Fig. 2

AC004540.4-inhibition reduces melanoma cell growth. a) AC004540.4 was targeted using Endoribonuclease prepared siRNA (esiRNA), which caused significant inhibition of cell growth in the melanoma cell lines D04 and MM415. Data were normalized to treatment with non-targeting pooled Control siRNA. b-d) AC004540.4 was targeted using three different siRNA sequences, which caused significant inhibition of cell growth in the melanoma cell lines D04, MM415, WM3629 and Sk-Mel-28. Data were normalized to treatment with non-targeting pooled Control siRNA, average cell growth inhibition with AC004540.4 siRNA treatment was 39% and is shown for AC004540.4 siRNA-1 in b), siRNA-2 in c) and siRNA-3 in d). e-g) Using three different AC004540.4-targeting ASO sequences caused significant inhibition of cell growth in the melanoma cell lines D04, MM415, WM1366, VMM39, Sk-Mel-2, WM3629, Sk-Mel-28, WM3211 and the primary derived melanoma cell lines Hs852T and AV5. Data were normalized to treatment with non-targeting Control ASO, average cell growth inhibition with AC004540.4 ASO-treatment was 80% and is shown for AC004540.4 ASO-1 in e), ASO-2 in f) and ASO-3 in g). AC004540.4-targeting oligonucleotide treatment had minimal impact on cell growth of primary human melanocytes (PHM), as shown by far-left bar-pairs in panels a–g). h) AC004540.4 ASO treatment inhibited colony formation in melanoma. Top: AC004540.4 ASO-1 treatment significantly reduced colony formation in the D04 (p = 0.002), MM415 (p = 0.004) and Sk-Mel-28 (p = 0.002) melanoma cell lines compared to treatment with non-targeting Control ASO. Bottom: Representative images of D04 colonies in 6 cm dishes after incubation with either AC004540.4 ASO-1 or Control ASO. i) All three AC004540.4 ASO constructs specifically and strongly reduced AC004540.4 RNA levels after 1 day of treatment, when compared to treatment with non-targeting Control ASO in the D04 cell line. j) Representative images of fluorescent signals received from D04 cell pellets treated with either AC004540.4 ASO-1, or Control ASO showed a strong reduction of fluorescent signal of AC004540.4-binding probes. Error bars represent standard deviation and significance is shown as p-values calculated by Student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Detailed information about cell line characteristics and cell growth reduction can be found in Suppl. Tables 1, 2