Fig 2: MIF activation of Class IB PI3Ks expression in HL60 neutrophil‐like cells. (A) Western blots show the expression of phosphorylated‐p85, total p‐85, p110α, p110β, p110γ and p101 monitored from 10 to 180 min. after the MIF activation of HL60 cells. All controls were measured at t = 0 without MIF activation. (B–D) Bar diagrams show fold change in intensity for p‐p85, p110γ and p101 respectively. The intensity data were analysed by ImageJ software and normalized to the same α‐tubulin as shown in A. The data presented here are representative sets from three independent experiments.

Fig 3: The chromatin landscape of activated B cell is modified by Zbtb18. (A) Principal components 1 and 2 (PC1 and PC2) ANOVA stabilized (VST) counts at differentially accessible peaks (FDR < 0.05) in control (RFP+ B220+ CD138− cells) and Zbtb18-overexpression groups (GFP+ B220+ CD138− cells) sorted by flow cytometry. Transduced cells were sorted on day 0 and day 2 after being cocultured in the presence of anti-CD40 and IL-4. (B) Number of differentially expressed genes at day 0 and 2 of B cells transduced with control or Zbtb18. (C) Heatmap of differentially accessible peaks color coded as in (A). Data are row scaled, and hierarchical clustering is based on Manhattan distances. (D) Heatmaps (top) of ATAC-seq read coverage (fragments per kilobase per million [FPKM]) at a 5-kb window centered over differentially accessible peaks. (E) Gene ontology analysis of genes that lose (blue) or gain (red) accessibility after Zbtb18 overexpression on day 2. (F) Normalized ATAC-seq coverage tracks (FPKM) for representative genomic regions at Pik3r5, Pik3r6, and Pik3r1, differentially accessible peaks highlighted by shading. Read density tracks from pooled replicates of one experiment performed (n = 2 for day 0, n = 3 for day 2).

Fig 4: Inhibition of Class I PI3Ks expression by MIF active site inhibitors. (A) Each inhibitor was dissolved in DMSO and was incubated with MIF for different length of time prior to HPP tautomerase enzyme assay. As a negative control, DMSO alone was incubated with MIF. The enzyme activity data shown here are a representative set among three repeats with triplicates. (B) Phosphorylation status of p85 was examined in THP‐1 cells after 180 min. of pre‐incubation with MIF in the absence and presence of active site inhibitors. Reduction in p110γ (C) and p101 (E) subunits expression by MIF inhibitors in THP‐1 cells was examined. Reduction in p110γ (D) and p101 (F) subunits expression by MIF inhibitors in HL60 cells was examined.

Fig 5: Zbtb18 directly binds and suppresses expression of PI3K class I genes. (A) Splenocytes were transduced with control (RFP+) or Zbtb18 (GFP+) vectors. Cells were analyzed by flow cytometry and mixed to achieve a 1:1 ratio of B220+-GFP+– and RFP+-expressing cells, followed by stimulation with anti-CD40 and IL-4. Two days after stimulation, RFP+ or GFP+ B220+ CD138– live cells were sorted by flow cytometry and subjected to Chip qPCR analysis of the ATAC-seq differential peaks of Pik3r1, Pik3r5, and Pik3r6 genes. Data are from pooled samples from three independent experiments performed (total n = 3). (B) De novo motif–enrichment analysis in repressed ATAC-seq peaks of Zbtb18 versus control (Ctrl) cells. Motif density distribution relative to the peak summit for Zbtb18, Pu.1 and Oct2 motifs in the repressed ATAC-seq peaks (bottom). (C) Chip qPCR analysis of interactions between Myc-tagged Zbtb18 (WT), or ΔZF with the indicated genes. Ctrl B cells transduced with empty GFP-expressing vector. Conserved Zbtb18-binding motif identified in the ATAC-seq peaks of the indicated genes are shown in red. Analysis was performed on transduced sorted cells (live B220+ GFP+ cells) 2 d after anti-CD40 and IL-4 stimulation. Data show pooled samples collected in three independent experiments performed (total n = 3, biological replicates). (D) Immunoblot analysis of p85a, p101, and GAPDH in Ctrl/RFP– or Zbtb18/GFP–transduced B cells 2 d after being cocultured with or without anti-CD40 and IL4 stimulation (left) [as in (A) above]. Cells were sorted prior to analysis on day 2 (gated on live B220+ CD138− GFP+ or RFP+ cells). Quantification of p85a or p101 expression normalized with GAPDH (right). Figure shows combined data collected in two independent experiments performed. Each circle represents data from one experiment. (E) qPCR analysis of the indicated genes in control- and Zbtb18-transduced B cells, sorted (as GFP+ B220+ CD138− live cells) 2 d poststimulation with anti-CD40 and IL-4. Expression of mRNA is presented relative to the abundance of GAPDH. Data in (E) show the results from one representative experiment out of three independent experiments performed. Each circle represents a technical replicate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.