Daudi, SUDHL-4 and JeKo-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 2 mM glutamine in a humidified incubator with 5% CO₂ and 95% air.

Cell viability was assessed by MTT assay. Daudi, SUDHL-4 and JeKo-1 cells were plated in 96-well plates at a density of 2×10³ cells per well containing 100 µl culture medium. ADR (1 mg/ml) or VCR (1 mg/ml) was added to 96-well culture plates with 10 µl per well and incubated for 72 h, followed by incubation with MTT. The control cells were treated only with PBS prior to incubation with MTT. Dimethyl sulfoxide was used to dissolve the formazan. The relative cell viability was calculated by measuring the absorbance value/well at 570 nm and using the following equation: Mean optical density (OD) of treated cells/mean OD of control cells.

The lentivirus particles of shATG5 LV-ATG5-RNAi (9513-1) were purchased from GENECHEM. The sequence for the synthesized shRNA targeting S100A8 was 5′-CCUGAAGAAAUUGCUAGAGTT-3′. The annealed oligonucleotide fragment was cloned into the lentivirus plasmid PLL3.7 (Addgene, Inc.) to establish the shRNA lentiviral vector. Recombinant lentiviruses expressing S100A8 shRNA or negative control (Shanghai GenePharma Co., Ltd.) were obtained by plasmid transformation. VSVG, pMDLg/pRRE, RSV-REV (Addgene, Inc.) and Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.) were used for lentivirus packaging in 293T cells. The supernatant of the 293T culture was collected and filtered using a 0.22-µm filter (EMD Millipore), followed by a concentration step. Medium containing lentivirus was stored at −80°C for future use.

Total RNA was isolated using TRIzol^® (Takara Bio, Inc.) and reverse-transcribed into cDNA using PrimeScript RT Master Mix (Takara Bio, Inc.). according to the manufacturer's instructions. RT-qPCR was performed using SYBR Green Real-time PCR Master Mix kit (Takara Bio, Inc.), according to the manufacturer's instructions. The following PCR conditions were used on the Light Cycler: 39 cycles at 95°C for 30 sec, 95°C for 5 sec, followed by 60°C for 30 sec in a 10-µl reaction volume. The relative expression was normalized to that of GAPDH, which was used as the internal control. The primers used were as follows: ATG5, 5′-CTCTGCAGTGGCTGAGTGAA-3′ forward and 5′-TCAATCTGTTGGCTGTGGGA-3′ reverse; S100A8, 5′-CCGAGCTGGAGAAAGCCTTG-3′ forward and 5′-AGGTCATCCCTGTAGACGGC-3′ reverse; GAPDH, 5′-AGAAGGCTGGGGCTCATTTG-3′ forward and 5′-AGGGGCCATCCACAGTCTTC-3′ reverse.

Cells were lysed in SDS lysis buffer (P0013G; Beyotime Institute of Biotechnology) containing 1% protease inhibitor PMSF (ST506; Beyotime Institute of Biotechnology). The extracted protein concentration was determined using the BCA protein assay kit (P0012S; Beyotime Institute of Biotechnology) and stored at −80°C. A total of 20 µg of each protein sample was separated by 12% SDS-PAGE and transferred to PVDF membranes (IPVH00010; EMD Millipore). Following blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with appropriate primary and secondary antibodies. Proteins were detected using an enhanced chemiluminescence kit (6305; Bio-Rad Laboratories, Inc.) under a gel electrophoresis imager (Bio-Rad Laboratories, Inc.). The primary antibodies for LC3 (source, rabbit; cat. no. ab192890; dilution, 1:1,000), P62 (cat. no. ab207305; source, rabbit; dilution, 1:2,000) and calnexin (source, rabbit; cat. no. ab13504; dilution, 1:2,000) were purchased from Abcam. The primary antibodies for B-cell lymphoma 2 (BCL2; cat. no. 12789-1-AP; source, rabbit; dilution, 1:500), BECN1 (cat. no. 11306-1-AP; source, rabbit; dilution, 1:500), PI3KC3 (cat. no. 20584-1-AP; source, rabbit; dilution, 1:100) and tubulin (cat. no. 10068-1-AP; source, rabbit; dilution, 1:2,000) were purchased from ProteinTech Group, Inc. The primary antibody for S100A8 (cat. no. HPA024372; source, rabbit; dilution, 1:500) was purchased from Merck KGaA. The primary antibodies for manganese-dependent superoxide dismutase 2 (cat. no. MAB3419; source, mouse; 0.5 µg/ml) and that for BNIP3 (cat. no. MAB4147; source, mouse; 0.5 µg/ml) were purchased from R&D. The secondary antibody for anti-rabbit (cat. no. SA00001-2; dilution, 1:2,000) was purchased from ProteinTech Group, Inc. The secondary antibody (anti-mouse; cat. no. SA00001-1; dilution, 1:2,000) was purchased from ProteinTech Group, Inc. The intensity of the western blot bands was quantified using ImageJ software (version 1.47; National Institutes of Health,). Only the blots with band intensity within the linear range were included.

Adenovirus expressing mCherry-GFP-LC3 fusion protein (Ad-mCherry-GFP-LC3, C3011) was purchased from Beyotime Institute of Biotechnology. Cells were plated in 6-well plates and allowed to reach 50–70% confluence at the time of Ad-mCherry-GFP-LC3 transfection. Adenoviral infection was performed according to the manufacturer's instructions. The presence of mRFP-LC3 puncta indicated the autolysosomes in red fluorescent images.

Cells were harvested and centrifuged at 1,000 × g for 10 min at room temperature. The cell pellet was fixed with 4% glutaraldehyde for 24 h at 4°C and 1% osmium tetroxide for 2 h at 4°C. Thereafter, the cell pellet was dehydrated in a graded series of alcohol and acetone, followed by embedding in Epon 816 (Electron Microscopy Sciences). Ultrathin sections were cut by a Leica ultramicrotome (Leica Microsystems, Inc.) and stained with uranyl acetate for 4 min and followed by lead citrate for 2 min at room temperature. The exact thickness of the ultrathin section of the cell samples under the transmission electron microscope was 40 nm. TEM was conducted using a JEM-1400Plus transmission electron microscope (JEOL, Ltd.). The mean number of autophagosomes in tumour cells was counted and quantified.

Cells were lysed at 4°C in ice-cold lysis buffer (Beyotime Institute of Biotechnology) and the cell pellet was prepared by centrifugation (12,000 × g for 10 min at room temperature). Immunoprecipitation was performed using anti-BECN1 antibody, which was captured with protein A+G agarose beads (Beyotime Institute of Biotechnology). The bead-bound proteins were then eluted by boiling in SDS sample buffer, subjected to PAGE and analyzed by WB.

Student's t-test was used when making comparisons in datasets containing two groups. One-way ANOVA test was used when making comparisons in datasets containing multiple groups, and Student-Newman-Keuls test was used as the post hoc test. All data were analyzed using GraphPad Prism software (version 8.3.1; GraphPad Software, Inc.) and presented as the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 were considered to indicate statistically significant differences.

ADR and VCR are effective antitumour agents with a broad antitumour spectrum. They exert strong inhibitory effects on RNA synthesis and the proliferation of BCL cells. First, the inhibitory effects of ADR and VCR on the viability of different BCL cells, including Daudi (Burkitt lymphoma cells), SUDHL-4 (diffuse large B-cell lymphoma cells) and JeKo-1 cells (mantle cell lymphoma cells) were verified. As expected, both ADR and VCR notably decreased the viability of Daudi, SUDHL-4 and JeKo-1 cells (Fig. 1A and B). Among these three cell lines, JeKo-1 cells exhibited the strongest drug resistance against ADR and VCR treatments, followed by SUDHL-4 and Daudi cells. Considering the association between autophagy and drug resistance, it was then verified whether the inhibition of autophagy counteracted the resistance of BCL cells to ADR and VCR. The results demonstrated that, when the cells were treated with the synthetic autophagy inhibitors 3-methyladenine and chloroquine diphosphate salt combined with ADR and VCR, the viability of BCL cells, particularly JeKo-1 cells, was significantly inhibited (Fig. 1C and D). To further confirm the association between autophagy and drug resistance, lentivirus infection was conducted to inhibit autophagy via stable ATG5 interference in Daudi, SUDHL-4 and JeKo-1 cells (Fig. 1E). Cell viability assays revealed that Daudi-shATG5, SUDHL-4-shATG5 and Jeko-1-shATG5 cells were markedly more susceptible to ADR and VCR, as compared with their respective control cells (Fig. 1F and G). These results demonstrated that the resistance of BCL cells to ADR and VCR is partially dependent on autophagy.

Since the drug resistance of BCL cells was found to be closely associated with autophagy and the drug resistance of BCL cells differed greatly, the autophagic activity in different BCL cells was examined. Three classic assays commonly used in autophagy research were conducted to characterize the status of autophagy: i) The protein level of LC3 and P62 was determined by WB; ii) mRFP-LC3 punta were examined by immunofluorescence; and iii) cytoplasmic accumulation of autophagosomes was detected by TEM. WB revealed that, among the three B-cell lymphoma cell lines, JeKo-1 cells exhibited the highest expression of LC3-II, followed by SUDHL-4 and Daudi cells; furthermore, Daudi cells exhibited the highest expression of P62, followed by SUDHL-4 and JeKo-1 cells (Fig. 2A and B). In the autophagy flux assays, there were markedly more mRFP-LC3 punta in the JeKo-1 cells compared with the Daudi and SUDHL-4 cells (Fig. 2C). The formation of autophagosomes and autolysosomes as analyzed by TEM was abundant in Jeko-1 cells and deficient in Daudi and SUDHL-4 cells (Fig. 2D and E). These results demonstrated that autophagy was activated in BCL cells exhibiting strong drug resistance.

S100A8 is particularly highly expressed in myeloid cells and plays an important regulatory role in the differentiation, autophagy and chemotherapy resistance of myeloid cells. To verify whether the drug resistance and autophagic activity of Daudi, SUDHL-4 and JeKo-1 cells was associated with S100A8, the expression of S100A8 was detected in the three B-cell lymphoma cell lines. RT-qPCR analysis revealed that JeKo-1 cells exhibited the highest mRNA expression of S100A8, followed by SUDHL-4 and Daudi cells (Fig. 3A). WB revealed a markedly higher protein expression of S100A8 in JeKo-1 and SUDHL-4 cells, as compared with that in Daudi cells (Fig. 3B and C). To further confirm the association between S100A8 and drug resistance, lentivirus infection was conducted to inhibit S100A8 expression in Daudi, SUDHL-4 and JeKo-1 cells (Fig. 3D). Cell viability assay revealed that Daudi-shS100A8, SUDHL-4-shS100A8 and JeKo-1-shS100A8 cells were markedly more susceptible to ADR and VCR, as compared with their respective control cells (Fig. 3E and F). These results demonstrated that the resistance of BCL cells to ADR and VCR was closely associated with the expression of S100A8.

Our previous study demonstrated that the chemotherapy resistance of BCL cells was largely dependent on autophagy, and the stronger drug resistance of BCL cells was associated with a higher S100A8 expression. Next, an attempt was made to elucidate the association between S100A8 and autophagy in BCL cells. WB revealed that Daudi-shS100A8, SUDHL-4-shS100A8 and JeKo-1-shS100A8 cells exhibited a markedly lower LC3-II and higher P62 protein expression, as compared with their respective control cells (Fig. 4A). In the autophagy flux assay, there were markedly more mRFP-LC3 punta in SUDHL-4-shS100A8 and JeKo-1-shS100A8 cells, as compared with their respective control cells (Fig. 4B). Finally, the formation of autophagosomes and autolysosomes as analyzed by TEM was markedly deficient in BCL cells with S100A8 interference (Fig. 4C and D), indicating that S100A8 promoted autophagy in BCL cells.

Autophagy is an effective method used by cells to cope with environmental stressors, such as starvation, and is greatly involved in the normal physiological and pathological processes of the body. BNIP3 is anchored to the outer mitochondrial and ER membrane via its C-terminus transmembrane domain, and it can interact with LC3. The binding of BNIP3 to LC3 on the autophagosome is crucial for the removal of both ER (ER-phagy) and mitochondria (mitophagy) via autophagy. To explore the molecular mechanism underlying S100A8-mediated autophagy, the BNIP3 expression was tested in ER and mitochondria following S100A8 interference. The results demonstrated that S100A8 interference significantly decreased the BNIP3 protein expression, both in the ER and mitochondria, in Daudi, SUDHL-4 and JeKo-1 cells (Fig. 5A and B). Next the early autophagic signalling event of BECN1-PI3KC3 and BECN1-BCL2 complex formation was analyzed. The co-immunoprecipitation assay revealed that S100A8 interference significantly reduced the formation of the BECN1-PI3KC3 complex in JeKo-1 cells (Fig. 5C). Furthermore, S100A8 interference significantly increased the formation of the BECN1-BCL2 complex in JeKo-1 cells (Fig. 5D). Collectively, these results indicated that S100A8 may activate autophagy by promoting the expression of BNIP3 and the BECN1-PI3KC3 complex and inhibiting that of the BECN1-BCL2 complex.