Fig 2: JMJD2B-silencing inhibits CRC cell autophagy. A. The positive association between JMJD2B and autophagy-related gene signatures was demonstrated using GSEA analysis in the TCGA (459 colon cancer) and GSE39582 (443 colon cancer) data sets (upper panel). The correlation between JMJD2B and autophagy-related gene signatures or the ubiquitin-proteasome system was conducted using GSEA analysis in gene microarrays with JMJD2B silencing (lower panel). B. Western blot of LC3B levels under normal growth conditions and glucose deprivation conditions in HCT116 and SW480 cells with downregulation of JMJD2B expression. C. Immunofluorescence staining of LC3B in HCT116 and SW480 cells transfected with NC-siRNA or JMJD2B-siRNA after glucose deprivation. Scale bar: 5 μm. D. Immunofluorescence staining of GFP-LC3B in HCT116 and SW480 cells after blockage of JMJD2B after glucose deprivation. E.F. Western blot of LC3B levels in HCT116 and SW480 cells co-cultured with CQ and transfected with NC-siRNA or JMJD2B-siRNA, with or without glucose. G. Transmission electron microscopy (TEM) analysis of HCT116 and SW480 cells silenced for JMJD2B expression after glucose deprivation.

Fig 3: JMJD2B promotes CRC cell survival partly via its effect on amino acids. A. Cell viability analysis of HCT116 and SW480 cells with JMJD2B knockdown in the presence or absence of three JMJD2B-dependent glucose deprivation-inducible amino acids (Asn, His, and Phe). Data are presented as the mean percentage ± SD from five independent samples (*P < 0.05). B.C. Apoptosis evaluated using flow cytometry showing the increased apoptosis of HCT116 and SW480 cells after transfection with JMJD2B siRNAs in glucose deprivation media. The increased apoptosis was abolished by Asn, His, and Phe treatment. Data from B were quantified in C. The results are shown as the mean ± SD of three independent experiments (*P < 0.05). D. Western blotting analysis of cleaved caspase 3, caspase 8, caspase 9, and PARP in JMJD2B-silenced HCT116 and SW480 cells, with or without Asn, His, and Phe treatment. E.F Clonogenic growth experiment of HCT116 and SW480 cells transfected with JMJD2B siRNAs or NC siRNAs, with or without Asn, His, and Phe treatment. Quantitative data are presented in the histogram F as the mean ± SD of three independent experiments (*P < 0.05).

Fig 4: The function of LC3B in the JMJD2B-mediated tumorigenesis in vivo. A-D Tumors from different groups were dissected and photographed (A, B), and the tumor volumes and weights were measured (C, D). Data are presented as the mean ± SD. (*P < 0.05, **P < 0.01, ***P < 0.001). E.F. JMJD2B and LC3B protein levels in the xenografts were detected using western blotting (E) and immunohistochemistry (F). G. Levels of amino acids in tumor lysates derived from xenografts were measured. Data are presented as the mean ± SD. (*P < 0.05, **P < 0.01). H. Representative immunohistochemistry of JMJD2B (upper) and LC3B (lower) proteins in CRC tissues. I. Schematic model showing that JMJD2B activates autophagy via epigenetic regulation of LC3B, resulting in the maintenance of intracellular Asn, His, and Phe levels, which consequently promote the survival of CRC cells upon glucose deprivation.

Fig 5: TGF-β promotes KDM4A recruitment to the DBC1 promoter. (A–I) IEC-6 cells were treated with TGF-β(2 ng/ml) and collected at indicated time points. ChIP assays were performed anti-acetyl H3, anti-trimethyl H3K4, anti-trimethyl H3K9, anti-trimethyl H3K27, anti-trimethyl H4K20, anti-KDM4A, anti-KDM4B, anti-KDM4C, or anti-KDM4D. All experiments were performed in triplicate wells and repeated three times. One representative experiment is shown.