P1585 1mg

For intracellular cytokine staining, cells were incubated for 4 hours in a humidified incubator with 5% CO₂, in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (15140-122, lot 1622132; Thermo Fisher Scientific) with 50 ng/mL phorbol 12-myristate-13-acetate (PMA) (P1585-1MG, lot SLBN5870V; Sigma-Aldrich), 500 ng/mL ionomycin (10634-1MG, lot 127M4002V; Sigma-Aldrich), 50 ng/mL recombinant mouse IL23 (14-8231-63, lot 4299410; eBioscience), and GolgiStop (51-2092KZ, lot 6308707; BD Bioscience). After incubation, FcγR blocking and surface antigen staining were performed as described earlier. After that, dead cells were stained with Fixable Viability Dye eFluor 780 (65-0865-14, lot 4333604; eBioscience) for 30 minutes at 4°C and protected from light. The cells were washed and incubated with fixation/permeabilization solution (00-5123-43, lot 1954346 and 00-5223-56, lot 4333317; Invitrogen) overnight at 4°C protected from light. Then intracellular cytokine was stained by fluorescence-conjugated monoclonal antibodies incubated for 30 minutes at 4°C and protected from light. The incubated cells were washed and analyzed by flow cytometry. For staining of Foxp3-positive cells, the same procedure but without stimulation with PMA, ionomycin, and IL23, was performed.

THP-1 cells used in this study are an authenticated cell line purchased from ATCC (TIB-202; human male). THP-1 cells were cultured at 37°C in 5% CO₂ in RPMI1640 (Gibco, 11875093) supplemented with 10% FBS (VWR) and 1% antibiotics (Gibco, 15240062). THP-1 cells were differentiated to macrophage-like cells by incubating them overnight in complete media containing 200 ng/mL PMA (Sigma-Aldrich, P1585-1MG), followed by 3 d incubation in fresh media without PMA. The resulting differentiated cells were stimulated with 200 ng/mL LPS (Sigma-Aldrich, L4391) and collected at four time points: 0 h post-stimulation (no stimulation), and 1, 2, and 4 h post-stimulation. Total RNA was extracted with TRIzol (Invitrogen, 15596018). To inhibit transcription, THP-1 cells were incubated in media with 10 µg/mL actinomycin D (Sigma Aldrich, A9415) for 15 min prior to stimulation with LPS.

Human monocytic THP‐1 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a 37°C incubator under 5% CO₂ in RPMI 1640 medium (Thermo, 11875093) supplemented with 10% foetal bovine serum (Thermo, 10099141C). To induce differentiation of macrophages, the cells were cultured in the presence of phorbol 12‐myristate 13‐acetate (100 ng/mL, Sigma, P1585‐1MG) for 24 hours. Lipopolysaccharide (100 ng/mL; SIGMA, L4391‐1MG) was applied for 24 hours to induce the differentiation of macrophages into an M1‐like phenotype. Interleukin 4 (20 ng/mL; PeproTech, 200‐04‐20) and interleukin 13 (20 ng/mL; PeproTech, 200‐13‐20) were applied for 24 hours to induce the differentiation of macrophages into an M2‐like phenotype. MGO (Sigma, M0252‐25ML) at a concentration of 10 μM was used to reflect the estimated MGO levels in the plasma of T2DM patients. PM (Sigma, P9158‐1G) at a concentration of 100 μM was used to quench MGO. MGO (10 μM) or MGO (10 μM) + PM (100 μM), using H₂O as a control, was applied to THP‐1‐derived macrophages in serum‐free RPMI 1640 for 24 hours.

K562s were cultured in RPMI media (Corning 10-040-CV) with 10% fetal bovine serum (FBS) (Gibco 26140079) and 1% penicillin-streptomycin (PS) (Gibco 15140122). For megakaryocyte differentiation, K562s were plated in either 6-well plates (RNA-seq, ATAC-seq) or T-175 flasks (Hi-C, CUT&RUN) at a density of 1 × 10⁵ cells/mL and treated with 25 nM PMA (Sigma-Aldrich P1585-1MG). After 24 h, the cells become semi-adherent. Cells were provided with fresh media and PMA after 24 h and 48 h. Cells were collected without treatment or after 6 or 72 h. For all treatments and library preparations, K562s were thawed and immediately split into two T-25 flasks to create biological replicates.