Methyl thiazolyl tetrazolium (mtt)

The raw material of jujube was produced from Xinjiang, China, and was purchased from Tai'an Market. The jujube with neat fruit shape was first soaked in water for 1 hr, then bagged and sealed, and then processed in a drying oven with a temperature of 75°C and a humidity of 80% for about 55 hr to produce blackened jujube, and the blackened jujube with a water content of about 26%, a color of dark brown with a sweet and sour taste. After blackening, they were cut into a fine flap, dried in the oven at 60°C, then ground to fine powders and sifted 40 mesh, put into the sealed bag for standby.
Standard oleanolic acid (purity ≥98%, HPLC) was provided by Shanghai Yuanye Biotechnology Co., Ltd. Macroporous resins including HPD‐100, X‐5, S‐8, D‐101, and AB‐8 were acquired from Cangzhou Baoan Technology Co., Ltd. MTT and penicillin–streptomycin (PS) were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. Hoechst 33258 reagents kit was bought from Nanjing Kaiji Biotechnology Co., Ltd. DMEM high glucose, fetal bovine serum (FBS), and dimethyl sulfoxide (DMSO) were purchased from Hyclone Company and Sigma‐Aldrich, respectively. All other reagents were of analytical grade, and all experiments were conducted with deionized water.

Cells were seeded into 96-well plate with a concentration of 3000 cells per well, and incubated at 37°C 4 days post infection. The number of viable cells was measured at daily intervals (hour 24, 48, 72 and 96). At each time point, 10 μl of 5 mg/ml MTT (Dingguo Biotechnology) was added, and incubation was continued for 4 h. Then the medium was removed carefully and 150 μl of DMSO was added at the end of incubation. The absorbance was measured at 592 nm on the spectrophotometer. For the colony formation assay, a total of 200 cells were seeded in 6-well plates in triplicate and maintained in the complete medium for 10 days. The natural colonies were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The colonies were then stained with Giemsa for 10 min, washed with water and air-dried. The total number of colonies with more than 50 cells was counted under fluorescence microscopy.

Cell growth was detected with cellomics high content screening (HCS). After transfection with either RPL34-siRNA lentivirus or NC-siRNA lentivirus, Saos-2 cells in exponential phase were seeded in 96-well plates with 1 × 10³ cells per well and then were cultured at 37 °C with 5% CO2 for 5 days. The Cellomics^®ArrayScan^® VTI HCS reader (Thermo Scientific™, Waltham, US) was used to scan the plates at the same time each day. The amount of cells expressing GFP was quantified with HCS Studio™ 2.0 Cell Analysis Software (Thermo Scientific™, Waltham, US). The cell growth curve was drawn for each group. For MTT assay, Saos-2 cells were collected 10 days after transfection with either RPL34-siRNA lentivirus or NC-siRNA lentivirus and then were cultured in 96-well plates at 2 × 10³ cells per well, followed by incubation at 37 °C with 5% CO2 for 5 days. 10 ul MTT (5 mg/ml, Beijing Dingguo Changsheng Biotechnology Co.Ltd, Beijing, China) was added to each well. The cells were incubated in the dark for 4 hours and then the medium was removed before adding 100 ul DMSO (Sinopharm Chemical Reagent Co.Ltd, Shanghai, China) to each well. The optical density was measured at 490 nm by Biotek Elx800 ELIASA (BioTek, Winooski, US).