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. Author manuscript; available in PMC: 2018 Feb 1.
Published in final edited form as: Mol Cancer Res. 2016 Nov 18;15(2):117–127. doi: 10.1158/1541-7786.MCR-16-0281-T

Figure 1.

Figure 1

OTUD6B depletion causes increased cap-dependent protein synthesis by affecting the step of initiation. A, immunoblot analysis of the indicated proteins 48 hours after transfection of H1299 cells with the indicated siRNAs. Similar results were obtained in A549 cells. B, AHA incorporation assay/FACS analysis of cells transfected with the indicated siRNAs. Left, representative experiment in H1299 cells. Right, histogram summarizing the results obtained from seven cell lines (one experiment in A549, H1299, H1437, H1734, ME16, HaCaT, WI38 cells). Columns represent averages, bars standard deviations. C, double luciferase assay in cells co-transfected with pcDNA3-RLUC-POLIRES-FLUC (translation of renilla luciferase is cap-dependent, while firefly luciferase translation is dependent on a polio virus IRES) and the indicated siRNAs. Bars represent the averages of a total 12 experiments (four experiments in each A549, H1299, H1347 cell line); bars represent standard deviations”. D, AHA incorporation/FACS analysis during the indicated time points in cells treated with completely inhibitory concentrations of antibiotics (left) and partially inhibitory concentrations (right), and transfected with the indicated siRNAs. On top is reported the silvestrol concentration. Points represent geometric means, while bars represent standard deviations. The graph displays the average of four experiments performed in H1299 cells. Similar results were obtained in A549 cells (two experiments). * represents the overlapping data of no AHA negative control, cell transfected with control siRNA and siRNA to OTUD6B exposed to 10 μg/ml cycloheximide or 100 nM homoharringtonine. No AHA: negative control for autofluorescence.