Fig 2: CRC immune evasion induced by IFN-γ is partially blocked by Evs carrying miR-15a. (A) The proportion of cells expressing PD-L1 by flow cytometry. (B) The mRNA expression of PD-L1 and Galentin-9 in CRC cells determined by RT-qPCR. (C) The protein expression of PD-L1 and Galentin-9 in CRC cells determined by western blot. (D) Measurement of specific lysis rate using an LDH kit. (E) miR-15a expression in cells determined by RT-qPCR. (F) The protein expression of KDM4B, H3K27me3, and HOXC4. The data in figures were all measurement data and presented as the mean ± standard deviation. Each assessment was done in triplicate with three–time repetition to ensure minimum deviation. Comparisons between multiple groups were analyzed by two-way ANOVA, followed by Tukey’s multiple comparison test. The experiment was repeated three times. *p < 0.05, **p < 0.01.

Fig 3: PITX1 controls HIF-1β levels at the JMJD2A, JMJD2B and JMJD2C promoters. (A) HeLa cells were transfected with control or PITX1 siRNA, prior to treatment with 1% O2 for 24 hours. Cells crosslinked and processed for chromatin immunoprecipitation, using HIF-1α and HIF-1β antibodies. Levels of these transcription factors were analyzed at the JMJD2B promoter by qPCR. Graph depicts mean and standard deviation of a minimum of 3 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. (B) HeLa cells were treated and processed as in A, but levels of HIF-1β present at the JMJD2A, JMJD2C and CA9 promoters were analyzed. Levels of HIF-1α are also presented for JMJD2A and JMJD2C. Graph depicts mean and standard deviation of a minimum of 3 independent experiments, Figure 5 (See previous page). except for JMJD2A, which presents the mean and standard deviation of 2 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. (C) HeLa cells were co-transfected with HIF-1β and empty vector or PITX1 expression constructs for 48 hours prior to crosslinked and lysed. Chromatin immunoprecipitation was performed using the indicated antibodies. Levels of HIF-1β were analyzed at the indicated promoters by qPCR. Graph depicts mean and standard deviation of a minimum of 3 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: Effect of G9a overexpression and KDM4B knockdown on TNF-α-induced VCAM1 expression.(a) VCAM1 mRNA levels in cells exposed to diverse doses of TNF-α for 24 h. (b) VCAM1 protein levels determined by western blotting using 30 μg of cell lysates. (c) Blocking of TNF-α-induced VCAM1 expression by G9a. Efficiency of MSCV-hydro-G9a transfection was confirmed by western blotting using 20 μg of cell lysate and 8% SDS-PAGE. (d) VCAM1 quantification was performed through densitometry (Image J software). Results were normalized to β-actin. Bars represent mean ± SEM (n = 3). The different characters denote significant differences (p < 0.05) among the groups. Uncropped blots are found in Supplementary Fig. S10.

Fig 5: KDM4B is essential for supporting cell survival, proliferation, migration, and invasion. A Morphology of cells was observed after knocking down KDM4B. B MTT curves of GBM cells after KDM4B knockdown. C Cells positive for BrdU staining after KDM4B knockdown are shown. D Analysis of colony formation in GBM cells after KDM4B knockdown. E The migration ability of KDM4B-knockdown GBM cells was measured by the wound healing test. F-G The migration and invasion ability of KDM4B-knockdown GBM cells were detected by transwell assays. **p < 0.01; ***p < 0.001; ns, not significant