Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Characters of ROBO1-NK cells. (A) Morphology comparison of PBMCs (upper panel), PBMC-NK cells (mid panel) and ROBO1-NK cells (bottom panel) under bright field. (B) Biomarkers determination of ROBO1-NK cells by flow cytometry. For the biomarkers CD34, CD45, CD3, CD56 and CD16, negative controls were used irrelevant IgG replacing primary antibody for incubating, presented as red peaks. For the ROBO1-CAR detection, negative control was used PBMC-NK incubating with anti-CAR antibody, presented as red peak. Samples in detection were presented as blue peaks.

Article Snippet: Subsequent to transfection, the NK cells were cultured for an additional 24 h and sorted using flow cytometry with the Anti-CAR antibody (0799-R271-F, Sinobiological, Beijing, CN).

Techniques: Comparison, Flow Cytometry, Negative Control

Journal: Viruses

Article Title: Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

doi: 10.3390/v14010013

Figure Lengend Snippet: Exposure to primary human neutrophils leads to increased AdV5 transduction, inhibition of neutrophil serine proteases ablates PMN mediated enhancement of epithelial AdV5 transduction, and addition of human neutrophil elastase alone enhances AdV5 transduction of MDCK-CAR Ex8 epithelia. ( A ) MDCK-CAR Ex8 epithelia were induced with doxycycline and 24 h later exposed to PMN. Cells were infected with AdV-LacZ, and beta galactosidase assay was performed the following day to measure AdV transduction. Data are presented as means ± standard errors (SEM) from quadruplicate samples in 13 independent experiments. *** p < 0.001 in two tailed Students t -test. ( B ) Freshly isolated PMN were treated with serine protease inhibitor AEBSF or vehicle control in HBSS at 37 °C or heat killed by placing in a boiling water bath for 30 min. Confluent monolayers of MDCK-CAR Ex8 epithelia in 24-well plates were then exposed to these PMN before infection with AdV5-LacZ and performing beta galactosidase assay the following day. ( C ) Confluent monolayers of MDCK-CAR Ex8 epithelia in 24-well plates were exposed to human neutrophil elastase, neutrophil proteinase 3, or vehicle control before infection with AdV5-LacZ and subsequent beta galactosidase assay. Error bars represent SEM of 3 independent experiments, each performed in quadruplicate. * p < 0.05 by one-way ANOVA and Bonferroni post-hoc test. NT, non-treated; all other treatments were compared to NT.

Article Snippet: Anti-CAR Ex8 antibody was generated by Genescript (Piscataway, NJ, USA) as previously described and used at a concentration of 1:3000 [ ].

Techniques: Transduction, Inhibition, Infection, β-Gal Assay, Two Tailed Test, Isolation, Protease Inhibitor, Control

Journal: Viruses

Article Title: Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

doi: 10.3390/v14010013

Figure Lengend Snippet: PMN exposure does not significantly change apical or total expression of CAR Ex8 in MDCK-CAR Ex8 epithelia. MDCK-CAR Ex8 cells were grown in 6-well transwell inserts until they were fully polarized and then exposed to PMN. Apical surface proteins were marked with sulfo-NHS-biotin and cell lysates were run through neutravidin beads to create an apical protein fraction. Cell lysates containing apical ( A ) and total ( C ) cell proteins were subjected SDS-PAGE, Ponceau staining, and subsequent Western blotting with antibody specific to CAR Ex8 . Band quantifications are depicted for apical ( B ) and total ( D ) CAR Ex8 staining relative to Ponceau loading control. Error bars represent SEM from 3 independent experiments. NT, non-treated.

Article Snippet: Anti-CAR Ex8 antibody was generated by Genescript (Piscataway, NJ, USA) as previously described and used at a concentration of 1:3000 [ ].

Techniques: Expressing, SDS Page, Staining, Western Blot, Control

Journal: Viruses

Article Title: Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

doi: 10.3390/v14010013

Figure Lengend Snippet: Inhibition of macropinocytosis and dynamin-dependent endocytosis pathways does not ablate PMN-mediated enhancement of epithelial AdV5 transduction. MDCK-CAR Ex8 cells were pretreated with amiloride or dynasore in serum-free media before exposure to PMN and subsequent infection with AdV5-LacZ, followed by beta-galactosidase activity assay the following day. Error bars represent SEM from 3 independent experiments each performed in triplicate. * p < 0.05 by one-way ANOVA and Bonferroni post-hoc test. NT, non-treated, ns, not significant.

Article Snippet: Anti-CAR Ex8 antibody was generated by Genescript (Piscataway, NJ, USA) as previously described and used at a concentration of 1:3000 [ ].

Techniques: Inhibition, Transduction, Infection, Activity Assay

Journal: Viruses

Article Title: Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

doi: 10.3390/v14010013

Figure Lengend Snippet: HNE treatment results in activation of autophagic flux in MDCK-CAR Ex8 cells. MDCK-CAR Ex8 cells were pretreated with cytochalasin D or bafilomycin A1 and subsequently treated with HNE. The MDCK-CAR Ex8 cells were then washed thoroughly before performing Western blotting on the samples. Representative Western blot data of ( A ) anti-p62-treated, ( C ) anti-LC3BII-treated blots, as well as ( E ) Ponceau staining are shown. Band quantifications of ( B ) p62 bands relative Ponceau staining and ( D ) LC3BII bands relative to Ponceau staining are shown. Error bars represent SEM from 3 independent experiments each performed in quadruplicate. NT, non-treated.

Article Snippet: Anti-CAR Ex8 antibody was generated by Genescript (Piscataway, NJ, USA) as previously described and used at a concentration of 1:3000 [ ].

Techniques: Activation Assay, Western Blot, Staining

Journal: Viruses

Article Title: Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

doi: 10.3390/v14010013

Figure Lengend Snippet: HBSS starvation results in activation of autophagic flux in MDCK-CAR Ex8 cells. MDCK-CAR Ex8 cells were starved in HBSS+/+ for 4 h and then treated with cytochalasin D or bafilomycin A1. The MDCK-CAR Ex8 cells were then washed thoroughly before performing Western blotting on the samples. Representative Western blot data of ( A ) anti-p62 treated and ( C ) anti-LC3BII treated blots as well as ( E ) Ponceau staining are shown. Band quantifications of ( B ) p62 bands relative Ponceau staining and ( D ) LC3BII bands relative to Ponceau staining are shown. Error bars represent SEM from 3 independent experiments each performed in quadruplicate. NT, non-treated.

Article Snippet: Anti-CAR Ex8 antibody was generated by Genescript (Piscataway, NJ, USA) as previously described and used at a concentration of 1:3000 [ ].

Techniques: Activation Assay, Western Blot, Staining

Journal: Viruses

Article Title: Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

doi: 10.3390/v14010013

Figure Lengend Snippet: Cytochalasin D partially ablates and bafilomycin A1 further exacerbates enhancement of epithelial AdV5 transduction. MDCK-CAR Ex8 cells were either ( A ) exposed to HBSS starvation and then treated with cytochalasin D before treatment with ( B ) HNE or ( C ) PMN. MDCK-CAR Ex8 cells were then either ( D ) exposed to HBSS starvation and concurrently treated with bafilomycin A1 or treated with bafilomycin A1 and then subsequently treated with ( E ) HNE or ( F ) PMN. In all cases, the MDCK-CAR Ex8 cells were subsequently infected with AdV5-LacZ. Beta-galactosidase activity assay was performed the following day to measure AdV5 transduction. Error bars represent SEM from 3 independent experiments each performed in quadruplicate. * p < 0.05, *** p < 0.001 by one-way ANOVA and Bonferroni post-hoc test. NT, non-treated.

Article Snippet: Anti-CAR Ex8 antibody was generated by Genescript (Piscataway, NJ, USA) as previously described and used at a concentration of 1:3000 [ ].

Techniques: Transduction, Infection, Activity Assay

Journal: Viruses

Article Title: Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

doi: 10.3390/v14010013

Figure Lengend Snippet: Summary model of how innate immune factors regulate epithelial susceptibility to AdV5 infection. (1) Under normal circumstances, polarized epithelia express very low levels of apical CAR Ex8 , making them relatively resistant to apical AdV infection. (2) AdV-5 triggers release of proinflammatory cytokine IL-8, which induces apical expression of CAR Ex8 and (3) transepithelial migration of neutrophils. (4) Once neutrophils arrive, release of HNE further enhances AdV-5 transduction of epithelia through a process that involves activation of epithelial autophagic flux.

Article Snippet: Anti-CAR Ex8 antibody was generated by Genescript (Piscataway, NJ, USA) as previously described and used at a concentration of 1:3000 [ ].

Techniques: Infection, Expressing, Migration, Transduction, Activation Assay