Journal: International Journal of Molecular Sciences

Article Title: The Chimeric Nuclease SpRYc Exhibits Highly Variable Performance Across Biological Systems

doi: 10.3390/ijms27010488

Figure Lengend Snippet: Genome-editing activity of SpCas9 and SpRYc in CEM-R5 T cells. ( A ) The localization of the tested target sites at the C–X–C motif chemokine receptor 4 ( CXCR4 ) and beta-2-microglobulin ( B2M ) loci. ( B ) CXCR4 KO efficiency measured with different PAMs. ( C ) B2M KO efficiency measured with different PAMs. To compare each experimental group at CXCR4 and B2M loci, a one-way ANOVA with Dunnett’s multiple comparison test was used. Data are presented as the mean ± SD. Significance: ns, not significant; **, p < 0.01; ****, p < 0.0001.

Article Snippet: For immunofluorescence staining, cells were washed once with PBS and incubated with phycoerythrin-labelled antibodies against CXCR4 (#E-AB-F1157D, Elabscience, Houston, TX, USA) or B2M (#316306, Biolegend, San Diego, CA, USA) diluted in PBS at 4 °C for 30 min. Then, cells were washed twice with PBS and analyzed using a CytoFLEX S flow cytometer (Beckman-Coulter, USA).

Techniques: Activity Assay, Comparison

Journal: Oncology Reports

Article Title: Hypoxia enhances CXCR4 expression by activating HIF-1 in oral squamous cell carcinoma

doi: 10.3892/or_00000275

Figure Lengend Snippet: Figure 2. Effect of hypoxia on CXCR4 expression in OSCC cell lines. Cells were cultured for indicated time under hypoxic conditions. Total RNA was analyzed by real-time quantitative RT-PCR for CXCR4 expression (A). Cells were cultured for 24 h under normoxic or hypoxic conditions. Cell lysates were analyzed by Western blotting for CXCR4 and HIF-1· expression (B). Luciferase activity increased under hypoxic conditions in cells transfected with plasmid carrying wild-type CXCR4 promoter. Normalization by luciferase activity derived from mutant type CXCR4 promoter trans- fectants (C).

Article Snippet: Non-specific reactions were then blocked with 5% dried milk in phosphate-buffered saline (PBS) for 1 h at room temperature and the sections incubated overnight at 4 ̊C with rabbit anti-human HIF-1· monoclonal antibody (1:50; Millipore, Bedford, MA) or rabbit anti-CXCR4 polyclonal antibody (1:100; ProSci, Poway, CA).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Derivative Assay, Mutagenesis

Journal: Oncology Reports

Article Title: Hypoxia enhances CXCR4 expression by activating HIF-1 in oral squamous cell carcinoma

doi: 10.3892/or_00000275

Figure Lengend Snippet: Figure 3. Inhibition of CXCR4 expression by siRNA specific for HIF-1· under hypoxic conditions in OSCC cell lines. Cells were transfected with siHIF-1· and then incubated in hypoxic conditions for 24 h. Cell lysates were prepared and analyzed by Western blotting for CXCR4 and HIF-1· expression. siHIF-1· significantly reduced the expression of CXCR4 and HIF-1· under hypoxic conditions (A). Total RNA was prepared and analyzed by real-time quantitative RT-PCR for CXCR4 mRNA expression (B). siHIF-1· inhibited the increase of reporter activity under hypoxic conditions in cells carrying CXCR promoter- luciferase. Control cells were transfected with siGFP. Normalization by luciferase activity derived from mutant type CXCR4 promoter transfectants (C).

Article Snippet: Non-specific reactions were then blocked with 5% dried milk in phosphate-buffered saline (PBS) for 1 h at room temperature and the sections incubated overnight at 4 ̊C with rabbit anti-human HIF-1· monoclonal antibody (1:50; Millipore, Bedford, MA) or rabbit anti-CXCR4 polyclonal antibody (1:100; ProSci, Poway, CA).

Techniques: Inhibition, Expressing, Transfection, Incubation, Western Blot, Quantitative RT-PCR, Activity Assay, Luciferase, Control, Derivative Assay, Mutagenesis

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Cell–cell fusion assays and recombinant envelope and receptor constructs. ( A ). Cell–cell fusion assays involved transfection of one set of cells with an Env expression construct (in red) plus a PM-targeted split GFP helix 1–10 expression construct (pcDNA3.1-PLCdeltaPH-GFP-H1-10; green), and transfection of a separate set of cells with expression vectors for CD4 (blue) and CXCR4 (pink) variants, plus a PM-targeted split GFP helix 11 expression construct (For pGFP-H11-PLCdeltaPH; green). One day post-transfection cells were seeded together onto coverslips, and scored for GFP+ syncytia two days later. ( B ). HIV-1 Env expression constructs included one that produced the WT HIV-1 NL4-3 envelope protein (Env-WT: pMDG-NL43-Env-WT or SVIII-Env-WT) and one that produced an Env protein (Env-753stop: pMDG-NL43-Env-753stop), truncated at Env residue 753, before the CT amphipathic helices. CD4 expression constructs included one that expressed a Myc-tagged WT human CD4 protein (CD4: pCAGGS-CD4-Myc) or a variant in which the CD4 transmembrane and cytoplasmic tail (TMCT) domains were replaced with a GPI anchor (CD4-GPI: pCAGGS-CD4-GPI). CXCR4 expression constructs included a WT human CXCR4 expression construct (CXCR4: pcDNA3-CXCR4), constructs with partial (CXCR4-318: pcDNA3-CXCR4-318Stop) or complete (CXCR4-309: pcDNA3-CXCR4-309Stop) CXCR4 cytoplasmic tail (CT) deletions, and constructs in which CT truncations were fused to the PLCδPH that preferentially binds to PI(4,5)P2 (CXCR4-318-PH and CXCR4-309-PH).

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Recombinant, Construct, Transfection, Expressing, Produced, Residue, Variant Assay

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: HIV-1 Env cell–cell fusion assays. Cell–cell fusion assays were performed with 293 cells as illustrated in . Panels ( A – F ) show coincubations of cells transfected with pGFP-H11-PLCdeltaPH plus CD4-GPI plus CXCR4-318 mixed with cells transfected with pcDNA3.1-PLCdeltaPH-GFP-H1-10 plus either Env-WT ( A , B ), Env-753Stop ( C , D ), or a mock ( E , F ). Panels ( A – F ) are matched photos imaged for DAPI nuclear stain ( A , C , E ) and GFP ( B , D , F ). Note that the size bar (panel ( F )) pertains to all fluorescent images ( A – F ). In panel ( G ), numbers of syncytia per 0.4 mm 2 are averaged with standard deviations from five images each of coincubations of cells co-transfected with split GFP constructs, plus the indicated Env or CD4 plus CXCR4 expression construct variants. Note that numbers of syncytia for Env-753 were approximately tenfold higher than syncytia for Env-WT, and that syncytia for Env-WT were approximately twenty-fold higher than syncytia for the Env-minus (No Env) control. Differences between the sets were highly significant ( p < 0.001), and were obtained by conversion of means and standard deviations to Z values for probability calculations.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Transfection, Staining, Construct, Expressing, Control

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Infection of Env-expressing cells via fusion in reverse. ( A ) Target 293 cells expressing different Env variants are infected with a β-gal-transducing, HIV-1-based, lentivirus vector pseudotyped with a GPI-anchored CD4 variant (CD4-GPI) plus a C-terminally-truncated CXCR4 protein (CXCR4-318). Receptor plus co-receptor binding to cellularly expressed Env activate the Env fusion machinery, resulting in virus infection. ( B ) Shown are β-gal activity-derived infectivities of cells expressing Env-753Stop, Env-WT, or no Env (mock), normalized to the Env-753Stop cells. Note that results are averaged from five independent experiments each, are plotted on a log scale graph to facilitate comparison, and that standard deviations for the Env-753Stop and mock cells were too small to be observed on the graph. ( C ) Cells expressing the indicated Env proteins were infected with CD4-GPI plus CXCR4-318 pseudotyped β-gal-transducing HIV-1 lentivirus vectors in the absence (753, WT) or presence (753 + T20, WT + T20) of HIV-1 Env fusion inhibitor T20. Results for pairs of infections are normalized to the infectivities of viruses in the absence of T20. Averages and standard deviations derive from three separate infections, and differences between 753 and WT and WT and mock were highly significant ( p < 0.001), as were differences between untreated and treated samples. Probabilities were obtained by conversion of means and standard deviations to Z values, which were used for calculations.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Infection, Expressing, Plasmid Preparation, Variant Assay, Binding Assay, Virus, Activity Assay, Derivative Assay, Comparison

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Fusion in reverse infectivities for different Env, receptor, and co-receptor combinations. 293 cells expressing the 753Stop ( A ) or WT HIV-1 Env ( B ) were infected with equivalent volumes of β-gal-transducing HIV-1 particles carrying the indicated receptor plus co-receptor combinations (CD4, CD4-GPI [gpi],—[mock], CXCR4, 318 [CXCR4-318], 309 [CXCR4-309], 318PH [CXCR4-318-PH], 309PH [CXCR4-309-PH]). At 72 h post-infection, cells were subjected to β-gal assays and β-gal activities are plotted as infectivities relative to viruses carrying the CD4-GPI/CXCR4-318 receptor/co-receptor combinations. Averages and standard deviations derive from the following numbers of experiments: 753/gpi/318, 9; 753/gpi/-, 2; 753/-/318, 2; 753/CD4/325, 2; 753/gpi/CXCR4, 4; 753/gpi/309, 3; 753/gpi/318PH, 5; 753/gpi/309PH, 5; WT/gpi/318, 9; WT/gpi/-, 2; WT/-/318, 2; WT/CD4/318, 3; WT/gpi/CXCR4, 6; WT/gpi/309, 4; WT/gpi/318PH, 5; WT/gpi/309PH, 5. Note that differences between the gpi/318 samples versus the gpi/-, -/318, and CD4/318 samples had probabilities of p < 0.001, as determined by conversion of means and standard deviations to Z values for probability calculations.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Expressing, Infection

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Infection of HIV-1-infected cells. Jurkat T cells, HIV-1-infected J1.1 Jurkat cells, and J1.1 cells induced with tumor necrosis factor alpha (J1.1 + TNFa) were subjected to immunoblot detection of Gag proteins ( A ) with an anti-CA antibody, immunoblot detection of Env proteins ( B ) with an anti-gp41(TM) antibody, and infection with a lentivirus vector ( C ) generated by transfection of 293 cells with psPAX2, pLVX-puro-Xho-ATG-β-gal, CD4-GPI, and CXCR4-328 plasmids. ( A , B ) Migration positions of PrGag, CA, gp160 and gp41 proteins are indicated, as are molecular weight standards (in kDa) that were electrophoresced in parallel. ( C ) Infectivity results represent normalized transduced β-gal activity readings normalized to results for induced J1.1 cells. Averages and standard deviations derive from duplicate (Jurkat, J1.1) or triplicate (J1.1 + TNFα) infections, and differences between Jurkat and J1.1, as well as J1.1 versus J1.1 + TNFα were highly significant ( p < 0.001), as determined by conversion of means and standard deviations to Z values that were used in probability calculations.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Infection, Western Blot, Plasmid Preparation, Generated, Transfection, Migration, Molecular Weight, Activity Assay

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Effects of lipid variations on fusion in reverse infections. ( A ) The HIV-1 viral membrane is highly enriched in cholesterol (CHOL), and has substantial amounts of ceramide (CER). The inner leaflets are enriched for phosphatidylserine (PS), phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2; PIP2), and phosphatidylethanolamine (PE), while the outer leaflet is enriched in sphingomyelin (SM), hexosylceramides (HEX), and saturated phosphatidylcholines (PCs). ( B – D ) Infectivities of equivalent volumes of virus were scored via β-gal activities, and were normalized either to untreated (−) samples, or for convenience in the panel ( D ) CD/CX infections, to viruses from CerS−/− cells. In all panels, CD/CX viruses refer to lentivirus vectors generated by co-transfection of 293 cells with a GagPol expression vector (psPAX2), a β-gal-transducing vector (pLVX-puro-Xho-ATG-β-gal), as well as CD4-GPI and CXCR4-318 plasmids. Also, in all panels, Env-WT and Env-753 targets cells refer to 293 cells transfected to express those proteins. For panels ( B , C ), WT and 753 viruses are the NL4-3 variants and were used to infect TZM-bl reporter cells; for panel ( D ), WT refers to viruses produced from cells transfected with psPAX2, pLVX-puro-Xho-ATG-β-gal, plus a WT Env expression vector, while targets were 293 cells transfected to express the CD4-GPI and CXCR4-318 proteins. In panel ( B ), AME refers to treatment of viruses with 10 mM AME prior to infections. In panel ( C ), TMEM refers to co-transfection of virus producing cells with (+) or without (−) the mTMEM-GY scramblase expression vector. In panel ( D ), CerS2−/− pertains to producing viruses either from 293 cells (−) or from 293-CerS2−/− knockout cells (+). For panels ( B – D ), averages and standard deviations are as indicated, noting that where error bars are missing, it is because the standard deviations were too small to be depicted. The following significant differences were obtained by conversion of means and standard deviations to Z values for probability calculations: Panel ( B ) WT/TZMBL, AME −/+, p < 0.001; Panel ( B ) CDCX/Env-WT, AME −/+, p < 0.001; Panel C all pairs p < 0.001; Panel ( D ) CDCX/Env-WT, CerS2−/−, −/+, p < 0.01; all other Panel D pairs, p < 0.001. Note that in panel ( D ), if normalizations were based on CDCX viruses produced from WT 293 cells and infections of Env-753Stop-expressing cells, infectivities of CDCX viruses from virus–cell combinations would be as follows: from WT 293 cells into Env-753Stop cells, 100%; from CerS2−/− cells into Env-753Stop cells 500%; from WT 293 cells into Env-WT cells, 8%; from CerS2−/− cells into Env-WT cells, 19%.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Membrane, Virus, Generated, Cotransfection, Expressing, Plasmid Preparation, Transfection, Produced, Knock-Out

Journal: World journal of gastroenterology

Article Title: CXCR4/SDF-1 axis is involved in lymph node metastasis of gastric carcinoma.

doi: 10.3748/wjg.v17.i19.2389

Figure Lengend Snippet: Figure 4 Expression of CXC chemokine receptor-4 in gastric carcinoma tissues and stromal cell-derived factor-1 in lymph nodes. A: CXC chemokine recep- tor-4 (CXCR4) protein was detected by immunohistochemistry in primary gastric carcinoma tissues; B: CXCR4 protein was not detected in normal mucous membrane; C: Stromal cell-derived factor-1 (SDF-1) protein was detected by immunohistochemistry in lymph nodes with gastric cancer cell metastasis; D: SDF-1 protein was not detected in normal lymph nodes (400 ×).

Article Snippet: Subsequently, the slides were pretreated with 1% bovine serum albumin in phosphate-buffered saline (PBS) and incubated with antiSDF-1 antibody (Boster; dilution 1:100) and anti-CXCR4 antibody (Boster; dilution 1:50) for 1 h at room temperature.

Techniques: Expressing, Derivative Assay, Immunohistochemistry, Membrane

Journal: World journal of gastroenterology

Article Title: CXCR4/SDF-1 axis is involved in lymph node metastasis of gastric carcinoma.

doi: 10.3748/wjg.v17.i19.2389

Figure Lengend Snippet: Figure 5 mRNA expression of CXC chemokine receptor-4 in gastric can- cer cells, tumors and normal mucous membranes and of stromal cell- derived factor-1 in lymph nodes. A: CXCR

Article Snippet: Subsequently, the slides were pretreated with 1% bovine serum albumin in phosphate-buffered saline (PBS) and incubated with antiSDF-1 antibody (Boster; dilution 1:100) and anti-CXCR4 antibody (Boster; dilution 1:50) for 1 h at room temperature.

Techniques: Expressing, Derivative Assay