Fig. 1: Genome-wide RNAi screening identified genes that regulate GSC survival and differentiation.

a Schematic diagram of genome-wide RNAi screening through reverse transfection. Single GS5 cells were plated on polyornithine coating 384-well plates, in which siRNA complexes were pre-assembled. Five days later, cells were stained with Hoechst 33342 (blue, nuclei staining) and anti-Nestin antibody⁵⁰, followed by imaging using an Opera high-content screening system. b Representative images of cells treated with RISC-free control siRNA, siNestin, or siUBC. Experiments were performed in triplicate. Scale bar, 20 μm. c The top 5 genes which demonstrated the greatest inhibitory effects on survival of total cells. d The top five genes which demonstrated the greatest effects on reducing Nestin⁺ cell population. For c and d, each gene was targeted by four individual siRNAs, instead of a pool of four siRNAs. AB(AB00477)-xxx represents the ID number of the specific siRNA coded by Dharmacon. Black line represent mean, dots indicate values. Data are presented as mean ± SD (n = 3). Statistical differences were determined by two-tailed student’s t-test.