Fig 2: Kdm4b and Znf217 protein levels predict differentiation state-specific sensitivity to JIB-04 and SP2509.a Relative cell viability in WM115 cells (left), WM902B cells (middle), and A2058 cells (right) following treatment with indicated doses of the combination of vemurafenib and trametinib or vehicle (DMSO) in the presence of pools of four siRNAs targeting either KDM4A, KDM4B, KDM5A, or KDM5B for 96 h. Viability data for each treatment condition were normalized to cells treated with DMSO and the non-targeting (control) siRNA. Data are presented as mean values ± s.d. calculated across n = 3 biologically independent experiments. Statistical significance was determined by two-sided t test. b Relative cell viability in Cas9-positive A2058 cells following treatment with three different types of KDM1A lentiviral single guide RNA (sgRNA) for 96 h. Viability data were normalized to cells treated with non-targeting (control) sgRNA. Data are presented as mean values ± s.d. calculated across n = 4 biologically independent experiments. Statistical significance was determined by two-sided t test. c Two-sided Pearson’s correlation analysis of variations in the difference between Ngfr and Axl protein levels and Znf217 (left panels) or Kdm4b (right panels) in comparison with the rest of the measured proteome (shown as histograms) across BRAF-mutant melanoma cell lines. Protein data are extracted from the Cancer Cell Line Encyclopedia (CCLE) proteomics database (measured by multiplexed mass spectrometry) and z-scored across all of BRAF-mutant melanoma cell lines present in the database. d Two-sided Pearson’s correlation between the baseline fractions of Znf217High cells and Kdm4bHigh cells and the fractions of NgfrLow/AxlHigh cells (top left), the fractions of NgfrHigh/AxlLow cells (top right), sensitivity to SP2509 at 1 μM (bottom left), and sensitivity to the combination of JIB-04 (at 0.2 μM), vemurafenib (at 100 nM) and trametinib (at 10 nM) (bottom right), as evaluated by normalized growth rates following 5 days of treatment. Protein data were measured by immunofluorescence microscopy, quantified following single-cell analysis, and averaged across n = 3 biologically independent samples. e Two-sided Pearson’s correlation between responses (normalized growth rates) to SP2509 (top) or JIB-04 in combination with vemurafenib and trametinib (bottom), measured for each of the 21 melanoma cell lines (x-axis) and corresponding responses fitted by multi-linear regression (MLR) analysis of 14 cell lines (training set; shown in gray) or responses predicted by the trained models for the remaining seven cell lines (validation set; shown in blue or red). Source data are provided as a Source data file.

Fig 3: RFX5-KDM4A pathway negatively regulates apoptosis in HCC. HCC cells with different treatment were subjected to FCAS analysis with Annexin-V and 7AAD staining to quantify the apoptotic cells. The frequency of Annexin-V positive cells was indicated as apoptotic cell population. (A–D) MHCC-97H (A,B) and HepG2 (C,D) cells transduced with a retroviral vector control, FLAG-RFX5 or KDM4A were treated with 5 nM ActD and subjected to apoptosis analysis (A,C) and one-way ANOVA (B,D). (E–H) MHCC-97H (E,F) and HepG2 (G,H) cells transduced lentiviral sgRNA targeting RFX5 together with retroviral control vector or KDM4A were treated with 5 nM ActD and subjected to apoptosis analysis (E,G) and one-way ANOVA (F,H). *P < 0.05, ***P < 0.001.

Fig 4: RFX5-KDM4A negatively regulates p53 and downstream pathway. (A,B) Total proteins from MHCC-97H (A) and HepG2 (B) cells transduced with either KDM4A, FLAG-RFX5 or lentiviral vector control were determined with Western blot. (C,D) Total proteins from MHCC-97H (C) and HepG2 (D) cells transduced with lentiviral sgRNA targeting RFX5 together with either retroviral control vector or KDM4A were determined with Western blot. (E) Total proteins from xenograft tumors were determined with Western blot analysis. (F) The mechanism underlying RFX5 and KDM4A in HCC progression.

Fig 5: Loss of Jmjd2/Kdm4 activity results in decreased growth combined with an increase in differentiation and cell death for MLL-AF9 transformed leukemic cells. (A) Western blot documenting efficient depletion of Jmjd2a, Jmjd2b, and Jmjd2c protein levels in MLL-AF9 transformed murine leukemic cells after 96 h of treatment with 500 nM OHT. (B) RT-qPCR from the indicated cells, confirming efficient depletion of full-length Jmjd2a, Jmjd2b, and Jmjd2c mRNA after OHT treatment. (C) Growth curve of three different leukemic cell lines (L-MA9-2c, L-MA9-2abc-1, and L-MA9-2abc-2) in liquid culture with or without 500 nM OHT. The cell lines were established from leukemic mice transplanted with preleukemic cells of the indicated genotypes. (D) Blast colony formation assay of MA9-2c and MA9-2abc leukemic cells in methylcellulose. Cells were plated after 4 d of treatment with 500 nM OHT in liquid medium. (E) FACS analysis of EdU incorporation in MA9-2abc cells showing an increased sub-G1 fraction, indicative of cell death. (F) The top panel represents cell cycle distribution of MA9-2abc cells, excluding the sub-G1 fraction. The bottom red panel shows the percentage of cells with a sub-G1 (<2N) DNA content. The cells were treated for 96 h with 500 nM OHT prior to the analysis. (G) FACS plot showing increased Mac1 staining in L-MA9-2abc cells treated with OHT for 96 h prior to the analysis. (H) May-Grünwald-Giemsa staining of L-MA9-2abc cells treated with OHT for 96 h. (I) Growth curve of c-kit+-enriched untransformed progenitors from 2abc;CreER and 2c;CreER in liquid culture with or without treatment of OHT. The graphs show mean ± SD of technical replicates and are representative of at least three independent experiments.