Fig 2: ZFP36L1 blocks neuroendocrine differentiation and proliferation in SCLC in part through its ability to bind and degrade the 3'UTRs of SOX2 and INSM1.a, b RT-qPCR and immunoblot analysis of CORL47 dCas9-VP64 cells that were infected with sgRNAs targeting the promoter of ZFP36L1 or a non-targeting sgRNA (sgControl). c Proliferation assays of cells in a. For a–c, n = 3 biological independent experiments. d Schematic showing the cysteine, C, sites in the zinc-finger RNA-binding domains of ZFP36L1 that were mutated to alanines, A, to disrupt the RNA-binding activity of ZFP36L1. e Immunoblot analysis of CORL47 cells stably infected with ZFP36L1WT-V5, ZFP36L1 mRNA-binding mutant-V5 (ZFP36L1MUT-V5), or the corresponding empty vector (EV). f Proliferation assays of cells in e. n = 4 biological independent experiments. g Immunoblot analysis of CORL47 cells stably infected with a doxycycline inducible (DOX-ON) lentivirus encoding ZFP36L1WT-FLAG, ZFP36L1MUT-FLAG, or EV. Cells were treated with DOX (1 µg/mL) for 3 days where indicated. h Proliferation assays of cells in g grown in +/– DOX (1 µg/mL) for the days indicated. n = 2 biological independent experiments. i Immunoblot analysis after immunoprecipitation (IP) of ZFP36L1WT-FLAG, ZFP36L1MUT-FLAG, or EV CORL47 cells treated with DOX (1 µg/mL) for 3 days relative to input. j RNA quantitation relative to input after RT-qPCR from IP experiments in i with primers specific to SOX2, INSM1, E2F1, CDK2, and GAPDH. n = 3 biological independent experiments. k Immunoblot analysis of 293T cells stably infected with ZFP36L1WT-V5, ZFP36L1MUT-V5, or EV. l, m 3'UTR luciferase reporter assay in 293T cells from k transfected with a SOX2-3'UTR luciferase reporter (l), a INSM1-3'UTR luciferase reporter (m), or the empty vector (EV), and bioluminescence (BLI) was measured 24 h later. BLI of each condition was first normalized to the cell count and then normalized to the plx304 EV of each reporter. For l, m, n = 3 biological independent experiments. n, o Immunoblot analysis of CORL47, NCI-H1876, and NCI-H69 cells transfected with siGenome SMARTPool targeting SOX2 (siSOX2) (n) or INSM1 (siINSM1) (o) or non-targeting siRNA control (siCON) at the times indicated after transfection. For n, o immunoblots are representative of 3 biological independent experiments. For all panels, data are mean values +/– SEM. Statistical significance was calculated using unpaired, two-tailed students t-test and p-values are indicated.