SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes

Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation (Haptoglobin, MCP-1, IL-6, PAI-1), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells.


Introduction
The pathogen SARS-CoV-2, a member of the coronavirus family, is the causative agent behind the worldwide COVID-19 pandemic.This disease is characterized by a spectrum of clinical manifestations, ranging from asymptomatic infection to severe forms, for which extreme inflammation and cytokine storm have been implicated as potential contributors to fatal outcomes [1].Notably, obesity, a condition often regarded as a global pandemic closely linked to poverty and socioeconomic disadvantage, emerges as an independent risk factor for severe and lethal forms of COVID-19 [2][3][4].While patients with obesity commonly exhibit a spectrum of other conditions typical of the metabolic syndrome and are predisposed to an elevated risk of cardiovascular disease [5], it is noteworthy that comorbidities linked to excess weight, such as hypertension and type 2 diabetes, confer an independent relative risk for severe forms of COVID-19, albeit lower than that associated with obesity [3,6,7].The excessive accumulation of fat mass could potentially serve as a trigger for the severe progression of COVID-19 itself.Notably, during obesity, fat mass undergoes substantial rearrangement, leading to a radical shift in the expression profile of adipocytes [8].This alteration results in the overproduction of chemoattractant factors that recruit immune cells from the circulation, eliciting a state of low-grade chronic systemic inflammation [9].This inflammatory condition primes the onset of the comorbidities associated with obesity.The critical entry point for the virus, the membrane-bound glycoprotein angiotensin-converting enzyme 2 (ACE2), is expressed in white adipose tissue (WAT).WAT has been implicated as a target for SARS-CoV-2 and a potential reservoir for prolonged viral shedding, suggesting a potential contribution to the outcome of "long COVID".Notably, SARS-CoV-2 has been detected in postmortem specimens of white adipose tissue from infected subjects [10][11][12].
The connection between COVID-19 and obesity extends beyond the virus' ability to enter adipocytes.Notably, embolic fat has been identified in the lungs of individuals with obesity who succumbed to COVID-19 [13].The authors posit that a substantial source of these lipids may originate from infected, dying adipocytes.This observation raises critical implications for understanding the effects of adipose tissue infection on the response to COVID-19 in distant regions, such as the respiratory apparatus.
A deeper comprehension of the interaction between the coronavirus and the adipocyte is pivotal for addressing the evolution of COVID-19, both in its acute phase and considering the increasingly recognized long-term effects that have become a critical issue to tackle.
Adipocytes are primarily characterized by the presence of lipid droplets (LD), vacuoles filled with heterogeneous triglycerides enclosed by a phospholipid monolayer and adorned with a specific set of proteins, with perilipin 1 (PLIN1) being the most abundant.While in vivo adipocytes typically feature a single lipid vacuole, cultured adipocytes never reach this condition, and instead exhibit the presence of multiple LDs.SARS-CoV-2 exhibits a heightened capacity for mediating cell-cell fusion, a notable distinction from the previously emerged SARS-CoV [14].This fusion is facilitated by the Spike (S) protein, a crucial target for antiviral interventions responsible for viral receptor binding and membrane fusion.Notably, the classification of SARS-CoV-2 variants also takes into account their membrane fusion kinetics, indicating potential differences in pathogenicity [15].
Taking the aforementioned into account, it is plausible that the increased susceptibility to adverse outcomes in COVID-19 among individuals with obesity results from a complex interplay between the virus and alterations in adipocytes.
This study aims to explore the suitability of the preadipocyte/adipocyte human cell line SGBS for investigating the effects of SARS-CoV-2 on the adipose organ.Our assessment will focus on morphological changes and alterations in gene expression.

SGBS Cells Are Permissive to SARS-CoV-2 Infection
SGBS cells are a non-immortalized cell strain originally isolated from the subcutaneous WAT of a patient with Simpson-Golabi-Behmel syndrome and reflecting key features of human primary adipose-derived stromal cells [16,17].In the undifferentiated stage (postinduction day 0, from now on referred to as PID0 or pre-adipocytes) they appear as normal fibroblasts and, when induced with an adipogenic cocktail, undergo changes in gene expression and cellular morphology that lead to the accumulation of triglycerides in the form of multiple lipid droplets (from now on referred to as adipocytes or PID10).Of note, staining of adipocytes with Oil Red O enables their efficient imaging by a confocal microscope by collecting far-red fluorescence emissions (Figure 1A,B).Given that their response to SARS-CoV-2 infection was never reported, we wanted to investigate if they were permissive to the virus infection.Our initial focus was on tracking the expression of ACE2, the main receptor for virus entry into the cell.ACE2 mRNA was weakly detectable in both adipocytes and preadipocytes.However, as illustrated in Figure 2A, while the ACE2 protein is not expressed in the latter, its expression is detectable in the former.This finding was confirmed by the confocal microscopy of cells immunostained for ACE2, which showed a large difference between the fluorescence signals of SGBS adipocytes and pre-adipocytes (Figure 1C,D).
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 3 of 15 accumulation of triglycerides in the form of multiple lipid droplets (from now on referred to as adipocytes or PID10).Of note, staining of adipocytes with Oil Red O enables their efficient imaging by a confocal microscope by collecting far-red fluorescence emissions (Figure 1A,B).Given that their response to SARS-CoV-2 infection was never reported, we wanted to investigate if they were permissive to the virus infection.Our initial focus was on tracking the expression of ACE2, the main receptor for virus entry into the cell.ACE2 mRNA was weakly detectable in both adipocytes and preadipocytes.However, as illustrated in Figure 2A, while the ACE2 protein is not expressed in the latter, its expression is detectable in the former.This finding was confirmed by the confocal microscopy of cells immunostained for ACE2, which showed a large difference between the fluorescence signals of SGBS adipocytes and pre-adipocytes (Figure 1C,D).Subsequently, we employed the Wuhan strain (B.1) of the coronavirus to infect SGBS cells at PID0 and at PID10, and measured the release of viral particles into the supernatant as a key indicator of productive infection.In Figure 2B, we note a distinctive pattern in the real-time cycle thresholds (Ct) that are inversely related to the abundance of SARS-CoV-2 mRNA.Notably, 24 h post-infection, there is a discernible drop in Cts in differentiated SGBS, but not in undifferentiated counterparts.This observation suggests that only the differentiated SGBS cells are actively engaged in the production of the virus, distinguishing them from the undifferentiated cells in this regard.
To gain further additional independent evidence in this direction, we conducted a Western blot analysis to examine the presence of the viral nucleocapsid protein (NC), which was consistently detected in infected adipocytes and in VERO E6 cells, the latter serving as a positive control [18,19] (Figure 2C).Positive infection of adipocytes was confirmed by confocal imaging: both Spike (Figure 3A, green color) and NC proteins (Figure 3C, yellow color) were detected in infected cells, as compared to control cells not exposed to the virus (Figure 3B,D).Super resolution imaging at mesoscale (100-140 nm) [20] by Image Scanning Microscopy (Airyscan mode) enabled the visualization of single viral particles [21], some of which were found in extremely close proximity to the lipid droplets (LDs) (Figure 3E,F, red Subsequently, we employed the Wuhan strain (B.1) of the coronavirus to infect SGBS cells at PID0 and at PID10, and measured the release of viral particles into the supernatant as a key indicator of productive infection.In Figure 2B, we note a distinctive pattern in the real-time cycle thresholds (Ct) that are inversely related to the abundance of SARS-CoV-2 mRNA.Notably, 24 h post-infection, there is a discernible drop in Cts in differentiated SGBS, but not in undifferentiated counterparts.This observation suggests that only the differentiated SGBS cells are actively engaged in the production of the virus, distinguishing them from the undifferentiated cells in this regard.
To gain further additional independent evidence in this direction, we conducted a Western blot analysis to examine the presence of the viral nucleocapsid protein (NC), which was consistently detected in infected adipocytes and in VERO E6 cells, the latter serving as a positive control [18,19] (Figure 2C).
Positive infection of adipocytes was confirmed by confocal imaging: both Spike (Figure 3A, green color) and NC proteins (Figure 3C, yellow color) were detected in infected cells, as compared to control cells not exposed to the virus (Figure 3B,D).Super resolution imaging at mesoscale (100-140 nm) [20] by Image Scanning Microscopy (Airyscan mode) enabled the visualization of single viral particles [21], some of which were found in extremely close proximity to the lipid droplets (LDs) (Figure 3E,F, red color).Positive infection of adipocytes was confirmed by confocal imaging: both Spike (Figure 3A, green color) and NC proteins (Figure 3C, yellow color) were detected in infected cells, as compared to control cells not exposed to the virus (Figure 3B,D).Super resolution imaging at mesoscale (100-140 nm) [20] by Image Scanning Microscopy (Airyscan mode) enabled the visualization of single viral particles [21], some of which were found in extremely close proximity to the lipid droplets (LDs) (Figure 3E,F, red color).Collectively, these findings establish that differentiated SGBS adipocytes are susceptible to SARS-CoV-2 infection, underscoring their potential utility as a model for investigating the virus's impact on the adipose organ.

The Effects of SARS-CoV-2 Infection on SGBS Morphology and Gene Expression
Next, we focused on differentiated SGBS only, and examined the influence of SARS-CoV-2 on the adipocyte phenotype and gene expression.The microphotographs captured at both low and high magnifications using light transmission and fluorescence, respectively, (Figure 4A-D) revealed a notable enlargement of LDs in infected cells as compared to controls.To verify this effect, we carried out a morphometric assessment on confocal images involving the intensity-based segmentation of single LDs.Analysis of over 1000 LDs indicated that exposure to SARS-CoV-2 leads to the significant enlargement of the LDs, with a fairly higher dispersion in infected cells (Figure 4E).Comparison of the area distributions clearly confirmed the statistically significant higher representation of larger LDs in the presence of infection (Figure 4F,G).Such enlargement may occur in cells that heighten their polarization toward adipogenesis, typically correlated with an increased abundance of transcripts marking the terminal differentiation of the adipocyte.These elements encompass the transcription factor PPARγ, the adipokines leptin and adiponectin, the crucial enzyme for triglyceride biosynthesis fatty acid synthase (FASN), the lipid transport protein FABP4, and the protein involved in LD homeostasis Perilipin 1 (PLIN1).However, droplet digital PCR (ddPCR) analysis of the abundance of these mRNAs revealed no increase; on the contrary, a significant decrease was observed in SARS-CoV-2-infected adipocytes for PPARgamma, FASN, leptin, FABP4, and PLIN1.This excludes their involvement in the observed enlargement of lipid vacuoles (Figure 4H).
of the LDs, with a fairly higher dispersion in infected cells (Figure 4E).Comparison of the area distributions clearly confirmed the statistically significant higher representation of larger LDs in the presence of infection (Figure 4F,G).Such enlargement may occur in cells that heighten their polarization toward adipogenesis, typically correlated with an increased abundance of transcripts marking the terminal differentiation of the adipocyte.These elements encompass the transcription factor PPARγ, the adipokines leptin and adiponectin, the crucial enzyme for triglyceride biosynthesis fatty acid synthase (FASN), the lipid transport protein FABP4, and the protein involved in LD homeostasis Perilipin 1 (PLIN1).However, droplet digital PCR (ddPCR) analysis of the abundance of these mRNAs revealed no increase; on the contrary, a significant decrease was observed in SARS-CoV-2-infected adipocytes for PPARgamma, FASN, leptin, FABP4, and PLIN1.This excludes their involvement in the observed enlargement of lipid vacuoles (Figure 4H).
Subsequently, we aimed to monitor the expression of genes typically upregulated in inflamed/dysfunctional WAT, a condition known to increase the risk of developing insulin resistance and other metabolic alterations.These genes include the macrophage chemotactic factors haptoglobin and MCP1/CCL2, the cytokine IL-6, and PAI-1.Intriguingly, all of them exhibited a higher transcript abundance in infected cells.Additionally, we found an increased expression of MnSOD, the primary superoxidescavenging enzyme that protects cells from the consequences of oxidative stress damage (Figure 4I).Subsequently, we aimed to monitor the expression of genes typically upregulated in inflamed/dysfunctional WAT, a condition known to increase the risk of developing insulin resistance and other metabolic alterations.These genes include the macrophage chemotactic factors haptoglobin and MCP1/CCL2, the cytokine IL-6, and PAI-1.Intriguingly, all of them exhibited a higher transcript abundance in infected cells.Additionally, we found an increased expression of MnSOD, the primary superoxide-scavenging enzyme that protects cells from the consequences of oxidative stress damage (Figure 4I).
The data suggest that SARS-CoV-2 infection has a remarkable impact on SGBS adipocyte expression, leading to the induction of an inflammatory profile and the repression of adipokine genes, as well as genes associated with triglyceride turnover.

The Role of Spike Protein in LD Enlargement
Having ruled out the virus-dependent induction of adipogenesis and/or triglyceride uptake/synthesis, we posited a hypothesis that the enlargement of LDs could be linked to the fusion of multiple organelles mediated by the viral spike protein, which, as mentioned above, has an established role in cell membrane fusion leading to the formation of pneumocytes and neuron syncytia [22][23][24].
To explore this possibility, we conducted transient transfections of SGBS cells during their adipogenic conversion (PID3) using a plasmid encoding the spike cDNA.Subsequently, we allowed the Spike(+) cells to progress until terminal differentiation (PID10).Following the verification of Spike protein expression through immunostaining, we conducted an LD morphometric analysis (Figure 5).
The data suggest that SARS-CoV-2 infection has a remarkable impact on SGBS adipocyte expression, leading to the induction of an inflammatory profile and the repression of adipokine genes, as well as genes associated with triglyceride turnover.

The Role of Spike Protein in LD Enlargement
Having ruled out the virus-dependent induction of adipogenesis and/or triglyceride uptake/synthesis, we posited a hypothesis that the enlargement of LDs could be linked to the fusion of multiple organelles mediated by the viral spike protein, which, as mentioned above, has an established role in cell membrane fusion leading to the formation of pneumocytes and neuron syncytia [22][23][24].
To explore this possibility, we conducted transient transfections of SGBS cells during their adipogenic conversion (PID3) using a plasmid encoding the spike cDNA.Subsequently, we allowed the Spike(+) cells to progress until terminal differentiation (PID10).Following the verification of Spike protein expression through immunostaining, we conducted an LD morphometric analysis (Figure 5).Remarkably, larger LDs were found to be associated with the presence of the Spike protein, with high statistical significance.Of note, transient transfection with EGFP did Remarkably, larger LDs were found to be associated with the presence of the Spike protein, with high statistical significance.Of note, transient transfection with EGFP did not induce LD enlargement (Supplementary Figure S1), suggesting that the increase in size of LDs cannot be attributed to the non-specific transfection of the cells.Collectively, these findings provide support for our hypothesis of a specific fusogenic effect of Spike proteins on LDs.

Discussion
Our study demonstrates that differentiated human SGBS cells effectively support SARS-CoV-2 infection and serve as a platform for virus replication.In contrast, undifferentiated SGBS cells show no permissiveness to active virus infection.These findings are in agreement with Martínez-Colón and colleagues [12], who observed viral particles in both adipocytes and stromal vascular cells (SVC) isolated from WAT biopsies.However, only adipocytes, not SVC, exhibited permissiveness for viral replication.The notable presence of the ACE2 protein, a crucial entry point for viral infection, identified by us in differentiated SGBS cells, but not in undifferentiated ones, may partially explain the observed outcomes.Our results diverge somewhat from previous reports which indicate low or negligible ACE2 levels in both pre-adipocytes and differentiated adipocytes [12,13] and contextually suggest alternative entry factors for SARS-CoV-2 infection, including CD147, dipeptidyl peptidase 4, neuropilin 1 (NRP1), and FURIN [25,26].The discrepancy in ACE2 expression between our study and others can be attributed to two key factors.Firstly, the use of different preadipocyte/adipocyte models, specifically human multipotent adipocyte stem cells (hMADS) or stromal vascular cells (SVC) versus SGBS cells.Secondly, the mentioned studies reported low ACE2 transcripts, consistent with our findings, while we were able to detect the ACE2 protein, a facet not explored in those studies.An aspect worth mentioning is the occurrence of virus RNA packaging within double-membrane vesicles originating from the endoplasmic reticulum [27,28].Intriguingly, LDs also emerge from the same cellular compartment [29].This shared origin prompts speculation about the potential influence of these vesicles or the underlying mechanisms of their formation, suggesting a possibility that they may favor virus packaging and replication specifically within adipocytes.By harnessing the capabilities of the SGBS human cell line, our research has unveiled intriguing insights into the influence of SARS-CoV-2 on adipocyte function.These revelations encompass not only morphostructural changes, impacting the size of LDs-the main reservoirs of triglycerides-but also transcriptional alterations affecting genes linked to metabolism and inflammation.Notably, the induction of inflammatory genes stands out as a highly anticipated outcome, aligning with findings documented in prior literature about the induction of an inflammatory response in SARS-CoV-2-infected WAT.An elevated infiltration of inflammatory cells has been documented in post-mortem samples of WAT from COVID-19 patients [11,13].The heightened expression of MCP1 and Haptoglobin in infected adipocytes, as observed in our study, could potentially play a pivotal role in driving this infiltration.Previous studies [9,30,31] highlight, in fact, their significance as chemoattractant proteins for leukocytes and macrophages, suggesting a plausible mechanism for the observed higher inflammatory cell infiltration in COVID-19 patients' WAT [11].Similarly, the infection-induced increase of PAI-1 may also contribute to macrophage infiltration [32].In addition, elevated PAI-1 levels have been linked to the impairment of insulin signaling in obesity and in the establishment of a chronic inflammatory state [33], similar to the role played by IL-6, also upregulated in our model.The data presented paint a picture of the response of adipocytes to a viral insult.On the one hand, it triggers the activation of the immune system and contributes to an increased systemic inflammatory status, while at the same time it strengthens its defenses against increased oxidative stress, as shown by the induction of MnSOD (superoxide dismutase) [34].The expression profile of SGBS adipocytes undergoes significant alterations post-infection, manifesting in noteworthy changes, such as a pronounced downregulation of leptin and genes associated with lipid synthesis, including PPARgamma, FASN, FABP4, and PLIN1.PPARgamma, a pivotal transcription factor crucial for adipogenesis, plays a key role in triglyceride accumulation within adipocytes by initiating the lipogenesis machinery [35,36].Consequently, the diminished expression of PPARgamma observed during SARS-CoV-2 infection appears to be a potential regulatory factor upstream of the observed decrease in the abundance of the other mentioned genes.These findings collectively suggest an overall impairment in adipocyte function induced by SARS-CoV-2 infection.Regarding leptin, it is worth mentioning that the "satiety hormone" has previously been reported to be elevated in both plasma and adipose tissue of COVID-19 patients [37,38].The published analyses within the field employ a predominantly cross-sectional approach, neglecting the established and direct correlation between leptin and adipose mass, or leptin and increased risk of cardiovascular diseases (as extensively documented) [39,40].Notably, adipose mass emerges as a determinant for the severe prognosis of COVID-19 [41], suggesting that the association with leptin may manifest as a secondary rather than a direct relationship.This observation holds significance, as leptin is recognized for its role in shaping Th cytokine production toward a proinflammatory phenotype (Th1, IFN) [42,43], recognized as crucial for effective control of SARS-CoV-2 [44], and leptin reduction may thus imply an impaired immune response.It is important to note that the current study has concentrated on representative adipokines and factors.However, the impact and relevance of other adipokines and molecules should not be dismissed and should be further investigated, especially in the case of factors for which a clear relationship between degree of obesity and abundance is documented (reviewed in [45]).
Lastly, it is essential to address the noticeable enlargement of lipid droplets (LD) within adipocytes.Given the information provided earlier, this enlargement cannot be linked to a more efficient accumulation of triglycerides which on the other hand are impaired.Other potential mechanisms can be considered.For instance, the ability of SARS-CoV-2 to induce cell membrane fusion and syncytia formation is noteworthy, as LDs are encased in a lipid membrane, and fusion among different vacuoles may lead to the creation of larger formations.Spike protein has been implied as the molecular mechanism driving cell membrane fusion: even if the mechanism of syncytia formation during infection is not clearly elucidated, the following mechanism has been postulated [46,47].During SARS-CoV-2 infection, the protein interacts with the ACE-2 receptor present on the target cell, and subsequently induces fusion of the viral and cellular membranes after undergoing a conformational modification.The presence of Nrp1 and TMPRSS2 proteins on cell surface, in particular, eases the cleavage of Spike protein and increases the syncytia formation.Our findings from SGBS adipocytes transfected with spike cDNA support the notion that the spike protein plays a pivotal role in mediating the fusion of lipid vacuoles.Remarkably, the mere ectopic expression of the gene is potent enough to replicate, albeit to a lesser degree, the LD enlargement effect observed during infection with the complete virus.
Many viruses were reported to interfere with the host cell's lipid metabolism.Among these, SARS-CoV-2 demonstrates the capacity to influence lipid synthesis and uptake pathways by upregulating the expression of transcription factors associated with lipogenesis and tri-acyl-glycerol synthesis in human monocytes, ultimately leading to the formation of new lipid droplets [48].Additionally, during viral infection, the core protein of human cytomegalovirus (HCMV) binds to the surface of lipid droplets (LDs) to promote the assembly of viral particles.To sustain elevated levels of constitutive lipid synthesis throughout the infection, HCMV induces the production and enlargement of lipid droplets in human fibroblasts [49,50].
In summary, our findings, leveraging SGBS cells as a valuable tool for investigating SARS-CoV-2's impact on adipocytes, uncover substantial transformations in this cell type during COVID-19.Notably, we identify an inflammatory surge the fueling systemic inflammation characteristic of the disease, coupled with a diminished endocrine function and triglyceride storage capacity, fostering a state of lipotoxicity and diminished insulin sensitivity.The observed enlargement and fusion of lipid droplets, induced by both active virus infection and Spike protein-only artificial expression, highlight the virus's ability, likely through its capsid protein, to instigate membrane fusion and generate dysfunctional tissue regions.

Cell Lines and Virus
Vero E6-TMPRSS2 cells and a clinical isolate of SARS-CoV-2, B.1, were kindly provided by Vita-Salute San Raffaele University Hospital.The manipulation and cell culture propagation of the SARS-CoV-2 isolate was performed at the Virology Unit, Pisa University Hospital, using personal protective equipment and in an approved facility that complied with biosafety level 3 regulations for laboratories, as required for the COVID etiologic agent (https://www.cdc.gov/labs/BMBL.html,accessed on 5 February 2024).Both Vero E6 and Vero E6-TMPRSS2 cells were cultured in DMEM-F12 supplemented with heatinactivated 10% fetal bovine serum (FBS) (Sigma-Aldrich, Milan, Italy), 2 mM L-glutamine, 100 I.U./mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, Milan, Italy), at 37 • C in the presence of 5% CO 2 .SARS-CoV-2 was propagated in Vero E6-TMPRSS2 cells.Briefly, cells were plated into T75 flasks, and, at about 80% of confluency, were infected with 500 µL of SARS-CoV-2 diluted in 5 mL of medium.Cells were incubated at 37 • C and 5% CO 2 for 2 h, shaking the flasks every 15 min.At the end of the incubation, the culture medium supplemented with 5% of serum was added, and the cells were incubated until full cytopathic effect was achieved.Cell lysates from the flasks were then centrifuged at 900 g for 10 min and filtered through a 45 µM filter.The supernatants containing the virus particles were aliquoted and stored at −80 • C. The viral titer was calculated from the cytopathic effect (CPE) induced by viral infection in experiments of limited dilution.The viral title was calculated by means of the Spearman-Karber method, and expressed by Median Tissue Culture Infectious Dose tissue/mL (TCID 50/mL).The viral stocks (p3) were titrated both by limited dilution methods and plaque assay.The viral titer calculated by the Reed and Muench method was, respectively, 7 × 10 8 TCID50/mL.

Western Blot
Cells were washed twice with PBS 1X and then were lysed in ice-cold 1× RIPA buffer (Millipore, USA).Lysates were centrifuged at 14,000× g for 20 min at 4 • C. Supernatants were resolved by SDS-PAGE (Criterion, Bio-Rad Laboratories, USA).Immunoblots were developed using the Clarity Western ECL Substrate (Bio-Rad Laboratories, USA) and with the ChemiDoc Imaging System (Bio-Rad Laboratories, USA).Densitometric analysis was performed using Image Lab Software v 6.1.0(Bio-Rad Laboratories, USA).

Immunostaining of Cells
Adherent cells (untransfected and transfected or infected) in WillCo dishes, after fixing and ORO staining, were rinsed 3 times with 1× PBS and 3 more times with 0.5% Bovine Serum Albumin (BSA) in PBS (PBB).Subsequently, cells were maintained for 40 min at RT in 2% BSA in PBS and rinsed 3 more times with PBB.
For direct immunostaining, SGBS cells (infected, transfected or untreated) were incubated with a primary antibody diluted in PBB, and they were maintained at 4 • C O/N.After rinsing three times with PBB, cells were incubated for one hour with αr488 diluted in PBB.Cells were then extensively rinsed with PBS.
For nuclear staining, immunostained cells were exposed for 5 min to 1 mg/100 mL of Hoechst 33,342 (Thermo Fisher Scientific, USA) in water.

Confocal and ISM Microscopy
Fluorescence was measured by a confocal Zeiss LSM 880 with Airyscan (Carl Zeiss, Oberkochen, Germany) supplied with GaAsP detectors (Gallium:Arsenide:Phosphide, Olympus, Tokyo, Japan).Samples were viewed with a 63× Apochromat NA = 1.4 oilimmersion objective.We adopted a 0.9× zoom for imaging multiple cells (1 pixel = 220 nm) and a 2-6× zoom for imaging single cells; ISM (Airyscan) imaging was carried out at zoom > 3. The pinhole size was set to 44 mm, which corresponds to 1 airy unit (AU) for the green acquisition channel.Pixel dwell time was adjusted to 1.52 µs, and 512 × 512 pixel or 1024 × 1024 images were collected.In confocal mode, we carried out the concomitant acquisition for all channels line-by-line, with the line-average set to 4. In airyscan mode, we carried out a sequential acquisition for all channels with the frame-average set to 4. The acquisition channels were set as follows: -Blue (Hoechst 33342): λex = 405 λem = 420-500 nm -Green (Alexa488): λex = 488, λem = 500-560 nm -Far-red (Oil red): λex = 640, λem = 650-700 nm The images were visualized and processed by the open-source software Fiji v 2.14.0/1.54f(NIH, Bethesda, MD, USA).

Figure 1 .
Figure 1.Phenotypes of SGBS pre-adipocytes and adipocytes, as revealed by confocal microscopy.(A,B) One SGBS pre-adipocyte (A) and one SGBS adipocyte (B) stained by Hoechst and Oil red, and confocally imaged at the medial plane by collecting fluorescence in the blue and far-red channels.(C,D) One SGBS pre-adipocyte (C) and one SGBS adipocyte (D) immunostained for ACE2 (primary Ab: rabbit antiACE2, secondary Ab: αr488) confocally imaged at the membrane plane by collecting fluorescence in the green channel.Fluorescence intensity was coded by a pseudo-color FireHot scale to pinpoint the strong difference in expression levels between the two cell types.In all cases, the cell's contours were highlighted by a white line.Scale bar: 10 µm.

Figure 1 .
Figure 1.Phenotypes of SGBS pre-adipocytes and adipocytes, as revealed by confocal microscopy.(A,B) One SGBS pre-adipocyte (A) and one SGBS adipocyte (B) stained by Hoechst and Oil red, and confocally imaged at the medial plane by collecting fluorescence in the blue and far-red channels.(C,D) One SGBS pre-adipocyte (C) and one SGBS adipocyte (D) immunostained for ACE2 (primary Ab: rabbit antiACE2, secondary Ab: αr488) confocally imaged at the membrane plane by collecting fluorescence in the green channel.Fluorescence intensity was coded by a pseudo-color FireHot scale to pinpoint the strong difference in expression levels between the two cell types.In all cases, the cell's contours were highlighted by a white line.Scale bar: 10 µm.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 4 of 15

Figure 3 .
Figure 3. SARS-CoV-2 infection of SGBS pre-adipocytes and adipocytes, as revealed by confocal and super-resolution microscopy.(A-D) Confocal imaging of SGBS adipocytes exposed (A,C) and not exposed (B,D) to SARS-CoV-2 and immunostained for the S protein ((A,B), green) and the N protein ((C,D), yellow).Red signal: lipid droplets (Oil red); blue signal: Hoechst.(E,F) Super-resolution (Image Scanning Microscopy/Airyscan) images of infected adipocytes (green: Spike protein, red: lipid droplets).The portion of the image surrounded by the white square in (E) is magnified in (F).Scale bar: 10 µm.

Figure 3 .
Figure 3. SARS-CoV-2 infection of SGBS pre-adipocytes and adipocytes, as revealed by confocal and super-resolution microscopy.(A-D) Confocal imaging of SGBS adipocytes exposed (A,C) and not exposed (B,D) to SARS-CoV-2 and immunostained for the S protein ((A,B), green) and the N protein ((C,D), yellow).Red signal: lipid droplets (Oil red); blue signal: Hoechst.(E,F) Super-resolution (Image Scanning Microscopy/Airyscan) images of infected adipocytes (green: Spike protein, red: lipid droplets).The portion of the image surrounded by the white square in (E) is magnified in (F).Scale bar: 10 µm.