Selected sgRNA targeting genes validated in the TFEB-GFP secondary screening and CREB5 expressing rescuing TFEB nucleus retention upon starvation. (a) Images selected from triplicates of 350 images per sgRNA knockdown (designated in black) made over an 18-h time course during starvation. Low probability values are shown in green for cytosolic GFP-TFEB, high probability values (red) for nuclear TFEB-GFP. TFEB-GFP–expressing U2OS cells treated with the designated sgRNA were starved in HBSS for 18 h and images were acquired every hour. Scale bar, 5 µm. (b) The mean CNN-based prediction values were calculated from 2,500 single-cell images per condition HBSS starved for 1–8 h. Mean ± SEM from three independent experiments. (c) Cell lines from b were analyzed by immunoblotting with antibodies against LC3B, LAMP1, p39, and loading control actin. n = 2. CREB5, pHAGE vector expressing full length CREB5; n.t., nontargeting sgRNA; vector, mock pHAGE vector expressing RFP only. ***P < 0.0001 (one-way ANOVA).