Fig 2: KDM6B interacts with HOXC9 for targeting neuronal genes for epigenetic activation.a ChIP-qPCR analysis of HOXC9, KDM6B, and H3K27me3 levels in the promoter region of NEFM in BE(2)-C_tetoff-myc-HOXC9 cells that were cultured in the presence of absence of Doxy for 6 days. b ChIP-qPCR analysis of KDM6B levels in the promoter region of NEFM in BE(2)-C cells with overexpression of HA-tagged KDM6B without (shGFP) or with HOXC9 knockdown by shHOXC9-9. All ChIP-qPCR data correspond to the mean of three technical replicates ±s.d. and are representative of two independent ChIP experiments. Data were analyzed by two-tailed Student’s t-test. ***p < 0.001. c Co-IP showing HOXC9 interaction with endogenous KDM6B in BE(2)-C_tetoff-myc-HOXC9 cells following HOXC9 induction in the absence of Doxy for 6 days. IgH immunoglobulin heavy chain

Fig 3: KDM6B is required for HOXC9-induced neuronal differentiation.a–b qRT-PCR (a) and immunoblot (b) analyses of KDM6B mRNA and protein expression in BE(2)-C_ and SK-N-DZ_tetoff-myc-HOXC9 cells infected with lentiviruses expressing shGFP or shKDM6B. Error bars represent s.d. (n = 3). α-tubulin levels are shown as loading control (b). c Growth curves of BE(2)-C_ and SK-N-DZ_tetoff-myc-HOXC9 cells expressing either shGFP or shKDM6B-36 that were cultured in the presence or absence of Doxy for the indicted times. Data correspond to the mean of four technical replicates ± s.d. and are representative of at least two independent experiments. Data were analyzed by two-way ANOVA with p values indicated. d qRT-PCR analysis of GFRA3, NEFM, and RET mRNA expression in BE(2)-C_tetoff-myc-HOXC9 cells expressing either shGFP or shKDM6B-36 that were cultured in the presence or absence of doxycycline (Doxy) for 9 days. Data were analyzed by two-tailed Student’s t-test. ***p < 0.001