Methyl thiazolyl tetrazolium (mtt)

Glioma cells were cultured in 96-well dishes (JET BIOFIL, China) at a density of 4000 cells/well. After treating the cells under different conditions, 10 μl of MTT (5 mg/ml) was added to each well, and the cells were cultured for 4 h at 37 °C in the presence of 5% CO₂. Next, the culture medium was replaced with 150 μl of dimethyl sulfoxide. Cell viability was measured by recording the absorbance at a wavelength of 490 nm using a BioTek ELx800 (USA) microplate reader according to the manufacturer’s instructions. MTT (Cat# HY-15924), chloroquine (CQ; Cat# HY-17589A) and hyaluronic acid sodium (Cat# HY-B0633) were purchased from MedChemExpress, and 4-MU (Cat# M1381) was purchased from Sigma-Aldrich. The CD44 antibody (Cat# 217594-100ULCN) was purchased from Millipore.

Human intestinal epithelial cells were treated with TNF-α (10 ng/ml, 12 h) (96-300-01A-10, PeproTech) and then shRIP3 plasmid or 2 μM RIP3 inhibitor GSK-872 (CAS No. 1346546-69-7, MedChemExpress) for 24 h. 20 μl of 5 mg/ml MTT was added to each well. After 4 h of treatment with MTT (Beyotime Biotech, Shanghai, China), the supernatant was discarded, and 100μL of dimethyl sulfoxide (DMSO) was added. Optical density value (OD value) was detected at wavelengths of 492 nm and 630 nm, and the cell survival rate was calculated according to the formula: cell survival rate (%) = [experimental group OD average value-background group OD average value]/[Blank control group OD average value-background group OD average value] × 100%

The viability of the L-929 cell line (ATCC^® Manassas, VA, USA) was assessed using an indirect MTT (3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide) colorimetric assay (MedChemExpress, Monmouth Junction, NJ, USA). Extracts of fragmented biomaterials (HGO1, HGO2, BGO1 and BGO2) were prepared by depositing them on inserts for 24 h in a DMEM culture medium. Subsequently, these extracts, along with a positive control (CP, 0.1% Triton/DMEM), were added to cells seeded in 96-well plates at densities of 1 × 10⁴/well, 5 × 10³/well and 2.5 × 10³/well. The cells were then incubated at 37 °C, 5% CO₂ in the DMEM medium for 24, 48, and 72 h, respectively. At the end of the incubation period, the medium was removed and 1 mg/mL MTT (Sigma Aldrich^®, USA) was added. The absorbance of formazan was measured at 570 nm using a microplate scanning spectrophotometer (BioTek, Winooski, VT, USA). Cell viability was calculated in comparison to untreated cell culture (negative control, CN) using the formula
$$Cell viability \%=\frac{OD sample}{OD negative control}\cdot 100\%$$
where OD sample is the absorbance of the sample at λ = 570 nm (average of 5 replicates) and OD negative control is the absorbance of the negative control at λ = 570 nm (average of 6 replicates).
According to the current ISO 10993-5:2009(E) norm [56 ], a material that does not show cytotoxicity is one for which the cell viability is at least 70%

The following reagents were purchased from the indicated manufacturers: Lipofectamine 2000 (Invitrogen); puromycin (Thermo); Dual-specific luciferase assay kit (Promega); SYBR (BIO-RAD); polybrene (Millipore); MTT (MedChemExpress); ELISA kit for human IFN-β and TNF-α (4A Biotech); mouse antibodies against Flag and β-actin (Sigma), His and HA (OriGene), MyC (9B11), and IKKβ (8943S) (Cell Signaling Technology); rabbit monoclonal antibodies against HA, phosphor-IKKα (Ser176)/β (Ser177) (2078S) (Cell Signaling Technology), phosphor TBK1 (ab109272) and TBK1 (ab40676) (Abcam); and rabbit polyclonal antibodies against MAVS (Bethyl). HEK293, HeLa and HEK293T cells were purchased from ATCC. HEK293 cells stably expressing ACE2 (HEK293-ACE2) were obtained by lentiviral transduction. SeV (Cantell strain) was obtained as previously described.^{40 (link)} SARS-CoV-2 (IVCAS 6.7512) was isolated from BALF collected from a patient with viral pneumonia in December 2019 in Wuhan, China.^{41 (link)} Isolated viral particles were propagated in Vero E6 cells.^{42 (link)} HEK293-ACE2 cells were infected with SARS-CoV-2 in a biosafety level 4 (BSL-4) laboratory (Wuhan).

We took 19 patient samples and categorized them into “lower miR-34a level” group (with the relative miR-34a expression < 0.5, n = 8) and “higher miR-34a level” group (with the relative miR-34a expression > 1.5, n = 11), based on the RT-qPCR results as described below. Tumor samples were minced into small pieces (~0.5 mm in diameter) and placed on a collagen sponge gel in a 24-well plate. Samples were incubated with RPMI-1640 medium containing 20 μg/mL cisplatin or 0.4 μg/mL SN-38 (both from Sigma-Aldrich, Shanghai, China) for 7 days. After removal of culture medium, Hank's Balanced Salt Solution (Sigma-Aldrich) containing 5 mg/mL MTT (MedChemExpress, Shanghai, China) was added into each well, and the samples were incubated for another 8 h, followed by final spectrophotometry measured at 540 nm (Bio-Rad, Hercules, CA, USA). The inhibitory index was calculated according to a previous report [22 (link)]: the inhibitory index (%) = (1 − mean absorbance of treated tumor/g)/mean absorbance of untreated tumor/g.