Decitabine

From September 2016 to June 2019, a total of 37 newly diagnosed MDS patients in the Hematology Department of the General Hospital of Tianjin Medical University were enrolled in the study. The study included 22 males and 15 females with a median age of 61 years (range 27–79 years) (details in Table 1). The patients were divided into two groups based on the revised International Prognostic Scoring System (IPSS-R), the relative low risk group (IPSS-R score less than or equal to 3.5, n=18) and the relative high risk group (IPSS-R score more than 3.5, n=19). The low risk MDS patients were treated with Recombinant Human Erythropoietin (Sansheng, China) and lemalidomide (BeiGene, China) (only for 5q- patient). The high risk MDS patients were treated with decitabine (Janssen, China).
Twenty-three healthy people were selected as controls in this study, including 13 men and 10 women with a median age of 52 (range 24–74 years).
The study was approved by the Ethics Committee of the General Hospital of Tianjin Medical University. Informed written consents have been obtained from all patients and control groups or their guardians according to the Helsinki Declaration.

The mouse CRC cell line CT26 was preserved by our laboratory and cultured in RPMI-1640 medium (PAN, Wimborne, UK) supplemented with 10% fetal bovine serum (Biowest-Uruguay, Nuaillé, France). Decitabine (DAC) was purchased from Janssen Pharmaceutical Ltd., Xian. An anti-PD-1 antibody (clone: J43) was kindly provided by Prof. Lianjun Shen from Hengrui Pharmaceutical Co. Ltd., Shanghai. Cytidine (Sigma, MO, USA) was purchased from Univ Biotech Co., Ltd. as a control drug.
CT26 cells were seeded at a density of 1 × 10⁵ cells/ml in a six-well culture plate. After 24 h, the medium was replaced with complete medium containing 1 μM DAC every 24 h for 3 days. After the third DAC treatment, the medium was replaced with fresh complete medium without DAC, and the cells were cultured for an additional 24 h and then used for subsequent experiments. Control groups were treated synchronously under the same conditions except for the absence of DAC.

MEDS433 and brequinar were synthesized as described in [4 (link)]. ATRA, Teriflunomide, Dilazep and uridine were purchased from Sigma-Aldrich. Reagents were dissolved in DMSO and diluted in culture medium before use. Final DMSO concentration did not exceed 0.1%. Dilazep was dissolved in water. Ara-C (Citarabina Hospira, Lake Forest, IL, USA), Idarubicin (Zavedos, Pfizer, New York, NY, USA), Dipyridamole (Persantin, Boehringer Ingelheim, Ingelheim am Rheim, Germany), Decitabine (Dacogen, Janssen-Cilag, Beerse, Belgium) were purchased.