Fig 2: KDM4B and c-Myc are co-recruited at the LDHA locus. (A-B) Transactivation activity of C4-2B cells (LKO, sh4B#1, or sh4B#2) transfected with a LDHA reporter construct (A) or C4-2B co-transfected with a LDHA reporter construct, a KDM4B vector [KDM4B wild-type (WT), inactive KDM4B mutant (H189A)], and/or c-Myc. (B) The Beta-Glo/One-Glo Assay System was applied to assess the luciferase activity. β-galactosidase was an internal control. (C-E) Chromatin immunoprecipitation (ChIP) analysis of KDM4B and c-Myc occupancy on the LDHA (C), ENO1 (D), and PFK (E) promoter loci. ChIP was performed with specific antibodies against KDM4B, c-Myc, and H3K9me3, respectively in control (LKO) and KDM4B-knockdown (sh4B#1 and sh4B#2) C4-2B cells. ChIP DNAs were analyzed by qPCR with primers that amplify genomic regions. Data were presented as the average of fold enrichment normalized to IgG ± SD from three independent experiments. Statistical analysis was assessed by Student's t-test.

Fig 3: Depletion of KDM4B alters the extracellular acidification rate (EACR) and oxygen consumption rate (OCR). The ECAR (A) and OCR (C) of KDM4B-knockdown (sh4B#1 or sh4B#2) or control (LKO) C4-2B were measured by the Seahorse analyzer. For the ECAR assay, adherent cells were incubated at glucose-free and NaHCO3-free medium, followed by injection of 10 mM glucose, 1 µM oligomycin (Oligo), and 50 mM 2-DG. For the OCR assay, adherent cells were incubated at NaHCO3-free medium with subsequent injection of 1 μM oligomycin (Oligo), 0.5 μM FCCP, and 50 μM rotenone/antimycin A (RA). (B) The glycolytic capacity was plotted based on the ECAR assay (A). (D-F) are metabolic parameters inferred from the OCR assay (C), basal respiration (D), ATP production (E), and maximal respiration (F). All data are presented as the mean ± S.D. and p values were calculated by two-sided Student's t-test.

Fig 4: Association of p65 and KDM4B is critical for osteoclastogenesis. a The most significantly enriched transcription factor (TF) motifs at KDM4B binding regions, as identified by de novo motif analysis using HOMER. b KDM4B interaction with p65. c, d Mapping the binding domain of KDM4B and p65. RHD Rel homology domain, TAD transactivation domain. e Heatmap of normalized tag densities on KDM4B, CCAR1, MED1, and p65 peaks (left panels). Quantifications of p65 tag counts are shown in the box plots (right panel). The boxes enclose the 25th to 75th percentile values. The whiskers extend to the 10th and 90th percentiles. The central horizontal bar indicates the median. ****P < 0.000 1 (Kolmogorov–Smirnov test). f ChIP assay of KDM4B occupancy at the Fosl2 or Tpm1 promoter in p65-depleted cells. g ChIP assay of p65 occupancy at the Fosl2 or Tpm1 promoter in KDM4B- or CCAR1-depleted cells. The data are presented as the mean ± SD values of three independent experiments [two-way ANOVA in (f, g)]. **P < 0.01; ***P < 0.001. See also Supplementary Fig. 6

Fig 5: Tumorigenicity assays in nude mice(A) Stable KDM4B knockdown LoVo cells and control cells were injected subcutaneously into nude mice (n = 10 for each group). The tumors were collected and shown. (B) Tumor development was monitored for 15 days. The length and width of tumors were measured every 3 days to determine tumor volume (mean ± sd, n = 10). (C) The mean tumor weight of each group was calculated(shControl vs shKDM4B = 0.030 ± 0.004 vs 0.009 ± 0.002 g). (P < 0.001) (D) Representative images of immunohistochemical (IHC) staining (using anti-KDM4B and anti-HAX1 antibodies) and hematoxylin and eosin (HE) staining of tumor tissues were shown. (E and F) Representative images of TUNEL staining in tumor tissues are shown (E). The percentage of TUNEL-positive cells in total cells is shown (F) (shControl vs shKDM4B = 3.23 ± 0.57 vs 7.77 ± 0.61). (P < 0.001) The arrow indicates TUNEL-positive cell.