Fig 2: Model for HOXC9-KDM6B cooperation in RA induction of neuronal differentiation in neuroblastoma cells.RA induces HOXD8, which, in turn, transcriptionally activates HOXC9 expression. HOXC9 then activates KDM6B transcription and interacts with KDM6B for targeting neuronal genes for epigenetic activation by removing the repressive chromatin marker H3K27me3

Fig 3: shRNA knockdown of H3K27 demethylases reduces senescence-associated biomarkers. (A) Representative fluorescence microscopy images of MRC5 cell lines grown in SCM and expressing shRNAs targeting Jmjd3 and Utx. The addition of doxycycline (Dox) at 2 µg/mL activated each shRNA, as indicated by turbo red fluorescent protein (tRFP) reporter expression. Representative images show SA-ß-gal staining at 48 h. (B) Quantification for SA-ß-gal staining shown in A. (C) RT-qPCR analysis for the expression levels of H3K27 demethylases and SASP genes in control, Jmjd3, and Utx shRNA cell lines at 72 h. (B,C) Student’s t-test; bars represent standard deviation and asterisks indicate statistical significance (n.s., not significant; *, p = 0.05; **, p = 0.01; ***, p = 0.001; n = 3).

Fig 4: 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 5: 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). a-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