Journal: PLOS Pathogens

Article Title: EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression through recruiting monocytes and regulating their directed differentiation

doi: 10.1371/journal.ppat.1011934

Figure Lengend Snippet: (A) The mRNA expression levels of GM-CSF, IL-8 and GRO-α were determined by real-time PCR in CNE2 cells that express the EBV lytic genes as indicated (n = 3). (B) Stable expression of ZTA in CNE2 cells confirmed by Western blot. (C) Cytokine antibody array identified the differences of cytokine secretion between the supernatants of CNE2-ZTA and CNE2-CTR. The spot signal densities were quantified by Image J. (D) The concentrations of GM-CSF, IL-8 and GRO-α in the supernatants of each CNE2-derived cell line were measured by ELISA (n = 3). (E) The number of immune cells recruited by CNE2-ZTA and CNE2-CTR (n = 4). (F) and (G) After treatment with the supernatants of CNE2-ZTA and CNE2-CTR, the difference in the yield of DCs was examined by CD14 and CD1a expression profile, the proportion of DC-d-Ms was examined by CD14 and CD68 (n = 4). (H) The proportion of M1 and M2 subtypes were determined by detecting CD86&CD163 (n = 4). (I) Neutralizing antibodies were used to block the recruitment of monocytes to CNE2-ZTA. The proportion of CD14 + cells was determined by flow cytometry (n = 4). (J) Neutralizing antibodies were used to block the ability of CNE2-ZTA to induce TAMs. The proportion of CD163 + cells was determined by flow cytometry (n = 4). Data are presented as the mean±SEM; * P < 0 . 05 , ** P < 0 . 01 , *** P < 0 . 001 , **** P < 0 . 0001 , NS , not significant .

Article Snippet: ELISA: Secreted cytokines in CNE2 supernatant were quantified by using Human GRO-α ELISA Kit (#E-EL-H0045C, Elabioscience), Human GM-CSF ELISA Kit (#RK00045, abclonal), Human IL-8 ELISA Kit (#EHC008.96, Neobioscience), based on the forward sandwich binding technique, each ELISA experiment was done following the protocol provided by the manufacturer.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Ab Array, Derivative Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Flow Cytometry

Journal: PLOS Pathogens

Article Title: EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression through recruiting monocytes and regulating their directed differentiation

doi: 10.1371/journal.ppat.1011934

Figure Lengend Snippet: (A) and (B) The CRISPR/Cas9-mediated knockout efficiency of ZTA was estimated by measuring ZTA mRNA expression (n = 3), as well as ZTA protein levels. (C) EBER1 expression levels in the knockout and control cells were quantified by RT-PCR. (D) The GFP profiles of EBV + CNE2 after ZTA deletion were detected by flow cytometry. (E) and (F) The mRNA expression levels of GM-CSF, IL-8, GRO-α in ZTA knockout and control EBV + CNE2 cells were detected by real-time PCR (n = 3), the concentrations of GM-CSF, IL-8 and GRO-α in the supernatants were measured by ELISA (n = 3). (G) The number of immune cells recruited by CNE2-EBV + SgCTR and CNE2-EBV + SgZTA (n = 4). (H) and (I) After treatment with the supernatants of CNE2-EBV + SgCTR and CNE2-EBV + SgZTA, the yield of DCs was examined by CD14 and CD1a expression profile. The proportion of DC-d-Ms was examined by CD14 and CD68 (n = 4). (J) M1 and M2 proportions were identified by CD86 and CD163 expression (n = 4). Data are presented as the mean±SEM. * P < 0 . 05 , ** P < 0 . 01 , *** P < 0 . 001 , **** P < 0 . 0001 , NS , not significant .

Article Snippet: ELISA: Secreted cytokines in CNE2 supernatant were quantified by using Human GRO-α ELISA Kit (#E-EL-H0045C, Elabioscience), Human GM-CSF ELISA Kit (#RK00045, abclonal), Human IL-8 ELISA Kit (#EHC008.96, Neobioscience), based on the forward sandwich binding technique, each ELISA experiment was done following the protocol provided by the manufacturer.

Techniques: CRISPR, Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay