Fig 1: ARID3B controls PD-L1 expression through epigenetic regulation and STAT3-mediated activation. A A scattering plot presenting the positive correlation between the immunoreactive score of ARID3B and PD-L1 intensity in 15 CRC patient samples. B Western blot of PD-L1 expression in the SW480 and HT-29 cells stably infected with ARID3B (SW480/HT29-ARID3B) versus a control vector (SW480/HT29-Vec) (left) and the HCT-15 cells depleted of ARID3B by CRISPR/Cas9 (HCT15-sg3B) versus control (HCT15-Ctrl) (right). #1 and #2 represent two subclones. L, long exposure; S, short exposure. β-actin was used as a loading control. C ChIP shows the occupancy of ARID3B and KDM4C on the regulatory region of CD274 in HT29-ARID3B vs. HT29-vector control (HT29-Vec) (left) and HCT-15 cells receiving CRISPR/Cas9 for targeting ARID3B (sg3B) versus a control vector (Ctrl) (right). The representative data were from three independent experiments. D ChIP assay shows the occupancy of ARID3B and KDM4C on the regulatory region of CD274 in HT29-ARID3B vs. HT29-vector control (HT29-Vec). The representative data were from three independent experiments. E Western blots indicated that the levels of Y705-phosphorylated STAT3 and T202/Y204-phosphorylated ERK were increased, whereas the total STAT3 and ERK remained unchanged in HT29-ARID3B vs. HT29-vector control (HT29-Vec). β-actin was used as a loading control. F Western blots showing that PD-L1 was suppressed by treatment with the indicated inhibitors with decreased Y705-phosphorylated STAT3 in HT29-ARID3B cells for 24 h. Western blots showing full-length or cleaved caspase 3; the inhibitor did not induce cell apoptosis during the assay. See also Figure S6, Table S12.
Fig 2: ARID3B recruits KDM4C for demethylating H3K9me3 at target genes. A Representative immunohistochemical staining results of ARID3B and KDM4C in CRC samples. Case 1, ARID3BhighKDM4Chigh; case 2, ARID3BlowKDM4Clow. Scale bar=200 μm. B Representative images showing the expression of ARID3B and KDM4C in PDXs. Scale=200 µm. C ChIP shows the occupancy of ARID3B and KDM4C on the regulatory region of target genes in HT29-ARID3B vs. HT29-vector control (HT29-Vec). Signals amplified by the ChIP primers. One representative experiment of three independent experiments is shown. D ChIP shows the occupancy of ARID3B and KDM4C on the regulatory region of the target gene in HCT15-sg3B versus HCT15-Ctrl. Signals amplified by the ChIP primers. One representative experiment of three independent experiments is shown. E ChIP for analyzing the enrichment of H3K9me3 on the regulatory region of target genes in HT29-ARID3B vs. HT29-vector control (HT29-Vec) cells is shown. Signals amplified by the ChIP primers. One representative experiment of three independent experiments is shown. F Sequential ChIP for analyzing the co-occupancy of ARID3B and KDM4C on the promoters of target genes in HT29-ARID3B vs. HT29-vector control (HT29-Vec). One representative experiment of three independent experiments is shown. See also Figure S5, Tables S10-11.
Fig 3: KDM4 inhibitors reverse ARID3B-induced target gene activation. A-C Left: Western blot for analyzing the expression of Notch intracellular domain (NICD) and HES1 in the HT29-ARID3B cells treated with the Notch inhibitor DAPT, Wnt inhibitor IWR1, and KDM4A/B inhibitor NSC636819 or KDM4C inhibitor SD70 for 24 h. The working concentration of DAPT was 16 μM, of IWAR1 was 64 μM, of WNT-59 was 4 μM, of SD70 was 16 μM, and of NSC636819 was 64 μM for 24 h. Among them, the SD70 inhibitor showed the most significant effect on suppressing the activation of ARID3B downstream targets. Right: RT-qPCR for analyzing the relative expression of intestinal stem cell genes and CD274 in the above conditions. Data represent the mean ± SD. n = 3 independent experiments (each experiment contained two technical replicates). **p < 0.01. *p < 0.05. See also Figure S7.
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