Inhibition of the SREBP pathway prevents SARS-CoV-2 replication and inflammasome activation

SARS-CoV-2 induces alterations in lipid metabolism

Viruses are intracellular parasites that can alter cell metabolism to favor their own maintenance and replication. Members of the Flaviviridae family (Boulant et al, 2007; Samsa et al, 2009; Carvalho et al, 2012; Viktorova et al, 2018; Lee et al, 2019) and the Coronaviridae family (Yuan et al, 2019; Dias et al, 2020; Ricciardi et al, 2022) are +RNA viruses that can modify lipid metabolism in different cells, triggering lipid droplet (LD) formation, using this host organelle for different steps of their replicative cycle. To evaluate the effects of SARS-CoV-2 infection on lipid metabolism, we used type II pneumocytes (Calu-3 cells) infected with SARS-CoV-2 at an MOI of 0.01.

Here, we observed decreased expression of the precursor form and augmented activation (mature form) of the lipid transcription factors SREBP1 and SREBP2 24 h after infection with SARS-CoV-2 in Calu-3 cells (Fig 1A). SREBP1 is involved in fatty acid metabolism, whereas SREBP2 controls cholesterol homeostasis (Horton & Shimomura, 1999; Goldstein et al, 2006). Accordingly, we observed that SARS-CoV-2 infection up-regulated pathways of LD biogenesis and maturation, such as DGAT1 and PLIN2, and pathways of both fatty acid (FASN) and cholesterol synthesis (ABCA1 and SOAT1) (Fig 1B and C). Moreover, SARS-CoV-2 infection up-regulated inflammatory pathways, with increases in IL-6, IL-1, and IL-10 (Fig 1C). As previously observed in other cells (Dias et al, 2020), SARS-CoV-2 infection of Calu-3 cells induced LD biogenesis (Fig 1D–G), with increased cholesterol (Fig 1H and I) and triacylglycerol accumulation (Fig 1J) after 48 h of infection. Altogether, SARS-CoV-2 infection of Calu-3 cells triggered SREBP activation along with up-regulation of genes and key proteins of lipid metabolism and increased cholesterol and triacylglycerol, which accumulated in LDs (Fig 1K).

SREBPs are master regulators of lipid metabolism during SARS-CoV-2 infection

To investigate the functions of SREBPs in SARS-CoV-2 infection, we knocked down SREBF1, SREBF2, and DGAT1 with siRNAs separately or in combination. The knockdown efficiency was confirmed by Western blotting (Fig S1A–C) and real-time PCR (Fig S1D–F). Knockdown of SREBF1 and -2 separately partially inhibited viral replication (Fig 2A) but failed to protect Calu-3 cells from death (Fig 2B). Combined knockdown of both SREBFs demonstrated a significant reduction in viral replication in relation to the control group with scramble (Fig 2A) and protected against cell death, decreasing LDH release into the supernatant (Fig 2B). These data suggest that both SREBPs are important for SARS-CoV-2 infection and replication in Calu-3 cells.

To gain insights into the contributions and mechanisms of SREBP1 and SREBP2 during SARS-CoV-2 infection in Calu-3 cells, we analyzed the expression of different genes involved in lipid metabolism regulated during SARS-CoV-2 infection after SREBP knockdown. SREBF1 knockdown down-regulated genes related to LD formation, such as DGAT1 and FASN, and inflammatory genes, such as IL-1β (Figs 2C and S2A and B). SREBF2 knockdown down-regulated genes involved in cholesterol metabolism, such as SOAT1 and inflammatory genes (Figs 2C and S2A and B). Moreover, the combined knockdown of SREBF1 and -2 down-regulated all the genes previously observed (Figs 2C and S2A and B). Altogether, these data reinforce the concept that both genes that encode SREBPs are important and complementary in modulating the lipid and inflammatory profiles during SARS-CoV-2 infection.

Previous findings have established an important role for LDs in SARS-CoV-2 infection, at least in part, because of their roles in the biogenesis of replication organelles (Dias et al, 2020; Ricciardi et al, 2022). To evaluate the role of SREBPs in LD formation, Calu-3 cells were stained with a lipidtox LD probe and J2 antibody for double-stranded RNA (dsRNA) labeling, and the fluorescent area of each marker was quantified. SREBF1, but not SREBF2, knockdown significantly reduced viral replication sites (Fig 2D–F) and LD accumulation (Fig 2D and G–I). The combined knockdown of SREBF1 and -2 was more effective in reducing the SARS-CoV-2 replication (Fig 2D–F) sites and LD accumulation compared with the cells infected with scramble (Fig 2D and G–I). Moreover, knockdown of the DGAT1 enzyme was evaluated, and we observed a reduction in LD biogenesis and viral replication sites, comparable with the effects of combined knockdown of SREBF1 and SREBF2 (Fig 2A, B, and D–I).

Altogether, these results indicate that during SARS-CoV-2 infection, the genes that encode the SREBPs are master regulators of lipid metabolism and participate in different processes, such as inflammatory cytokines, cell death, viral replication, and LD accumulation, in Calu-3 cells. Indeed, the mechanisms that control DGAT1 and LD accumulation are downstream and largely dependent on the activation and transcriptional regulation of SREBPs.

Fatostatin inhibits both SREBPs’ activation and reduces the replication sites of SARS-CoV-2

To further analyze the role of the SREBPs during SARS-CoV-2 infection, we used fatostatin, a pharmacologic inhibitor of the ER–Golgi translocation of SREBPs that binds to their escort protein, SREBP cleavage-activating protein (SCAP) (Kamisuki et al, 2009). Of note, activation of all SREBP isoforms is controlled by SCAP. Fatostatin has been demonstrated to inhibit the viral replication of several viruses from the Flaviviridae family, including WNV and ZIKV (Merino-Ramos et al, 2017).

First, we investigated whether fatostatin inhibits SREBPs in our model. We pretreated Calu-3 cells with fatostatin for 2 h before SARS-CoV-2 infection and maintained the treatment for all times of infection until the analyses of the experiment. Fatostatin treatment inhibited the processing and activation of both SREBP1 and SREBP2 during infection, leading to the accumulation of the precursor form of SREBPs in Calu-3 cells (Fig 3A and B). Furthermore, the blockage of SREBP1 and SREBP2 activation by treatment with fatostatin during SARS-CoV-2 infection reduced cholesterol accumulation (Fig 3C and D), as observed by filipin III labeling and cholesterol quantification, and triacylglycerol accumulation (Fig 3E). As shown in Fig S3A, treatment with fatostatin was devoid of cytotoxicity at the doses used.

To confirm that the inhibition of SREBPs by fatostatin treatment could affect LD accumulation, Calu-3 cells were pretreated with fatostatin, and LD accumulation was analyzed 48 h after infection. As shown in Fig 3, treatment with fatostatin reduced LD accumulation after SARS-CoV-2 infection, similar to the DGAT1 inhibitor A922500 (Fig 3F–H), as previously observed in other cell types (Dias et al, 2020). Indeed, we observed a reduction in the protein expression related to the LD form and maturation (DGAT1 and PLIN2) in Calu-3 cells treated with fatostatin (Fig 3I and J). Thus, this confirms that SREBPs are master regulators in the process of LD accumulation in Calu-3 cells infected with SARS-CoV-2 through the increase in the DGAT1 enzyme and PLIN2, favoring LD biogenesis.

SARS-CoV-2 may explore host lipid metabolism to favor its replication using LDs as an energy source for its own replication (Dias et al, 2020; Ricciardi et al, 2022). To investigate the role of SREBP in the formation of SARS-CoV-2 replication sites, we labeled Calu-3 cells with a J2 clone for dsRNA and BODIPY for LDs and quantified the labeled area of each marker. First, we observed an increase in dsRNA and BODIPY during SARS-CoV-2 infection, and when we treated the cells with fatostatin or A922500, we observed a reduction in the labeled area (Fig 4A and B). Moreover, almost all cells were positive for dsRNA when they were infected with SARS-CoV-2, and both treatments reduced the number of dsRNA-positive cells by up to 20% (Fig 4C). Considering the double-positive cells (dsRNA and BODIPY labeling), we observed that almost all cells infected with SARS-CoV-2 presented a close association with LDs (Fig 4D and E), but treatment with fatostatin or A922500 reduced the double-positive cells up to 10% (Fig 4D and E). To determine whether DGAT2 also plays a role in LD formation during SARS-CoV-2 infection, Calu-3 cells were pretreated with DGAT2 inhibitor PF-06424439 and were stained with a lipidtox LD probe and J2 antibody for dsRNA labeling, and the fluorescent area of each marker was quantified. The treatment with DGAT2 inhibitor did not alter the LD accumulation and dsRNA during SARS-CoV-2 infection (Fig S4A–F); also, DGAT2 inhibitor did not affect the SARS-CoV-2 replication (Fig S4G).

We observed through electron microscopy that SARS-CoV-2–infected cells presented an increase in LDs (*) and a close association of the viral particles (arrow) with LDs (Figs 4F and S5B–D), as previously shown (Dias et al, 2020; Ricciardi et al, 2022). Moreover, SARS-CoV-2 infection induced a clear signal of cell injury in Calu-3 cells, as indicated by the presence of myelin figures (arrowhead) (Fig S5B–D), in comparison with control cells (Fig S5A). Furthermore, fatostatin or A922500 treatment reduced LD accumulation and the presence of viral particles in comparison with the cells infected and treated with vehicle (DMSO) (Fig 4F–I).

Altogether, our data suggest that SARS-CoV-2 modulates the lipid metabolism of Calu-3 cells in favor of increasing the activation of SREBPs, inducing LD accumulation, and promoting a close association with dsRNA and viral replication sites.

Fatostatin protects Calu-3 cells from death during SARS-CoV-2 infection

Viral infections can cause alterations in cell homeostasis, leading the virus to use cellular compounds for its own benefit, increasing viral replication that can induce a process of cell death using cellular resources or by a heightened inflammatory response. SARS-CoV-2 infection has the capacity to induce cell death in different cells, such as human monocytes, by the release of LDH into the extracellular space (Dias et al, 2020; Fintelman-Rodrigues et al, 2020 Preprint; Ferreira et al, 2021). Here, we observed the cell morphology and measured LDH release in Calu-3 cells infected with SARS-CoV-2 after 48 h of infection. Our data showed that infection with SARS-CoV-2 was able to alter the cell monolayer, causing damage to the cellular membrane and increasing LDH release into the supernatant (Fig 5A and B). Treatment with fatostatin alone did not interfere with cell morphology or LDH release, whereas treatment during SARS-CoV-2 infection was able to decrease damage, block cell death, and reduce LDH release in Calu-3 cells (Fig 5A and B).

As previously observed with the knockdown of both genes that encode SREBPs, treatment with fatostatin, which inhibits the activation of both SREBPs, was able to reduce viral replication by two logs (Fig 5C), presenting viral inhibition of almost 90% and with a 50% antiviral concentration (IC50) of 14.15 μM (Fig S3A). Similarly, pretreatment with A922500 inhibited viral replication (Fig 5C), with almost 100% viral inhibition and an IC50 of 3.88 μM (Fig S3B).

To analyze other inhibitors of SREBP, we evaluated two other inhibitors, betulin and AM580. Neither of the inhibitors presented any cytotoxicity in Calu-3 cells (Fig S3C and D). To observe the effects of these inhibitors during SARS-CoV-2 infection, we analyzed the protection from cell death and viral replication. The inhibitor betulin did not protect against cell death, as observed by LDH release, and did not affect viral replication (Fig S3C), but the inhibitor AM580 was able to reduce viral replication without altering cell death (Fig S3D).

SREBP inhibition blocks inflammasome complex activation and pyroptosis death during SARS-CoV-2 infection

Several studies have associated cholesterol metabolism and SREBP activation with the inflammasome, culminating in the release of proinflammatory cytokines, such as IL-1β (Li et al, 2013; Guo et al, 2018). In previous work, we already demonstrated that human monocytes infected with SARS-CoV-2 activate caspase-1 and promote IL-1β release with activation of GSDMD1, which suggests a process of pyroptosis (Ferreira et al, 2021). In addition, other works supporting these data have already demonstrated inflammasome NLRP3 assembly and activation in human primary monocytes infected with SARS-CoV-2 and PBMCs from COVID-19 patients (Rodrigues et al, 2021).

To investigate the link between SREBP activation, viral replication, and pyroptosis-mediated cell death during SARS-CoV-2 infection in lung epithelial cells, we evaluated caspase-1 activation by staining Calu-3 cells with FAM-YVAD-FLICA. Indeed, infected cells presented an increase in activated caspase-1 during SARS-CoV-2 infection, and when SREBPs were inhibited by treatment with fatostatin (Fig 5D and E) or A922500 (Fig S6A), a reduction in activated caspase-1 was observed by fluorescence microscopy and flow cytometry, which was also confirmed by Western blotting (Fig S6B and C).

Moreover, we observed that the cells infected with SARS-CoV-2 presented an increase in GSDMD1 activation, a protein related to membrane pore formation that is activated during the process of pyroptosis. Furthermore, treatment with fatostatin was able to reduce the activation of GSDMD1 (Fig 5F). Altogether, these data suggest that activation of SREBPs during SARS-CoV-2 infection is associated with an increase in activated caspase-1, contributing to the formation of membrane pores by GSDMD1.

It is already well known that SARS-CoV-2 promotes an exacerbated inflammatory response that aggravates cell damage and consequently cell death with the release of several cytokines (Dias et al, 2020; Ferreira et al, 2021). Here, we observed an increase in the main inflammatory cytokines produced by the activation of caspase-1, such as IL-1β and IL-18 (Fig 5G). We also observed an increase in other pro-inflammatory cytokines, such as TNFα and IL-6, and the chemokine CxCL-10 (Fig S6D and E). Consistent with prior data, the inhibition of SREBPs by fatostatin (Figs 5G and S6D) or DGAT1 by A922500 (Fig S6E) was able to reduce all cytokines and chemokines previously observed. Altogether, our data suggest that SREBPs participate in the inflammatory process during SARS-CoV-2 infection in Calu-3 cells.

Cells and reagents

The human lung epithelial adenocarcinoma cell line (Calu-3 - ATCC/HTB-55) and African green monkey kidney (Vero subtype E6) were cultured in high-glucose DMEM supplemented with 10% FSB (HyClone) and 100 U/ml penicillin‒streptomycin (P/S; Gibco) and were incubated at 37°C in 5% CO₂.

Virus infection and virus titration

Nasopharyngeal swab samples were collected from confirmed cases from Rio de Janeiro/Brazil (GenBank accession no. MT710714). SARS-CoV-2 was amplified in Vero-E6 cells in high-glucose DMEM supplemented with 2% FBS for 2–4 d of infection and incubated at 37°C in 5% CO₂. Virus titers were determined by the tissue culture infectious dose at 50% (TCID₅₀/ml), and the virus stocks were kept in −80°C freezers. All specimens were handled under the laboratory biosafety guidance required involving SARS-CoV-2 by the World Health Organization at a biosafety level 3 multi-user facility from Fundação Oswaldo Cruz/Fiocruz.

All cells were infected with SARS-CoV-2 at a MOI of 0.01 with or without pretreatment for 2 h with pharmacological inhibitors of SREBP activation, such as fatostatin (13562; Cayman), AM580 (A8843; Sigma-Aldrich) and betulin (11041; Cayman), and the pharmacological inhibitor of DGAT1, A922500 (A1737; Sigma-Aldrich), and of DGAT2, PF-06424439 (PZ0233; Sigma-Aldrich). The treatment with all inhibitors was continued after the infection. During knockdown experiments, the cells were infected at the same MOI, and 24 h before infection, the cells were transfected with the siRNAs for SREBF1 (s129), SREBF2 (s27), DGAT-1 (16567), and the negative control with scramble RNA (4390844) according to the manufacturers’ instructions (Thermo Fisher Scientific). For virus titration, we performed a plaque-forming assay in Vero-E6 cells seeded in 96-well plates. Cell monolayers were infected with different dilutions of the supernatant containing the virus for 1 h at 37°C. The cells were overlaid with high-glucose DMEM containing 2% FBS and 2.4% carboxymethylcellulose. After 3 d, the cells were fixed with 10% formaldehyde in PBS for 3 h at room temperature. Cell monolayers were stained with 0.04% crystal violet in 20% ethanol for 1 h. The viral titer was calculated from the count of plaques formed in the wells corresponding to each dilution and expressed as PFU/ml.

Cell viability assay

Calu-3 cells were seeded in 96-well plates. Then, the cells were treated with a range of concentrations of the inhibitors for 24 and 48 h. Then, the cells were fixed using 3.7% formaldehyde for 20 min. Cell monolayers were stained with 1% crystal violet in 20% ethanol for 10 min. The cells were washed with water, and crystal violet was extracted using methanol. The crystal violet was read in a spectrophotometer at a wavelength of 595 nm.

Ultrastructural analysis of cells by transmission electron microscopy

For ultrastructural analysis, the infected and noninfected Calu-3 cell monolayers were trypsinized at 24 and 48 h post infection or cultivation, respectively. Cell suspensions were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (0.2 M, pH 7.2), postfixed in 1% buffered osmium tetroxide, dehydrated in acetone, embedded in epoxy resin, and polymerized at 60°C over the course of 3 d (Barreto-Vieira et al, 2010; Barth et al, 2016). Ultrathin sections (50–70 nm) were obtained from the resin blocks. The sections were picked up using copper grids (300 mesh) and observed using a Hitachi HT 7800 (Hitachi) transmission electron microscope. The LD area analysis was performed using ImageJ software. For each group and time of kinetics, the LD area and total area of four cells of TEM images were manually gauged. The values obtained from all images were grouped and the mean value was calculated. Viral particles attached to the cell membrane and within the cytosol were counted in five microscopic fields (magnification = x5.0k). The counts were performed with the use of the ImageJ software.

Lipid droplet staining

Calu-3 cells were seeded on coverslips. Cells infected or not infected were fixed using 3.7% formaldehyde, and the LDs were stained with 0.3% Oil Red O (diluted in 60% isopropanol) at room temperature for 2 min. The coverslips were mounted on slides using antifade mounting medium (VECTASHIELD). DAPI staining (1 μg/ml) for 5 min was used for nuclear recognition. Fluorescence was analyzed by fluorescence microscopy with a 100x objective lens (Olympus). The numbers of LDs were automatically quantified from 15 random fields by ImageJ software analysis.

Immunofluorescence staining

Calu-3 cells were seeded on coverslips and fixed using 3.7% formaldehyde after 48 h of infection for 20 min at room temperature. Cells were rinsed three times with PBS containing 0.1 M CaCl₂ and 1 M MgCl₂ (PBS/CM) and then permeabilized with 0.1% Triton X-100 plus 0.2% BSA in PBS/CM for 10 min (PBS/CM/TB). Double-RNA was labeled by the mouse monoclonal antibody J2 clone Scicons (Schönborn et al, 1991; Dias et al, 2020) at a 1:500 dilution overnight, followed by a mouse anti-IgG-DyLight 550 at a 1:1,000 dilution for 1 h with 0.2 μg/ml BODIPY493/503 dye for 5 min for LD staining. In addition, mouse anti-IgG-DyLight 488 was used at a 1:1,000 dilution for 1 h with LipidTox Neutral Red dye (H34476; Thermo Fisher Scientific) at a dilution of 1:1,000 for 30 min for LD staining. Slides were mounted using antifade mounting medium (VECTASHIELD). Nuclear recognition was based on DAPI staining (1 μg/ml) for 5 min. Fluorescence microscopy was analyzed with a 100x objective lens (Olympus). The number of LD was counted in random microscopic fields and the LD diameter was measured using ImageJ software. Total LD area per cell was evaluated by ImageJ software analysis by measuring the fluorescent area of LDs. In addition, the dsRNA puncta and total dsRNA area were evaluated using ImageJ software.

Triglyceride and cholesterol measurements

Calu-3 cells were harvested after 48 h of SARS-CoV-2 infection using ice-cold lysis buffer pH 8.0 (1% Triton X-100, 2% SDS, 150 mM NaCl, 10 mM HEPES, and 2 mM EDTA in the presence of protease inhibitor cocktail; Roche). For analysis of triglyceride and cholesterol levels, 80 μg of protein/sample of the cell lysates was extracted with chloroform/methanol/water 1:2:0.8 (vol/vol/v) in glass tubes. The samples were vortexed for 5 and 5 min for 1 h. Then, they were centrifuged at 1,500g for 20 min. The aqueous phase of each sample was collected and transferred to another glass tube, the pellet was resuspended in chloroform/methanol/water 1:2:0.8 (vol/vol/v), and the last steps were repeated. The aqueous phase was obtained and transferred to the same glass tubes as the first aqueous phase. Then, chloroform/water 1:1 (vol/vol) was added and vortexed for 10 s. After this step, the samples were centrifuged at 1,500g for 30 min, and two phases were obtained. A lower phase was collected in a glass tube, evaporated with nitrogen gas, and resuspended in chloroform/methanol 1:2 (vol/vol). The triglyceride levels were quantified using Triglycerides Liquiform (87-2/100; Labtest kit) and the cholesterol levels were quantified using Cholesterol Liquiform (76-2/250; Labtest kit) according to the manufacturer’s instructions.

For cholesterol staining, Calu-3 cells were seeded in coverslips and after 48 h were fixed using 3.7% formaldehyde for 20 min at room temperature. Then, the cells were washed three times with PBS and incubated with 20 μM glycine for 10 min. Next, the cells were stained with 50 μg/ml filipin III (#70440; Cayman) for 2 h at room temperature in the dark. For nuclear recognition, the cells were labeled with 1 μM TO-PRO-3 (T3605; Thermo Fisher Scientific) for 10 min. Slides were mounted using antifade mounting medium (VECTASHIELD). Fluorescence was analyzed by fluorescence microscopy with a 40x objective lens (Olympus).

SDS‒PAGE and Western blot

After 24 h of SARS-CoV-2 infection, Calu-3 cells were harvested using ice-cold lysis buffer (pH 8.0) containing 1% Triton X-100, 2% SDS, 150 mM NaCl, 10 mM HEPES, and 2 mM EDTA in the presence of a protease inhibitor cocktail (Roche). The protein levels were measured by a bicinchoninic acid assay protein kit (Thermo Fisher Scientific). 30 μg of the protein/sample was heated at 100°C for 5 min in Laemmli buffer pH 6.8 (20% β-mercaptoethanol; 370 mM Tris base; 160 μM bromophenol blue; 6% glycerol; 16% SDS) and resolved by electrophoresis on an SDS-containing 10% polyacrylamide gel (SDS‒PAGE). Next, the separated proteins were transferred to nitrocellulose membranes and incubated in blocking buffer (5% nonfat milk, 50 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween 20). Membranes were probed overnight with the following antibodies: anti-SREBP1 (14088-1-AP; Proteintech), anti-SREBP2 (28212-1-AP; Proteintech), anti-DGAT1 (11561-1-AP; Proteintech), anti-PLIN2 (15294-1-AP; Proteintech), anti-GSDMD1 (97558; Cell Signaling), and anti-GAPDH (60004-1-1 g; Proteintech). After washing, the membranes were incubated with IRDye-LICOR or HRP-conjugated secondary antibodies for 2 h at room temperature. All antibodies were diluted in blocking buffer. Signal detection was performed by Supersignal Chemiluminescence (GE Healthcare) or fluorescence imaging using the Odyssey system. The densitometries were analyzed using Image Studio Lite Ver 5.2 software.

Quantitative real-time RT‒PCR assay

Monolayers from Calu-3 cells after 24 h of SARS-CoV-2 infection were harvested, and the total RNA from each sample was extracted using an SV total RNA isolation system kit according to the manufacturer’s protocol (Promega). RNA concentration and purity were determined by a spectrophotometer (NanoDrop 2000) measuring absorbance at A260 and A280 nm, and RNA was stored at −70°C in nuclease-free water. Total RNA (2 μg) was reverse transcribed in a 20-μl reaction mixture using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s protocol. The cDNA was amplified in 10 μl of 1× TaqMan Universal PCR master mix with Predeveloped TaqMan assay primers and probes Perilipin-2 (PLIN2) Hs00605340_m1; DGAT1, Hs01020362_g1; Fatty Acid Synthase (FASN) Hs01005622_m1; SREBF1 Hs01088691_m1; SREBF2 Hs01081784_m1; patatin-like phospholipase domain containing 2 (PNPLA2) Hs00386101_m1; Sterol O-Acyltransferase 1 (SOAT1) Hs00162077_m1; ATP-binding cassette transporter-1 (ABCA1) Hs01059118_m1; IL-6 Hs00985639_m1; IL-10 Hs00961622_m1; IL-1β Hs01555410_m1, and GAPDH Hs99999905_m1 was used as an endogenous control according to the manufacturer’s instructions (Thermo Fisher Scientific). Quantitative RT‒PCR was performed in a StepOne Real-Time PCR System (Thermo Fisher Scientific). PCR products were analyzed in a comparative manner relative to the endogenous control GAPDH (ΔΔCt).

Measurements of inflammatory mediators and LDH activity

Calu-3 cell supernatants were obtained after 24 h of SARS-CoV-2 infection with or without treatment with inhibitors. Cytokines and chemokines were measured in the supernatant by ELISA following the manufacturer’s instructions (Duo set; R&D). Cell death was determined according to the activity of lactate dehydrogenase (LDH) in the culture supernatants using a CytoTox Kit according to the manufacturer’s instructions (Promega).

Assessment of activated caspase-1

After 48 h of infection, Calu-3 cells were stained to detect caspase-1 activation using fluorescent-labeled inhibitors of caspase-1 activity (FAM-YVAD-FMK/FLICA) according to the manufacturer’s instructions (Bio-Rad). The fluorescence of caspase-1 activity, expressed as the percentage of activated cells, was evaluated by flow cytometry (FACSCalibur), and the generated data were analyzed with FlowJo. In parallel, the cells were seeded on coverslips; after 48 h of infection, the cells were labeled with FAM-YVAD-FMK/FLICA according to the manufacturer’s instructions. Then, the cells were fixed with 3.7% formaldehyde for 20 min at room temperature. The nuclei were stained with DAPI (1 μg/ml) for 5 min, and the coverslips were mounted using the antifade mounting medium (VECTASHIELD). Fluorescence was analyzed by fluorescence microscopy with a 100× objective lens (Olympus).

Statistical analysis

Data are expressed as the mean ± SEM of three and a maximum of six independent experiments. The paired two-tailed t test was used to evaluate the significance of differences between two groups. Multiple comparisons among three or more groups were performed by one-way ANOVA followed by Tukey’s multiple comparison test. P-values < 0.05 were considered statistically significant when comparing SARS-CoV-2 infection with the uninfected control group (*) or SARS-CoV-2 infection with inhibitor groups (pharmacological inhibitors) or knockdown groups (siSREBF1 and -2 and DGAT1) (#).