HeLaP4 cells were transduced with a dilution series of vector expressing a miRNA-based shRNA against YB-1 (miRY1, red, miRY2, blue) or against DSRed (miRctrl, dark grey). YB-1 expression levels were measured with (A) RT-qPCR and with (B) immunocytochemistry (ICC). YB-1 was detected with a specific antibody (green). DNA was stained using DAPI. (C) The stable cell lines were infected with VSV-G pseudotyped HIV_YFP and harvested 48–72 hours post infection. % YFP positive cells (upper panel) and the % YFP positive x mean fluorescence intensity (MFI) (lower panel) were measured using flow cytometry. Shown is a representative experiment out of 3 independent experiments. Standard deviations for triplicates within one experiment are shown. The various YB-1-depleted cell lines were compared to the miRctrl (black stars) condition using one way ANOVA followed by the Bonferroni multiple comparison test. (D) HeLaP4 cells depleted for YB-1 were transduced with different concentrations of vector expressing miRNA-resistant YB-1 (miRY2+BC, green) or control vector (miRY2+ctrl, light blue) and RNA expression levels were determined with RT-qPCR. (E) Experiment performed as in panel C. VSV-G pseudotyped HIV_YFP infection efficiency was partially restored upon YB-1 backcomplementation, as measured by flow cytometry. Shown is a representative experiment out of 3 independent experiments. Standard deviations of triplicate data points are shown. Differences were determined using one-way ANOVA, followed by the Bonferroni multiple comparison test. The miRctrl and the BC dilution series were compared to the miRY2 (dark blue stars) and miRY2+ctrl (light blue stars) conditions. *p <0.05,**p < 0.01, ***p < 0.001.

https://doi.org/10.1371/journal.pone.0241893.g001