Fig 1: Hypoxia induces epigenetic inactivation of immune effector expression.a RT-qPCR analysis assessing IFNG expression in T/NK cells in an epigenetic-drug screening. Both T cells and NK cells were cultured under 1% O2 with indicated treatments. Data were presented as the log2 fold change of IFNG mRNA level normalized to vehicle control, mean ± SD of technical triplicates, representative of two independent experiments (n = 2). b, c Representative histograms (left panel) and flow cytometric quantifications (right panel) of IFN? expression in human CD8+ T cells (b n = 4) and NK cells (c n = 3) with indicated treatments. Quantification data were presented as the mean ± SD of samples from three to four donors. P values were determined by two-way ANOVA with Turkey’s test. d ChIP-qPCR analysis of HDAC1, HDAC2, HDAC3, EZH2, and SUZ12 occupancy on IFNG promoter of human T cells. Four primers were designed to span the promoters of IFNG, with P1 at -1448 to -1354b, P2 at -707 to -628b, P3 at -257 to -171b, P4 at +350 to +461b, relative to TSS. For ChIP analysis of EZH2 and SUZ12 occupancy, RPL30 serves as the negative control and CCND2 as the positive control. e, f ChIP-qPCR analysis of H3K27ac and H3K27me3 enrichment on IFNG promoter of human T cells under indicated conditions. All ChIP-qPCR data (d–f) are presented as fold enrichment relative to IgG and expressed as mean ± SD of technical triplicates, representative of two independent experiments (n = 2). For ChIP-qPCR data of d, e, statistics were performed to analyze bindings of indicated markers across different sites in IFNG promoter (RPL30 and CCND2 excluded) between hypoxia and normoxia. P values were determined by two-way ANOVA analysis. g RT-qPCR analysis of human T cell with indicated gene knockdown. Data were presented as the fold change of mRNA level normalized to the control group under normoxia (1% O2), mean ± SD of technical triplicates, representative of two independent experiments (n = 2). Source data are provided as a source data file.
Fig 2: TNF α does not alter Sp3 binding to the AQP3 promoter in HT‐29 cells. Chromatin immunoprecipitation (ChIP) was performed on HT‐29 cells treated with TNF α (25 ng/mL) for 4 h. ChIP experimental success was verified using IP of Histone H3 bound to the Ribosomal protein L30 (RPL30, 160 bp) promoter in untreated samples (A). Sp3 antibody was used to IP chromatin, with an equivalent amount of rabbit IgG isotype antibody used as a negative control for nonspecific IP, and PCR was used to amplify the AQP3 promoter (AQP3, 101 bp) from the precipitate (B). Two percent of the total input into the ChIP reaction was used as a loading control to assess the equivalence of sample input, whereas a no template control (NTC) was used to ensure specific signal amplification in the PCR reaction. Relevant molecular weight markers from a 1 kb ladder (L) are shown on the left of the gels. A representative image for the ChIP results obtained is shown (n = 3).
Fig 3: Pulldown/binding assays for ZIC2-ABCC4(A) ChIP RTPCR to show the enrichment of ABCC4 promoter region using ZIC2 pulldown. H3 pulldown for enrichment of RPL30 exon 3 was used as a positive control and anti-rabbit IgG was used as a negative control. Three independent set of experiment was performed. (B) Mobility shift assay, confirming the binding of ZIC2 at ABCC4 promoter region. (C) ChIP-PCR under PCAT92 knockdown a reduction in the enrichment of ABCC4 promoter region was observed indicating a possible interaction between PCAT92 and ZIC2 to help the later in forming a stable complex at the promoter region. H3 pulldown for enrichment of RPL30 exon 3 was used as a control. The bars in the graph are plotted based on the band intensity observed in the immunoblot gel. Three independent set of experiment was performed. (*** P<=0.001, ** P<=0.01 and ns P>0.05).
from Cell Signaling Technology for SimpleChIP ® Human RPL30 Exon 3 Primers