Fig. 10: DHA interferes with transcription factor c-myc access to the PD-L1 promoter.

A Schematic diagram of the strategy to demonstrate the interaction of DHA and the promoter of PD-L1 (left panel). Oligo binding to DHA with the expected mobility shift on PAGE (SSGS, right panel). The transcription start site (TSS) is marked by the bent arrow. ATG; translation start code. B 10 pmol of synthetic DNA oligo PD-L1p8, PD-L1p9, and PD-L1p10 (60 mer/each) corresponding to the sequence on the promoter of PD-L1 incubated with DHA (1 µM) or EPA (1 µM) for 30 min at 37 °C. The oligos separated on 15% native PAGE and visualized with ethidium bromide. The red arrow indicates oligo migration shifted. C The shorter synthetic DNA oligos PD-L1p9A to PD- L1p9F (20 mer/each) correspond to the sequence of PD-L1p9. Representative PAGE for the oligos PD-L1p9B to PD-L1p9D with or without DHA. D Oligo PD-L1p9C incubated with gut supernatants (S) from hFB colonization mice without or with GELN treatment (S + GE). Representative SSGS showed the mobility shift of oligo PD-L1p9C and oligo mutant. Oligo PD-L1p9C mutant was used as the control. E Surface plasmon resonance (SPR) analysis of the interaction of biotin-labeled oligos PD-L1p9C and mutant PD-L1p9C-Mut with DHA (1 µM.) (P = 0.0062, two-way t test, n = 5). F The promoter sequences of PD-L1 and mutant inserted into a luciferase reporter p pEZX and transfection of microglia. Luciferase activity assessment 12 h after treatment with DHA (P = 0.0087, P = 0.6154, two-way t test, n = 5). G Representation of a 15% PAGE for the oligo PD-L1p9C, as well as mutants indicated in the figure. Each oligo contained a single base mutation. The size (kb) of the DNA length is indicated near the figure. The base in pink replaced by a different base, caused the abolishment of the DHA binding shift. H The sequence of mouse and human PD-L1 promoter indicating the distance from the TSS containing the DHA potential binding motif (pink) and c-myc binding site (box). I The analysis of luciferase activity for the c-myc KO B16F10 cells transfected with the luciferase plasmid containing mouse (m) and human (h) PD-L1 promoter sequence (P = 0.0005, P = 0.0023; P = 0.0065, P = 0.0078, two-way ANOVA test, n = 6). Treatment of DHA and/or recombinant c-myc protein is indicated in the figure. J 3D predicted structures of the interaction between DHA and oligo mPD-L1p9C or hPD-L1p (G- Red; T- Yellow; C- Green; A- Cyan) at position G-4 and G-11 by hydrogen bond respectively. K SPR analysis of the interaction between c-myc protein and biotinylated oligo PD-L1p9C covalently immobilized onto the sensor chip with or without DHA (P = 0.0002, two-way t test, n = 3). L Biotinylated oligo PD-L1p9C (WT) and mutant transfected into microglia for 12 h and incubated with gut supernatant for an additional 6 h. Metabolite analysis with LC-MS after pull-down with streptavidin beads (P = 0.0022, two-way t test, n = 6). Data are representative of three independent experiments. The box & whisker plots with the box representing the SD, the middle line within the box representing the median, and the bars representing the range of minimum and maximum. Source data are provided as a Source Data file.