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. 2025 Dec 9;17:594. doi: 10.1038/s41467-025-67297-0

Fig. 10. Mutations in the kink pocket induce functional inhibition of MITF.

Fig. 10

a Functional impact of individual MITF mutations in UACC-257 (top) and 92.1 cells (bottom), engineered to express dox-inducible shMITF and constitutive MITF, either WT or mutated in residues N278D, R217A, Q261A, R263A, A264C, respectively. Control cells express only dox-inducible shMITF. Cells were seeded at low density and treated with dox for 14 days (UACC-257) or 7 days (92.1), before fixation, staining by crystal violet and image acquisition. Numbers refer to % of cells detected in the dox-treated samples and quantified using the resazurin assay, compared to the respective control (- dox). The blots displayed are representative of at least 2 independent biological replicates. b Direct binding of (WT or mutated) MITF to an immobilized DNA oligonucleotide containing 6-time repeats of the MITF-specific M-box recognition element. Proteins were expressed using a HeLa-derived in vitro transcription-translation (IVTT) kit and DNA binding was quantified by a customized TransAM assay, adapted to use of an anti-MITF antibody for protein:DNA detection (see Methods). A mutated DNA oligo sequence was used to normalize data based on background signals. The data is reported as mean ± s.d. of n = 3 independent replicates. Mutations in the kink are indicated in blue and validated loss-of-function mutations (DNA binding) are in red. c Direct binding of cellular MITF (WT or A264C) to chromatin around transcription starting sites (TSS) of MITF KD differentially expressed genes (DEG) and MITF signature genes, in engineered UACC-257 cells as in panel (a) (see Methods). d Gene set enrichment analysis of differentially expressed pathways in UACC-257 cells, expressing dox-induced MITF KD (Control) in combination with constitutive MITF (WT or A264C). Each sample is compared to the respective no-DOX UACC-257 cells. e Transcription-factor motif enrichment analyses for MITF peaks within 3Kb of TSS. All peaks were ranked and binned by RNA expression fold change of closest genes in MITF KD. Source data for panels a and b are provided as a Source Data file.