Fig. 3

Restriction is circumvented by high MOI or CSA treatment. a LV particles pseudotyped with VSVg (LV-VSVg, n = 3), amphotropic (LV-Ampho, n = 3) or ecotropic MLV envelopes (LV-Eco, n = 2) were titrated on permissive HT1080eCat fibroblasts and titers were determined by flow cytometry as transducing units per mL (TU/mL from independently produced viral supernatants) (left panel). The different pseudotyped LV particles were applied to iPSC at an MOI of 10 and the percentage of EGFP and SSEA1 double positive cells was assessed by flow cytometry (right panel). b iPSC were transduced at indicated MOI. EGFP and SSEA1 double positive cells from independent replicates were collected by flow cytometry. MOI 10, n = 5; MOI 100, n = 4; MOI 250, n = 5; MOI 500, n = 5; MOI 1000, n = 5; MOI 2000, n = 4. c Pre-transduction (Pre-Td) experiments in iPSC were performed by Pre-Td with LV.Red or GV.Red at an MOI of 1000 (n = 3; independently produced viral supernatants) followed by a second transduction with LV at an MOI of 100, 6 h after Pre-Td. Data of the second transduction are shown relative to no Pre-Td (left panel). Transduction of LV.Red and GV.Red are shown as the Pre-Td control (right panel). d iPSC transduced with LV at an MOI of 100 in the presence of MG132 at indicated concentrations. Data from 3 independently produced viral supernatants were determined by flow cytometry. e LV (left graph) or GV (right graph) were applied to iPSC at an MOI of 100 in the presence of CSA at the indicated concentrations. Data from 3 to 4 independently produced viral supernatants were determined by flow cytometry. One-way ANOVA with Dunnett post hoc test was used for statistical analyses. LV 0 versus 10, *** p ≤ 0.001; LV 0 versus 20, ***p ≤ 0.001; GV 0 versus 10, ns p = 0.999; GV 0 versus 20, ns p = 0.112