Journal: BMC cancer

Article Title: Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility.

doi: 10.1186/s12885-015-1201-5

Figure Lengend Snippet: Figure 3 CXCR7 expression and localization are modulated by CXCL11 and CXCL12. (A) Immunofluorescence staining of CXCR7 in AD-LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (100 nM), or CXCL12 (100 nM) for 30 min. Nuclei and F-actin are labeled with DAPI and Texas-red phalloidin, respectively. (B) Western blot of cytosolic, membrane, and nuclear protein fractions isolated from LNCaP cells, cultured as described in (A) with pAbCXCR7 antibody. (C) Immunofluorescence staining of CXCR4 in AD-LNCaP cells as described in (A). (D-E) Western blot of cytosolic, membrane, and nuclear protein fractions isolated from LNCaP cells, cultured as described in (A), with antibodies to (D) CXCR4, and (E) AR, GM130, and histone H3. Silver staining demonstrates equivalent loading across samples. The densitometry values were normalized to BSA-treated samples for each subcellular compartment and labeled below the blots.

Article Snippet: The following reagents were purchased from the indicated vendors: AR agonist R1881 (methyltrienolone) (Perkin Elmer Life Sciences, Waltham, MA); CXCL11 (672-IT) and CXCL12 (2716-SD) ligands (R&D Systems, Minneapolis, MN); double-stranded experimentally validated siRNAs for scrambled control (1027281), AR (SI02757258), CXCR4 (SI02664235), CXCR7 (SI02660644) (Qiagen, Valencia, CA), and CXCR7 (109229) (Life Technologies, Chicago, IL); RNeasy Mini kit, RT2 qPCR primers for AR (PPH01016A), CXCR7 (PPH01182F), CXCR4 (PPH0 0621A), PSA (PPH01002B), FASN (PPH01012B), NK X3.1 (PPH02267C), TMPRSS2 (PPH02262C) (Qiagen); Oligofectamine Transfection Reagent, 4%-12% SDSpolyacrylamide gels, Superscript III enzyme, CyQUANT Cell Proliferation Assay Kit (Life Technologies); iQ SYBR-Green Supermix, Precision Plus Prestained Protein Standards, goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody, goat anti-rabbit HRP-conjugated secondary antibody (BioRad, Hercules, CA); mouse monoclonal AR antibody (AR441), rabbit polyclonal AR antibody (N-20), mouse monoclonal SBP antibody (SB19-C4) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal CXCR7 antibodies (ab38089 [a.a. 1–100], and ab72100 [a.a. 106–117, QHNQWPM GELTC]), rabbit polyclonal CXCR4 antibody (ab2074) (Abcam, Cambridge, MA); rabbit polyclonal CXCR4 antibody (PAB9849) (Abnova, Taipei, Taiwan); mouse monoclonal GM130 antibody (BD Transduction Laboratories, San Jose, CA); rabbit polyclonal PSA antibody (DAKO, Carpinteria, CA); mouse monoclonal PSMA antibody (Meridian Life Science Inc, Memphis, TN); rabbit polyclonal Histone H3 antibody, rabbit monoclonal GAPDH antibody (14C10) (Cell Signaling Technology, Beverly, MA); BCA Protein Assay Kit and ECL Western Blotting Substrate Kit (ThermoFisher Scientific, Waltham, MA); Hyperfilm ECL film (GE Healthcare, Piscataway, NJ); fetal bovine serum, charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT); GeneRuler 1 kb DNA Ladder (MBI Fermentas, Hanover, MD); Protein Deglycosylation Mix (P6039S, New England BioLabs, Ipswich, MA); Synthetic peptides to CXCR7 (a.a. 348–362, RVSET EYSALEQSTK) and AR (a.a. 299–315, KSTEDTAEYS PFKGGY) were synthesized by Alpha Diagonistics (San Antonio, TX).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Western Blot, Membrane, Isolation, Cell Culture, Silver Staining

Journal: BMC cancer

Article Title: Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility.

doi: 10.1186/s12885-015-1201-5

Figure Lengend Snippet: Figure 6 CXCR7 modulates AR transcriptional activity. (A) Luciferase assay testing the effects of CXCR7 overexpression on the AR-target promoter probasin in LNCaP cells. LNCaP cells were co-transfected with the pGL4.10-Luc2-probasin and pRLSV40 Renilla vectors along with increasing amounts (30 ng experimental + 270 ng pcDNA3, 100 ng experimental + 200 ng pcDNA3, 300 ng experimental + 0 ng pcDNA3) of CXCR7 or ACTN4 cDNA mammalian expression vectors. The maximal amount (300 ng) of the pcDNA3 mammalian expression vector served as the positive control. Cells were subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) and tested for dual luciferase activity. Student’s t-test was used to calculate significant differences (*p ≤0.05, n = 3) between control and experimental cells within the androgen-treatment group. (B) Luciferase assay testing the effects of CXCR7 siRNA knockdown and treatment with CXCR7 ligand on the AR-target promoter probasin. LNCaP cells were co-transfected with the pGL4.10-Luc2-probasin and pRLSV40-renilla vectors, along with control or experimental siRNAs (50 nM). Next, cells were pre-treated with indicated ligands (BSA, CXCL11, or CXCL12) for 30 min. Cells were then subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) for 18 hrs and tested for dual luciferase activity. Student’s t-test was used to calculate significant differences (*p ≤0.05, n = 3) between control cells and experimental cells within the androgen-treatment group. (C) RNA isolated from LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (10 nM), or CXCL12 (10 nM) for 30 min and subsequently treated with vehicle or androgen (1 nM R1881) for 18 hrs were subjected to qPCR analysis for AR, FASN, NKX3.1, PSA, and TMPRSS2 gene expressions. Student’s t-test was used to calculate significant differences (*p ≤0.05, n = 3) between control and chemokine ligand-treated cells.

Article Snippet: The following reagents were purchased from the indicated vendors: AR agonist R1881 (methyltrienolone) (Perkin Elmer Life Sciences, Waltham, MA); CXCL11 (672-IT) and CXCL12 (2716-SD) ligands (R&D Systems, Minneapolis, MN); double-stranded experimentally validated siRNAs for scrambled control (1027281), AR (SI02757258), CXCR4 (SI02664235), CXCR7 (SI02660644) (Qiagen, Valencia, CA), and CXCR7 (109229) (Life Technologies, Chicago, IL); RNeasy Mini kit, RT2 qPCR primers for AR (PPH01016A), CXCR7 (PPH01182F), CXCR4 (PPH0 0621A), PSA (PPH01002B), FASN (PPH01012B), NK X3.1 (PPH02267C), TMPRSS2 (PPH02262C) (Qiagen); Oligofectamine Transfection Reagent, 4%-12% SDSpolyacrylamide gels, Superscript III enzyme, CyQUANT Cell Proliferation Assay Kit (Life Technologies); iQ SYBR-Green Supermix, Precision Plus Prestained Protein Standards, goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody, goat anti-rabbit HRP-conjugated secondary antibody (BioRad, Hercules, CA); mouse monoclonal AR antibody (AR441), rabbit polyclonal AR antibody (N-20), mouse monoclonal SBP antibody (SB19-C4) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal CXCR7 antibodies (ab38089 [a.a. 1–100], and ab72100 [a.a. 106–117, QHNQWPM GELTC]), rabbit polyclonal CXCR4 antibody (ab2074) (Abcam, Cambridge, MA); rabbit polyclonal CXCR4 antibody (PAB9849) (Abnova, Taipei, Taiwan); mouse monoclonal GM130 antibody (BD Transduction Laboratories, San Jose, CA); rabbit polyclonal PSA antibody (DAKO, Carpinteria, CA); mouse monoclonal PSMA antibody (Meridian Life Science Inc, Memphis, TN); rabbit polyclonal Histone H3 antibody, rabbit monoclonal GAPDH antibody (14C10) (Cell Signaling Technology, Beverly, MA); BCA Protein Assay Kit and ECL Western Blotting Substrate Kit (ThermoFisher Scientific, Waltham, MA); Hyperfilm ECL film (GE Healthcare, Piscataway, NJ); fetal bovine serum, charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT); GeneRuler 1 kb DNA Ladder (MBI Fermentas, Hanover, MD); Protein Deglycosylation Mix (P6039S, New England BioLabs, Ipswich, MA); Synthetic peptides to CXCR7 (a.a. 348–362, RVSET EYSALEQSTK) and AR (a.a. 299–315, KSTEDTAEYS PFKGGY) were synthesized by Alpha Diagonistics (San Antonio, TX).

Techniques: Activity Assay, Luciferase, Over Expression, Transfection, Expressing, Plasmid Preparation, Positive Control, Control, Knockdown, Isolation

Journal: bioRxiv

Article Title: Identification of CD317-Positive Pro-inflammatory Immune Stromal Cells in Human Mesenchymal Stromal Cell Preparations

doi: 10.1101/2022.02.10.479972

Figure Lengend Snippet: (A) Comparative mRNA expression of ICAM-1 in MSC lines and primary cells sorted by CD317 expression (RNA was extracted from 3 different donors or 5 cell line passages; qPCR performed in triplicate, mean shown ± SEM). (B) Mean fluorescence intensity of ICAM-1 expression on the cell surface of MSC lines and primary MSCs differentially gated by CD317 staining (MSCs from 5 different donors or 4 different passages of MSC lines were stained for flow cytometry, mean shown ± SEM). (C)/(D) Comparative (mean ± SEM) mRNA expression of CXCL10 (red) and CXCL11 (blue) in MSC lines/ primary MSCs sorted for CD317 expression (RNA was extracted from 7 different donors/7 different cell passages; experiments were performed in triplicate). (E/F) CXCL10 secretion by MSC lines prior to IFN-γ priming and after priming with baseline (unprimed) secretion subtracted (mean ± SEM, n=2). (G/H) Comparative mRNA expression of 8 IFN-γ signature genes in MSC lines/primary MSCs sorted by CD317 expression (RNA was extracted from 5 different donors/5 different cell passages; experiments were performed in triplicate, mean shown ± SEM). (I)/(J) IFN-γ score for MSC lines/primary MSCs sorted by CD317 expression (n=5)*/** = significance at P<0.05/0.01 using an appropriate statistical test.

Article Snippet: To detect secreted proteins, supernatants from 100,000 cells incubated in 2.5 ml of serum free DMEM for 24 hours was analysed for secreted proteins by enzyme-linked immunosorbent assays (ELISA) using ELISA kits for CXCL10, CXCL11 (BioLegend); CCL2 (eBioscience); and SAA4 (Stratech) following manufacturers instructions.

Techniques: Expressing, Fluorescence, Staining, Flow Cytometry

Journal: Cancer immunology research

Article Title: Epigenetic Repression of STING by MYC Promotes Immune Evasion and Resistance to Immune Checkpoint Inhibitors in Triple-Negative Breast Cancer.

doi: 10.1158/2326-6066.CIR-21-0826

Figure Lengend Snippet: Figure 2. MYC inhibition promotes a T cell–inflamed tumor microenvironment A, Hallmark gene sets significantly enriched or downregulated in MDA-MB-436 and SUM159PT Tax-R cells transfected (24 h) with each of two MYC siRNAs compared to control siRNA (FDR<0.05). B, Transwell migration of Jurkat CXCR3 cells with CM from MDA-MB-436 and SUM159PT Tax-R cells ± neutralizing CCL5, CXCL10 or CXCL11 antibodies. Each bar represents the mean ± SEM (n=3; two-tailed unpaired t-tests). C- D, Flow cytometry quantification of tumor-infiltrating lymphocytes isolated from PBMC- humanized mice bearing SUM159PT Tax-R tumors (C) or immunocompetent mice bearing syngeneic 4T1 tumors (D) treated with or without MYCi975. Each bar represents the mean ± SEM (n=5, n=5; two-tailed unpaired t-tests). E, Hallmark gene sets significantly enriched

Article Snippet: An antibody neutralizing CCL5 (R&D systems, #MAB678SP), CXCL10 (R&D systems, #MAB266-SP) CXCL11 (R&D systems, #MAB672-SP), IFN alpha (R&D systems, #211001), or IFN beta (R&D systems, #MAB814-SP) was added to the CM in the bottom chamber.

Techniques: Inhibition, Transfection, Control, Migration, Two Tailed Test, Flow Cytometry, Isolation