Fluorouracil 5 fu

Azoxymethane (Sigma Chemical Co., St Louis, MO, USA) was dissolved in sterile 0.9% normal saline and was given subcutaneously at 15 mg/kg body weight once a week for 2 weeks to induce ACF35 (link). Fluorouracil (5-FU) (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in 0.9% normal saline and was administered at a concentration of 35 mg/kg body weight intraperitoneally twice a week36 (link).

A431 and MDA-MB-231 cells were purchased from ATCC (VA, USA) and cultured in DMEM (Thermo Fisher Scientific, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific) at 37 °C with 5% CO2. CKI (total alkaloid concentration of 26.5 mg/ml) was provided by Zhendong Pharmaceutical Co.Ltd (China) and used at a final concentration of 1 mg/ml. Fluorouracil (5-Fu) and doxorubicin were purchased from Sigma-Aldrich (MO, USA) and used at final concentrations of 10 ug/ml and 1 ng/ml, respectively. MYD88 inhibitor and control peptides were synthesised by GenScript (Hong Kong, China) with the following amino acid sequences with purity >95%^{29 (link),30 (link)}; inhibitor: DRQIKIWFQNRRMKWKKRDVLPGT and control peptide: DRQIKIWFQNRRMKWKK.
For all in vitro assays 6-well or 96-well plates were used. The seeding density for both A431 and MDA-MB-231 cells was 4 × 10⁵ cells/well for 6-well plates. For 96-well plates, A431 cells were seeded at 8 × 10⁴ cells/well and MDA-MB-231 cells were 1.6 × 10⁵ cells/well. After seeding, cells were cultured overnight before being treated.

The colorectal adenocarcinoma cell lines HCT-116 (p53 wild type) and HCT-116 p53 null (Horizon Discovery Ltd., Cambridge, UK) were grown under standard conditions (37 °C and 5% CO₂ in a humid atmosphere) using RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco; Invitrogen, Carlsbad, CA, USA). For the experiments that required cell synchronization, cells were cultured in serum-free medium for 6 h prior to the initiation of the experiment [1 (link)]. Next, the cells were cultured in media with serum in the conditions required for the experiments carried out.
Melatonin, agomelatine, fluorouracil (5-FU), and the remaining reagents employed in this study were obtained from Sigma-Aldrich (Sigma Chemical Co., St。 Louis, MO, USA).

NPECs were immortalized by transfecting Bmi-1 as described previously and were cultured in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA, USA)^{31 (link)} and labeled NPEC1 Bmi-1 and NPEC2 Bmi-1. NPECs N01-N09 were derived from primary cultures of fresh nasopharyngeal tissues. NPC cell lines including CNE1, CNE2, 6-10B, 5-8 F, HONE1, SUNE1 and HNE1 were obtained and cultured in RPMI-1640 medium (Gibco, USA) with 10% fetal bovine serum (Gibco) as described in our laboratory previously.^{23 (link), 31 (link)} All of the above NPC cell lines were cultured in 37 °C with 5% CO₂. Cisplatin (DDP) and fluorouracil (5-FU) were purchased from Sigma (Shanghai, China).

Human breast carcinoma cells (luminal types: MCF7, T47D, and ZR75; basal types: MDA-MB-231 and HS578T) were obtained from the Bioresource Collection and Research Center (BCRC; https://www.bcrc.firdi.org.tw/12092013 (accessed on 30 May 2022)). Cells were maintained in DMEM (Gibco) with 5% CO₂ at 37 °C in a humidified incubator. All cell culture media were supplemented with 10% FBS (Biological Industries) and 1% PSA (penicillin G/streptomycin/amphotericin B; Biological Industries). Kinase inhibitor wortmannin (20 μM; Sigma) and kinase activator SC79 (5 μM; Sigma) were used to investigate the Akt signaling pathway. Three chemotherapy drugs, fluorouracil (5FU) (Sigma), paclitaxel (Sigma), and doxorubicin (Sigma), were used to study chemoresistance in CD44-overexpressing and knockdown cells.

PBS or Clodronate loaded liposomes (Liposoma B.V., Amsterdam, The Netherlands, ClodronateLiposomes.org) were injected retro-orbital at 10 ml/g mouse and animals sacrificed 36 h later^{10 (link)}. Fluorouracil (5-FU) (Sigma-Aldrich Corp., St. Louis, MO) was injected intraperitoneal at a dose of 150 mg/kg and animals sacrificed 4 days later^{81 (link)}.