Techniques: Western Blot, Generated, Membrane

Journal: Kidney international

Article Title: Renal CD14 expression correlates with the progression of cystic kidney disease

doi: 10.1038/ki.2010.175

Figure Lengend Snippet: Panel a shows representative micrographs of anti-CD14 antibody stained sections of fetal RPKD and adult ADPKD kidneys. CD14-positive interstitial cells (presumed mature monocytes and macrophages) are present in both RPKD and ADPKD kidneys. Cystic as well as non-cystic epithelial cells were CD14-positive, resembling staining of cpk mouse kidneys. However, staining of renal tubular cells in fetal RPKD kidneys was comparable in strength to CD14-positive interstitial cells. CD14 content in cell membrane and cytoplasm closely resembled known CD14 distribution in macrophages. Although not frequent, a similar pattern of CD14 expression was observed also in some non-cystic tubular cells (an arrow) and epithelial cell lining in end-stage ADPKD kidneys. The CD14-like staining pattern was absent in corresponding mouse and human tissues stained with pre-immune sera and/or control antibodies. Panel b shows that similar to cpk mouse kidneys, CD14 content is decreased in lysates from RPKD and ADPKD kidneys. A possible explanation for the PKD-associated reduction in renal CD14 content is its shedding by activated cells. After their activation, these cells shed a GPI-anchor attached CD14 from cell membrane in the form of its 48 kDa CD14 fragment as well as yet unprocessed 55 kDa CD14 protein that are both present in urine from RPKD and ADPKD patients (panel c). Column numbers (panels b and c) designate sample assignment of unique individuals.

Article Snippet: These were xylene-deparafinized, rehydrated, and stained using rabbit polyclonal anti-human CD14 (M-305) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and species-specific secondary antibody conjugated with biotin (Molecular Probes, Eugene, OR).

Techniques: Staining, Membrane, Expressing, Control, Activation Assay

Journal: Kidney international

Article Title: Renal CD14 expression correlates with the progression of cystic kidney disease

doi: 10.1038/ki.2010.175

Figure Lengend Snippet: Urinary CD14 levels correlate with rates of two-year total kidney volume (TKV) change in ADPKD patients. While the overall correlation was marginal (r=0.43, p=0.09), it was moderately strong in males (designated by black diamonds; r=0.74, p=0.02). The correlation of the TKV change and GFR determined by iothalamate was not significant (r=(−0.22), p>0.20).

Article Snippet: These were xylene-deparafinized, rehydrated, and stained using rabbit polyclonal anti-human CD14 (M-305) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and species-specific secondary antibody conjugated with biotin (Molecular Probes, Eugene, OR).

Techniques: