Streptomycin solution

L. donovani (MHOM/SD/001S-2D) promastigotes were cultured as described previously [78] . Hamsters were infected by intracardial injection of 10⁶ peanut agglutinin purified metacyclic promastigotes [78] . For in vitro infections, stationary phase promastigotes were washed with PBS and used immediately to infect hamster BMDMs. Cells were infected at a promastigote to macrophage ratio of 2∶1 and cultured thereafter in complete medium (CM) composed of DMEM supplemented with 1 mM sodium pyruvate (Gibco), 1× MEM amino acids solution (Sigma), 10 mM HEPES buffer (Cellgro), and 100 IU/mL penicillin/100 mg/mL streptomycin solution (Cellgro), which was supplemented with 2% heat inactivated fetal bovine serum (HIFBS). When infecting BMDMs at this ratio all parasites were internalized so that no extracellular parasites could be observed by light microscopy at 24 hrs post-infection.

Hamsters were sacrificed at 28 days post-infection by CO₂ inhalation and the spleens were collected in complete DMEM (Gibco), supplemented with 2% fetal bovine serum (FBS), 1 mM Sodium pyruvate (Gibco), 1X MEM amino acids solution (Sigma), 0.02% v/v/ EDTA, 10 mM HEPES buffer (Cellgro) and 100 IU/mL Penicillin/100mg/mL Streptomycin solution (Cellgro). Adherent spleen cells were isolated after infiltrating the whole organ with the injection of 2 mL of Collagenase D (Sigma) at 2 mg/mL. The spleen tissue was cut into small pieces and incubated for 20 minutes at 37°C resuspended in the enzyme solution. The cell suspension and remaining tissue fragments were suspended in culture medium and gently passed using a syringe plug through a 100 μm cell strainer (B-D) to obtain a single cell suspension. Cells were cultured in a 75cm flasks and were allowed to adhere for 4 hr at 37°C and the non-adherent cells were removed after washing the monolayers with pre- warmed PBS (10 times), before the RNA was isolated.

Rhein (batch No. 5045, purity >98.0%) was purchased from Shanghai Standard Biotech Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), 0.25% trypsin, penicillin, and streptomycin solution were obtained from Corning (Corning, NY, USA). RPMI 1640 medium, dimethyl sulfoxide (DMSO), PBS, and MTT were purchased from Solarbio (Beijing, China). The LDH Assay Kit, Annexin V-FITC Apoptosis Assay Kit, DAPI Assay Kit, MMP Assay Kit, ROS Assay Kit, and Cell Cycle Assay Kit were obtained from Beyotime (Nanjing, China). Reduced glutathione (GSH) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against Fas (#4233S), Bax (#5023T), Bcl-2 (#15071), p53 (#2524T), p21 (#2947T), cyclin A (#4656T), CDK 2 (#2546T), cleaved caspase-3 (#9661T), cleaved caspase-9 (#9501T), cytochrome c (#4280T), and PARP (#9542T) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against caspase-8 (#ab25901) was purchased from Abcam (Shanghai, China) (#ab25901).