Penicillin streptomycin

HCT-8 cells were preserved and propagated in our laboratory. HCT-8 cells were cultured with RPMI 1640 medium (Biological Industries, Israel) containing 10% FBS and 1% penicillin streptomycin (Biological Industries, Israel) at 37 °C in 5% CO₂. When grown to 70–80% confluence, the cells were subjected to passaging or plating.
Cryptosporidium parvum (gene subtype: IIa A15G2R1) was preserved and propagated in our laboratory. Cryptosporidium oocysts, originally obtained from our laboratory, were used to infect 5-day-old Holstein calves (1×10⁸/animal). Following the onset of oocyst shedding, feces were collected daily, mixed with an equal volume of 5% potassium dichromate and stored at 4 °C. Oocyst isolation was through discontinuous sucrose gradients according to previous methods [33 (link)]. For purification of sporozoites, oocysts were resuspended in 0.8% sodium taurocholate (Sigma, MO, USA) with 2.5% trypsin (BOSTER, Wuhan, China), then incubated at 37 °C for 30 min, washed three times in PBS and resuspended in RPMI 1640 medium with 2% FBS and 1% penicillin-streptomycin. The excystation rate of sporozoites is about 70% using a cell counting plate, and the number of oocysts required is calculated as a ratio of 1:2 of sporozoites to HCT-8 cells.

NCTD, obtain from Selleck Chemicals (Houston, United States) and dissolved in DMSO. Alpha modification of Eagle’s medium (a-MEM) and penicillin/streptomycin were gained from Boster Bio (Wuhan, China), while fetal bovine serum (FBS) was purchased from Gibco-BRL (Sydney, Australia). Primary antibodies against ERK, JNK, p38, phosphorylated p-ERK, p-JNK, p-p38, IkBa, p-IkBa, AKT, p-AKT, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC) and GAPDH were supplied from Cell Signaling Technology (Cambridge, MA, United States); while primary antibodies specific for NFATc1 and Cathepsin K were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

Human embryonic kidney 293T (HEK-293T) cells were cultured in DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin (1:100, Boster, China) at 37°C in a humidified 5% CO₂ incubator. HEK-293T cells were transfected with wild-type or mutant AVPR2 plasmids or empty vector using Lipo3000 Reagent (Invitrogen, USA) according to the manufacturer's instructions.

293T and U251 cells were obtained from ATCC and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hycolone, Watham, MA, USA) supplemented with 10% fetal bovine serum (FBS, TransGen, Beijing, China) and 1% Penicillin/Streptomycin (BOSTER, Wuhan, Hubei, China).

Isoliquiritigenin (ISL, purity≥98%) and sodium caseinate (Cas, purify≥90%) were purchased from Adamas (Shanghai, China). The zein was provided by Tokyo Chemical Industry (Tokyo, Japan). Dextran sulfate sodium (DSS, 36–50 kDa) was offered by Seebio Biotechnology Co., Ltd. (Shanghai, China). DiR iodide (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide) was purchased from Meilunbio^® (Dalian, China). Cell culture supplies, including Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM) medium, RPMI 1640 medium, and fetal bovine serum provided by Gibco (United States). Penicillin-streptomycin was supplied by Boster Biological Technology Co., Ltd. (United States). Trypsin was obtained from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) kits were purchased from MULTI SCIENCES (LIANKE) BIOTECH Co., Ltd. (Hangzhou, China). Detergent Compatible Bradford Protein Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China).

HEK293T were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific), supplemented with 10% (v/v) fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Boster Biological Technology Co. Ltd.), and maintained at 37°C with 5% CO₂. Cells were seeded onto 96-well plate (BIOFIL). Twenty-four hours after seeding, cells at a confluence of ∼70–80% were transfected with 0.7 ul of Transeasy™ (Forgene) and 276 ng of PE2 plasmid DNA, 62 ng of pegRNA1 plasmid DNA and 62 ng of pegRNA2 plasmid DNA (for Bi-PE and Cas9 nickase transfections); 276 ng of PE2 plasmid DNA, 93 ng of pegRNA plasmid DNA and 31 ng of sgRNA plasmid DNA (for PE3 transfections); or 276 ng of Cas9 plasmid DNA and 62 ng of sgRNA 1 plasmid DNA and 62 ng of sgRNA2 plasmid DNA (for paired Cas9 nuclease and Cas9 nickase transfections), according to the manufacturer's protocol.