Mouse VSMCs (MOVAS cell line; CRL-2797) were purchased from the American Type Culture Collection, and were cultured in DMEM/F-12 (HyClone; Cytiva) supplemented with 10% fetal bovine serum (Shanghai ExCell Biology, Inc.) as described previously (14). Thereafter, the VSMCs were cultured at 2×10⁶ cells/dish for 24 h at 37°C and then starvation was induced with serum-free DMEM for 24 h at 37°C before the experimental treatment. In some experiments, the VSMCs were pretreated with lycorine (20 µM; MedChemExpress) for 12 h before 3 mmol/l PI treatment for 24 h at 37°C. For some experiments, SARS-CoV-2 N protein-overexpressing VSMCs were pretreated with 1.0 mmol/l phosphonoformate sodium (PFA; CAS. no. 63585-09-1; Shanghai Aladdin Biochemical Technology Co., Ltd.) for 24 h before PI treatment for 24 h at 37°C. PFA is not only an antiviral drug, but also a specific cell membrane sodium phosphate transporter (pit1/2) inhibitor that prevents extracellular phosphate from entering the cell (13). Phosphate stock solution contained NaH₂PO₄ and Na₂HPO₄ (pH 7.40).

VSMCs were transduced with 50 µl SARS-CoV-2 N protein lentivirus or negative control empty vector (Lenti-EF1α-2019-nCOV-N-Flag/His-CMV-Puro; 1×10⁸ IU/ml; cat. no. C3021; Beyotime Institute of Biotechnology) for 24 h with a multiplicity of infection of 50 at 37°C. A total of 48 h post-transduction, the VSMCs were cultured for 4–7 days with 2 µg/ml puromycin (Beyotime Institute of Biotechnology) at 37°C. Subsequently, the VSMCs were lysed and the specific protein expression in VSMCs was confirmed by western blotting.

The VSMCs or SARS-CoV-2 N protein-overexpressing VSMCs were transfected with 100 pmol SREBP2 small interfering RNA (siRNA) or negative control (NC) siRNA using 5 µl Lipo8000^™ (cat. no. C0533; Beyotime Institute of Biotechnology) for 24 h at 37°C, according to the manufacturer's instructions. Subsequently, the SARS-CoV-2 N protein-overexpressing VSMCs were further cultured for 12 h and then treated with or without 0.2 mmol PI for 24 h at 37°C. The sequence of the SREBP2-targeted siRNA was 5′-GCGGACAACACACAAUAUCAU-3′ and the sequence of the scrambled siRNA control was 5′-UUCUCCGAACGUGUCACGUTT-3′. SREBP2 siRNA and the scrambled siRNA were constructed by Sangon Biotech Co., Ltd.

Total protein was extracted from VSMCs using ice-cold RIPA Lysis Buffer (cat. no. P0013B; Beyotime Institute of Biotechnology), containing a protease and phosphatase inhibitor cocktail (cat. no. P1045; Beyotime Institute of Biotechnology). Protein concentrations were measured using a BCA protein assay kit (cat. no. P0010S; Beyotime Institute of Biotechnology) and 80 µg protein/lane was separated by SDS-PAGE on a 10 or 12% gel. The separated proteins were then transferred to a PVDF membrane and blocked for 2 h. The PVDF membranes were incubated with the following antibodies overnight at 37°C: SARS-CoV-2 N protein (cat. no. AF0325; 1:500; Beyotime Institute of Biotechnology), SCAP (cat. no. NBP2-04113; 1:1,000; Novus Biologicals, LLC), mature N-terminal SREBP2 (N-SREBP2; 1:1,000; 70 Kd, cat. no. ab30682; Abcam), NLRP3 (cat. no. WL02635; 1:1,000; Wanleibio Co., Ltd.), procaspase-1 (cat. no. WL02996; 1:800; Wanleibio Co., Ltd.), cleaved caspase-1 (cat. no. WL03450; 1:800; Wanleibio Co., Ltd.), IL-1β (cat. no. 16806-1-AP; 1:600; Wuhan Sanying Biotechnology), IL-18 (cat. no. 10663-1-AP; 1:500; Wuhan Sanying Biotechnology) and β-actin (cat. no. 81115-1-RR; 1:5,000; Wuhan Sanying Biotechnology). After overnight incubation, the blots were incubated with goat anti-rabbit IgG-HRP antibody (1:5,000; cat. no. A0208; Beyotime Institute of Biotechnology) for 2 h at 37°C. Finally, the bands were visualized using the ECL reagent kit (cat. no. WBKlS0100; MilliporeSigma) and the densities of the bands were analyzed using ImageJ 1.47i software (National Institutes of Health) for semi-quantification.

The total RNA was extracted from the cells with TRIzol^® reagent (cat. no. 15596-026; Invitrogen; Thermo Fisher Scientific, Inc.) and reverse-transcribed into cDNA using a high-capacity cDNA synthesis kit (cat. no. BL696A; Biosharp Life Sciences). The RT protocol was as follows: 25°C for 10 min, 55°C for 15 min and 85°C for 5 min. qPCR analysis was performed using the SYBR Green PCR Mix kit (cat. no. BL697A; Biosharp Life Sciences) and the ABI7500 System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min; followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec, with a final extension at 72°C for 30 sec. The specific primer sequences are listed in Table I. The relative fold changes in gene expression were normalized to the mRNA expression levels of β-actin and were quantified using the 2^−ΔΔCq method (16).

VSMCs were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.3% Triton X-100 for 5 min at 37°C. Thereafter, the VSMCs were treated overnight with anti-SCAP (1:100; cat. no. NBP2-04113; Novus Biologicals, LLC), anti-NLRP3 (1:200; cat. no. NBP1-97601; Novus Biologicals, LLC) and anti-Golgin 97 (1:150; cat. no. PA5-30048; Thermo Fisher Scientific, Inc.) at 37°C. Then, the VSMCs were stained with goat anti-rabbit IgG (TRITC; 1:100; cat. no. SA00007-2; Proteintech Group, Inc.) or goat anti-mouse IgG (FITC; 1:100; cat. no. SA00003-1; Proteintech Group, Inc.) for 45 min at 37°C. The nuclei were stained with DAPI for 8 min at 37°C and the images were viewed with a confocal laser scanning microscope (Zeiss GmbH).

Data are presented as the mean ± SD. The significance between two groups was assessed using unpaired Student's t-test. Differences among multiple groups were analyzed by one-way analysis of variance with Tukey's post hoc test. All statistical analyses were performed using SPSS software (version 18, SPSS, Inc.). All of the experiments were performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

SCAP transports SREBP2 to the Golgi apparatus, and then SREBP2 becomes transcriptionally active when its N-terminal DNA-binding fragment is proteolytically cleaved to its mature, active form (mature N-terminal SREBP2, N-SREBP2) (17,18). Subsequently, the cytoplasmic N-SREBP2 translocates to the nucleus to bind to DNA and initiate gene transcription. This process can be assessed by measuring the mature N-SREBP2 protein in the whole cell lysate (17–19). Our previous study indicated that SARS-CoV-2 N protein activates the dissociation of SCAP from the endoplasmic reticulum (ER), resulting in SREBPs activation, increased lipogenic gene expression and NLRP3 inflammasome activation (20). To examine the effect of PI on the activation of SCAP-SREBP2, SARS-CoV-2 N protein-overexpressing VSMCs were incubated with PI (3 mmol/l) for 24 h. Firstly, SARS-CoV-2 N protein was significantly increased in SARS-CoV-2 N protein-overexpressing VSMCs (Fig. 1A). Secondly, compared with those in the SARS-CoV-2 N protein group, PI increased the mRNA expression levels of SREBP2-targeted genes, i.e. HMG-CoA reductase (HMGCoAR), low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) in SARS-CoV-2 N protein-overexpressing VSMCs (Fig. 1B). In addition, the mRNA expression levels of SREBP1c-targeted genes, i.e. recombinant fatty acid synthase (FASN), stearyl coenzyme A desaturase 1 (SCD1), acetyl-CoA carboxylase α (ACACA) and ATP-citrate lyase (ACLY) were significantly increased in SARS-CoV-2 N protein-overexpressing VSMCs (Fig. 1C). In addition, the results indicated that PI significantly increased SCAP and SREBP2 protein expression levels in SARS-CoV-2 N protein-overexpressing VSMCs compared with in the non-phosphate-treated control (Fig. 1D). Likewise, PI significantly increased the relative mRNA expression levels of NLRP3 and IL-1β (Fig. 1E), and the protein expression levels of NLRP3, procaspase-1, cleaved caspase-1 and IL-1β compared with those in the control group (Fig. 1F). These results indicated that PI could amplify SARS-CoV-2 N protein-induced NLRP3 inflammasome activation and lipogenesis, which may be mediated by SCAP-SREBPs.

PFA is a specific inhibitor of intracellular phosphate uptake (21). In VSMCs overexpressing SARS-CoV-2 N protein, PFA significantly decreased the relative protein expression levels of SCAP and N-SREBP2 compared with those in the PI-treated group, suggesting that PFA reversed PI-induced SCAP-SREBP2 activation (Fig. 2A). Likewise, PFA significantly decreased the mRNA expression levels of SREBP2-targeted genes, i.e. HMGCoAR, LDLR and PCSK9 in PI-treated and SARS-CoV-2 N protein-overexpressing VSMCs (Fig. 2B). In addition, PFA significantly decreased the relative mRNA expression levels of NLRP3 and IL-1β (Fig. 2C), and the relative protein expression levels of NLRP3, procaspase-1, cleaved caspase-1 and IL-1β (Fig. 2D) compared with those in the phosphate-treated controls. These data indicated that PFA ameliorated the effect of PI and SARS-CoV-2 N protein on SCAP-SREBP-induced lipogenesis and NLRP3 inflammasome activation.

It has previously been shown that SCAP-SREBP2 binds with NLRP3 to form a ternary complex that promotes its translocation to the Golgi, thereby facilitating NLRP3 inflammasome activation (22). Consistent with previous studies (14), we found that NLRP3 (Fig. 3A) or SCAP (Fig. 3B) proteins were separately localized to Golgin 97 after PI stimulation, compared with that in the non-phosphate-treated controls.

Furthermore, the relative protein expression levels of NLRP3, procaspase-1, cleaved caspase-1, IL-1β and IL-18 were significantly increased by PI compared with those in the non-phosphate-treated control group (Fig. 3C). Therefore, these data demonstrated that PI could induce NLRP3 inflammasome activation by promoting SCAP and NLRP3 translocation to the Golgi.

It was demonstrated that the SCAP-SREBP inhibitor lycorine decreased the protein expression levels of SCAP and N-SREBP2 in VSMCs treated with PI (Fig. 4A). Compared with in the control group, PI also increased SREBP2-mediated cholesterol biosynthesis (i.e. SREBP2, HMGCoAR, LDLR and PCSK9 mRNA expression) (Fig. 4B) and SREBP 1c-mediated fatty acid synthesis (i.e. SREBP1c, FASN, ACACA and SCD1 mRNA expression) (Fig. 4C). Moreover, compared with in the PI-treated VSMCs, lycorine significantly decreased SEBP2-mediated cholesterol biosynthesis (i.e. SREBP2, HMGCoAR, LDLR and PCSK9 mRNA expression) and SREBP 1c-mediated fatty acid synthesis (i.e. SREBP1c, FASN, ACACA and SCD1 mRNA expression) (Fig. 4B and C). These results indicated that PI induced lipogenesis through SCAP-SREBPs.

VSMCs were treated with lycorine to confirm whether SCAP was involved in PI-induced NLRP3 inflammasome activation, it was shown that in PI-treated VSMCs, lycorine significantly decreased the NLRP3 (Fig. 5A) and SCAP (Fig. 5B) Golgi co-localization compared with the PI-only control.

Likewise, lycorine significantly decreased the relative protein expression levels of NLRP3, procaspase-1, cleaved caspase-1, IL-1β and IL-18 compared with those in the PI-only control (Fig. 5C). Therefore, PI was predicted to induce NLRP3 inflammasome activation by promoting SCAP-SREBP ER-to-Golgi translocation.

SCAP-SREBP2 has previously been identified as serving a key role in inflammation and cholesterol metabolism (22). The present results demonstrated that lycorine significantly decreased SREBP2-mediated cholesterol biosynthesis (i.e. SREBP2, HMGCoAR, LDLR and PCSK9 mRNA expression) and SREBP1c-mediated fatty acid synthesis (i.e. FASN and ACLY mRNA expression) (Fig. 6A and B), compared with in PI-treated SARS-CoV-2 N-protein overexpressing VSMCs. Furthermore, SREBP2 siRNA significantly decreased the protein expression levels of SREBP2 in untreated VSMCs compared with those in the control siRNA group (Fig. 6D). Likewise, it was demonstrated that in PI-treated SARS-CoV-2 N-protein overexpressing VSMCs, lycorine and SREBP2 siRNA markedly reduced NLRP3, procaspase-1, cleaved caspase-1 and IL-1β expression compared with those in the control groups (Fig. 6C and E). These findings suggested that SCAP-SREBPs are necessary for lipogenesis and NLRP3 inflammasome activation in PI and SARS-CoV-2 N protein-overexpressing VSMCs.