Figure 5.
IFITM2/3 proteins restrict LV transduction and are downregulated by caraphenol A. (A) HEK 293T cells stably expressing pQCXIP-FLAG-IFITM1, pQCXIP-FLAG-IFITM2, pQCXIP-FLAG-IFITM3, or pQCXIP-FLAG-IFITM3 Δ17-20 were used for evaluating LV IFITM restriction. Each 293T IFITM cell line was transduced with LV at MOI 13, then analyzed for EGFP expression 24 hours later by flow cytometry. EGFP levels were normalized to EGFP of LV transduced pQCXIP-FLAG 293T (no IFITMs) control set to 100 and results presented as bar graphs (mean ± SD; n = 5 independent experiments). (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of cell lysates generated from HeLa cells treated with caraphenol A (30 μM) or DMSO (0.06%) for the indicated period. Immunoblotting was performed with α-IFITM3-specific antibody and α-GAPDH as a loading control. Numbers indicate location and size (kD) of protein standards in ladder. Image provided is a representative blot of 3 experiments. (C) TZM-bl (wild-type and IFITM3 KO, n = 2 cultures from 2 independent experiments) were seeded and transduced with LV at MOI 10 in the presence of the indicated dose of caraphenol A (green lines) or resveratrol (orange lines) for 8 hours before LV and compound removal. Cells were evaluated for EGFP expression by flow cytometry 5 days later. Wild-type cells (closed circles) demonstrated significant transduction enhancements response to caraphenol A (DMSO vs caraphenol A, P < .0001, 2-tailed Student t test), but not resveratrol (not significant, Student t test). IFITM3 KO cells showed no significant difference compared with DMSO with caraphenol A treatment. (D) TZM-bl wild-type (solid lines) and IFITM3 KO (dotted lines) cells were treated for 4 hours with caraphenol A (30 μM, green) or DMSO (0.06%, blue), then fixed, permeabilized, and analyzed for intracellular IFITM2/3 expression by flow cytometry (n = 3 independent experiments). Expression reported as median fluorescence intensity of IFITM2/3+ cells. (E) UCB-CD34+ cells (n = 9 donors pooled) were prestimulated and then treated with either caraphenol A (30 μM) or DMSO (0.06%) for 4 hours. Cells were then fixed, permeabilized, and immunostained for IFITM2/3 expression before analysis by flow cytometry. Expression reported as percentage of IFITM2/3+ cells compared with isotype control and median fluorescence intensity of IFITM2/3+ cells. (F) mPB-CD34+ cells (n = 2 donors) were prestimulated (see "Methods") and then treated with caraphenol A (30 µM, green line), rapamycin (20 µg/mL red line), or 0.06% DMSO (blue line) for 4 hours followed by a 2-hour addition of 15 MOI integration-deficient LV (Figure 4C). Cells were then fixed, permeabilized, and immunostained for IFITM3 expression before analysis by flow cytometry. Expression reported as median fluorescence intensity of IFITM2/3+ cells.

IFITM2/3 proteins restrict LV transduction and are downregulated by caraphenol A. (A) HEK 293T cells stably expressing pQCXIP-FLAG-IFITM1, pQCXIP-FLAG-IFITM2, pQCXIP-FLAG-IFITM3, or pQCXIP-FLAG-IFITM3 Δ17-20 were used for evaluating LV IFITM restriction. Each 293T IFITM cell line was transduced with LV at MOI 13, then analyzed for EGFP expression 24 hours later by flow cytometry. EGFP levels were normalized to EGFP of LV transduced pQCXIP-FLAG 293T (no IFITMs) control set to 100 and results presented as bar graphs (mean ± SD; n = 5 independent experiments). (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of cell lysates generated from HeLa cells treated with caraphenol A (30 μM) or DMSO (0.06%) for the indicated period. Immunoblotting was performed with α-IFITM3-specific antibody and α-GAPDH as a loading control. Numbers indicate location and size (kD) of protein standards in ladder. Image provided is a representative blot of 3 experiments. (C) TZM-bl (wild-type and IFITM3 KO, n = 2 cultures from 2 independent experiments) were seeded and transduced with LV at MOI 10 in the presence of the indicated dose of caraphenol A (green lines) or resveratrol (orange lines) for 8 hours before LV and compound removal. Cells were evaluated for EGFP expression by flow cytometry 5 days later. Wild-type cells (closed circles) demonstrated significant transduction enhancements response to caraphenol A (DMSO vs caraphenol A, P < .0001, 2-tailed Student t test), but not resveratrol (not significant, Student t test). IFITM3 KO cells showed no significant difference compared with DMSO with caraphenol A treatment. (D) TZM-bl wild-type (solid lines) and IFITM3 KO (dotted lines) cells were treated for 4 hours with caraphenol A (30 μM, green) or DMSO (0.06%, blue), then fixed, permeabilized, and analyzed for intracellular IFITM2/3 expression by flow cytometry (n = 3 independent experiments). Expression reported as median fluorescence intensity of IFITM2/3+ cells. (E) UCB-CD34+ cells (n = 9 donors pooled) were prestimulated and then treated with either caraphenol A (30 μM) or DMSO (0.06%) for 4 hours. Cells were then fixed, permeabilized, and immunostained for IFITM2/3 expression before analysis by flow cytometry. Expression reported as percentage of IFITM2/3+ cells compared with isotype control and median fluorescence intensity of IFITM2/3+ cells. (F) mPB-CD34+ cells (n = 2 donors) were prestimulated (see "Methods") and then treated with caraphenol A (30 µM, green line), rapamycin (20 µg/mL red line), or 0.06% DMSO (blue line) for 4 hours followed by a 2-hour addition of 15 MOI integration-deficient LV (Figure 4C). Cells were then fixed, permeabilized, and immunostained for IFITM3 expression before analysis by flow cytometry. Expression reported as median fluorescence intensity of IFITM2/3+ cells.

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