Figure 4.
Concurrent caraphenol A treatment improves LV endosomal escape into the cytoplasm. (A) UCB-CD34+ cells (n = 3 donors) were transduced in the presence of caraphenol A (Cara, 30 μM, green symbols) or DMSO (0.06%, blue symbols) with LV, MOI 15, carrying the enzyme-Vpr protein (LV-Vpr). After a 6-hour transduction, cells were loaded with 7-hydroxycoumarin cephalosporin fluorescein–acetoxymethyl (CCF2-AM) substrate, and LV entry was quantified by flow cytometric detection of cells exhibiting cleaved CCF2. Transduction was measured by nerve growth factor receptor (NGFR) expression 7 days later. Data presented as dot plots (mean ± SD). Fusion, **P = .0039; transduction, *P = .016 by 2-tailed Student t test. (B) UCB-CD34+ cells (n = 3 donors) were LV-Vpr-transduced in the presence of DMSO or caraphenol A, as above, and then transferred to 4°C at indicated points before loading with CCF2-AM substrate at 12°C for overnight, then analyzed as earlier. Data presented as linear plots (mean ± SD). Combo plot indicates mean ± SD of 3 donors combined. Small plots (closed circles) indicate mean ± SD of technical replicates (n = 2 cultures) from each donor. Combination figure (*P = .0406, slopes of linear regression significantly different). (C) mPB-CD34+ cells (n = 2 donors) were pretreated with either caraphenol A (30 µM, green open circles; 50 µM, green closed symbols), rapamycin (20 µg/mL red closed symbols), or 0.06% DMSO (blue closed symbols) for 4 hours and transduced with varying doses of integration-deficient LV. Percentage EGFP expression was measured 3 days later by flow cytometric analysis. Data are shown as linear plots (mean ± SD). ****P < .0001 by 2-tailed Student t test, comparing percentage EGFP expression in 30 µM caraphenol A and 0.6% DMSO-treated cells. (D) HeLa cells (n = 5 cultures) were treated with LV at MOI 10 for 8 hours and caraphenol A (30 μM, green bars) was added at indicated points after LV addition. DMSO (0.06%, blue bar) with LV only was added as a separate control. Compounds and LV were washed out 8 hours after LV addition, and cells were analyzed for EGFP expression by flow cytometry 5 days later. Data presented as bar graphs (mean ± SD). (E) Cells were pretreated with caraphenol A (30 μM, green bars) for indicated lengths of time before washout of compound and exposure to LV-GFP for an additional 8 hours. After transduction, LV and any remaining compound was removed, and cells were analyzed as in panel D. Bar graphs presented (mean ± SD).

Concurrent caraphenol A treatment improves LV endosomal escape into the cytoplasm. (A) UCB-CD34+ cells (n = 3 donors) were transduced in the presence of caraphenol A (Cara, 30 μM, green symbols) or DMSO (0.06%, blue symbols) with LV, MOI 15, carrying the enzyme-Vpr protein (LV-Vpr). After a 6-hour transduction, cells were loaded with 7-hydroxycoumarin cephalosporin fluorescein–acetoxymethyl (CCF2-AM) substrate, and LV entry was quantified by flow cytometric detection of cells exhibiting cleaved CCF2. Transduction was measured by nerve growth factor receptor (NGFR) expression 7 days later. Data presented as dot plots (mean ± SD). Fusion, **P = .0039; transduction, *P = .016 by 2-tailed Student t test. (B) UCB-CD34+ cells (n = 3 donors) were LV-Vpr-transduced in the presence of DMSO or caraphenol A, as above, and then transferred to 4°C at indicated points before loading with CCF2-AM substrate at 12°C for overnight, then analyzed as earlier. Data presented as linear plots (mean ± SD). Combo plot indicates mean ± SD of 3 donors combined. Small plots (closed circles) indicate mean ± SD of technical replicates (n = 2 cultures) from each donor. Combination figure (*P = .0406, slopes of linear regression significantly different). (C) mPB-CD34+ cells (n = 2 donors) were pretreated with either caraphenol A (30 µM, green open circles; 50 µM, green closed symbols), rapamycin (20 µg/mL red closed symbols), or 0.06% DMSO (blue closed symbols) for 4 hours and transduced with varying doses of integration-deficient LV. Percentage EGFP expression was measured 3 days later by flow cytometric analysis. Data are shown as linear plots (mean ± SD). ****P < .0001 by 2-tailed Student t test, comparing percentage EGFP expression in 30 µM caraphenol A and 0.6% DMSO-treated cells. (D) HeLa cells (n = 5 cultures) were treated with LV at MOI 10 for 8 hours and caraphenol A (30 μM, green bars) was added at indicated points after LV addition. DMSO (0.06%, blue bar) with LV only was added as a separate control. Compounds and LV were washed out 8 hours after LV addition, and cells were analyzed for EGFP expression by flow cytometry 5 days later. Data presented as bar graphs (mean ± SD). (E) Cells were pretreated with caraphenol A (30 μM, green bars) for indicated lengths of time before washout of compound and exposure to LV-GFP for an additional 8 hours. After transduction, LV and any remaining compound was removed, and cells were analyzed as in panel D. Bar graphs presented (mean ± SD).

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