SPRED1 inactivation modestly alters melanoma growth in vitro and in vivo. (A) Driver lesions in tumor-suppressor genes occurring with SPRED1 alterations in human cutaneous melanoma (total, 363 samples). (B) Western blot analysis of SPRED1 levels in A375 melanoma cells stably transduced with shRNAs directed against SPRED1. shRNA#2 and #4 were selected for SPRED1 down-regulation experiments. Representative of two independent experiments. (C) Proliferation of BRAF-driven A375 human melanoma cells expressing either a control shRNA (shCTRL) or shRNAs against SPRED1 (shSPRED1#2 and #4). Mean ± SD of five independent experiments. P > 0.05 (not significant) compared with shCTRL, paired two-tailed t test. (D) Tumor-free survival curves of casper zebrafish injected with vectors expressing BRAFV600E and targeting either tp53 or both tp53 and spred1. Pooled data of three independent experiments. P = 0.08 (n.s., not significant), log-rank test. (E) Proportion of spred1 mutant alleles in 10 primary BRAF/tp53/spred1 zebrafish melanomas, as measured by deep sequencing of the CRISPR target loci. Indels are indicated as the number of base pairs altered followed by the type of indel: insertion (I) or deletion (D). (F) Tumor-free survival curves of casper zebrafish injected with vectors expressing BRAFV600E and targeting either cdkn2a or both cdkn2a and spred1. Pooled data of three independent experiments. **, P = 0.001, log-rank test.