Functional screening of circRNAs using a loss-of-function approach in vitro. (A) Workflow of the strategy used for circRNA prioritization and functional screening. (B) Results of the semimassive siRNA screening: the heat map on the left shows the average relative apoptotic rate with respect to sirNEG (measured as relative percentage of annexin and propidium iodide (PI) marked, ie, the sum of annexin-positive/PI-positive and annexin-positive/PI-negative; ∗indicates LFC > 1.5) upon silencing of 13 circRNAs with high siRNA efficiency (right columns; at least 75% of circRNA expression reduction in SEM and RS4;11 cell lines; relative circRNA expression measured by qRT-PCR and expressed as 2^-ΔΔCt using sir_NEG as calibrator and GAPDH as reference gene). (C) Relative circular RNA (top) and linear transcript (bottom) expression in the silenced samples compared to the sirNEG (gray bar). (D) Percentage of annexin-PI marked cells and (E) relative cell proliferation rate according to EdU assay upon circRNA silencing at 24 hours and 48 hours. For each bar, data are reported as the mean ± standard error of the mean of at least 3 independent replicates in RS4;11.