Fig. 6

c-MYC rescues general transcription and prevents senescence in the absence of MITF. a Immunoblot analysis of 501 mel cells under retrovirus-driven c-MYC expression compared to EV and subsequent siMITF₁ vs. siSCR transfection. b MITF, c-MYC, and CDK7 mRNA expression in 501 mel cells under conditions analogous to a. Relative expression was measured by qRT-PCR, normalized to GAPDH and given as mean ± SD from technical triplicates (two-tailed unpaired t-test). c Transcriptional activity measured as EU incorporation in 501 mel cells in analogy to a and b. Graphs indicate mean ±SEM of fluorescence intensity in ≥250 nuclei (two-tailed unpaired t-test). d Proliferation of 501 mel cells in analogy to a–c. Graphs represent mean ± SD of crystal violet absorbance from technical triplicates (two-tailed unpaired t-test). e SA-β-gal positivity in 501 mel cells at day 3 in analogy to d. Data represent mean ± SD from technical triplicates (two-tailed unpaired t-test). Micrographs display SA-β-gal signals and Hoechst 33342 nuclear staining under corresponding conditions (scale: 50 µm). f Regression analysis of MITF and MYC single-cell mRNA expression from a panel of metastatic melanomas (GSE72056). g ChIP‐seq tracks of MITF-binding signals at FUBP2/KHSRP gene locus in primary melanocytes, and 501 mel and COLO829 BT168F melanoma cell lines. Green arrowheads indicate MITF-binding consensus sequences (E box) at the promoter and first intronic region of the FUBP2/KHSRP gene. GEO accession numbers are listed under Materials and Methods. h MITF, c-MYC, and FUBP2 mRNA expression in 501 mel cells after siSCR, siMITF₁, or siFUBP2 transfection. Relative expression was measured by qRT-PCR, normalized to GAPDH and given as mean ± SD from technical triplicates (two-tailed unpaired t-test). i Immunofluorescence and immunoblot analyses of MITF, MYC, and FUBP2 protein expression in analogy to h. DRAQ5 used for nuclear staining and actin used as loading control (two-tailed unpaired t-test of c-MYC fluorescence intensity in 100 nuclei is indicated). a–e, h, i Experiments were repeated twice with comparable results