6. FIGURE LEGENDS
Figure 1 . SARS-CoV-2 replication kinetics .(A) Representation of the experimental timeline schedule.(B) Kinetics of SARS-CoV-2 replication using ancestral (Wh; left) and Omicron (BA.5; right) measured by mRNA of nucleocapsid (top) and ORF1ab (bottom) in culture supernatants via qPCR during osteoclast formation. (C) Kinetics of SARS-CoV-2 replication using ancestral (Wh; left) and Omicron (BA.5; right) measured by mRNA of nucleocapsid (top) and ORF1 (bottom) intracellularly in cell lysates via qPCR during osteoclast formation (normalized to human RNase P gene).(D) The efficiency of SARS-CoV-2 infection (measured by flow cytometry as intracellular expression of viral nucleocapsid) at 3 dpi; expression in the infected VeroE6 cell line was used as a positive control. (E) SARS-CoV-2 (Wh strain) titration in culture supernatants reported as the number of plaque-forming units per ml (PFU/ml). dpi, days post-infection. n: 2-5 donors were used.
Figure 2. Effects of SARS-CoV-2 on cell viability and mROS production. (A) Assessment of programmed cell death, presented as the percentage of cells stained with Annexin V/7-AAD, following SARS-CoV-2 infection with the ancestral variant at two distinct time points, 3 dpi and 12 dpi. (B) Representative dot plots obtained through flow cytometry, illustrating Annexin V/7-AAD staining at the time points indicated in (A). (C) Quantification of mitochondrial ROS (mROS) production, expressed as the percentage of cells stained with MitoSOX measured by flow cytometry, at various time intervals. (D) Representative dot plots for cellular mROS measurement obtained through flow cytometry, illustrating MitoSOX staining at the time points indicated in (C). Wh, ancestral variant. Ni, non-infected controls. hpi, hours post-infection. ns = not significant. n: 2-5 donors were used.
Figure 3. SARS-CoV-2 increases ACE2 expression in infected macrophages. (A) Evaluation of ACE2 surface expression in macrophages at 3 dpi, measured through flow cytometry (values connected are paired samples obtained from the same donor). (B)Representative histograms obtained by flow cytometry illustrating ACE2 expression as depicted in (A). (C) Quantification of relative ACE2 protein expression via Western blot analysis. Values connected are paired samples obtained from the same donor. (D) Representative blots of ACE2 and Na+/K+ ATPase. Wh, ancestral variant. Ni, non-infected controls. —: Jurkat cells; +: Huh7.5 cells, CM: cardiomyocytes, n: 5-8 donors were used.
Figure 4. SARS-CoV-2 positively modulates osteoclast differentiation. (A) Osteoclast quantification presented as a percentage (%) of OC/total cells at 12 dpi. (B) Representative images corresponding to (A) at 200x magnification. (C)Osteoclast quantification displayed as a percentage (%) of OC/total cells at 12 dpi, infected with three different multiplicities of infection (MOI). Values connected are paired samples obtained from the same donor. (D) Representative images corresponding to (C) at 200x magnification. Ni, non-infected controls. Wh, ancestral variant. BA.5, Omicron variant. Scale bar: 200 μm. *p < 0.05, **p < 0.01, and ***p < 0.001. ns, not significant. n: 2-5 donors were used.
Figure 5. SARS-CoV-2 enhances osteoclast maturation and bone-resorbing activity. (A) CD51/61 expression in osteoclasts at 12 dpi, measured by flow cytometry (values connected are paired samples obtained from the same donor). (B) Representative dot plots obtained by flow cytometry depicting CD51/61 expression (A).(C) Resorption area expressed as a percentage of the total analyzed field observed under a light microscope (200x magnification) and analyzed using Image-J. (D) Top: Representative data of TRAP-positive (red) osteoclasts at 12 dpi. Bottom: Bone resorption pits formed by osteoclasts, staining slices at 12 dpi with toluidine blue. Yellow arrowheads indicate the presence of pits. Zoomed-in views of yellow dashed squares provide a more detailed observation. Ni, non-infected controls. Wh, ancestral variant. BA.5, Omicron variant. Scale bar: 200 μm. *p < 0.05. n: 2-3 donors were used.
Figure 6. mRNA Levels of RANK, NFATc1, MMP9, and DC-STAMP during osteoclastogenesis after SARS-CoV-2 infection. The mRNA levels (expressed as 2-ΔCt) of: (A) RANK at 3 dpi,(B) NFATc1 at 6 dpi, (C) MMP9 at 12 dpi, and(D) DC-STAMP at 12 dpi, were quantified using real-time quantitative PCR (RQ-PCR) in paired samples obtained from the same donor. The values of mRNA levels are relative to GAPDH (a housekeeping gene). Ni, non-infected controls. Wh, ancestral variant. *p < 0.05. n: 4-6 donors were used.
Figure 7. SARS-CoV-2 infection of macrophages induces a profile switch from M1 to M2. (A) Representative images of macrophage morphology at two different time points, 1 dpi, and 3 dpi (100x). Scale bar 50 μm. (B) Kinetics of membrane cell markers of M1 and M2 macrophages measured by flow cytometry during SARS-CoV-2 infection. Mean fluorescence intensity (MFI) median values are shown. (C)Representative histograms of flow cytometry in (B). (D)Kinetics of cytokines production in cell supernatants measured by ELISA during SARS-CoV-2 infection. (E) Measurement of mRNA of M2-related cytokines (TGF-β and IL-10) and M1-related cytokines (TNF-α and IL-1β) by RT-qPCR at 3 dpi in paired samples obtained from the same donor. The values of mRNA levels are relative to GAPDH (a housekeeping gene). Ni, non-infected controls. Wh, ancestral variant. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. ns = not significant. n: 5-8 donors were used.
Figure 8. SARS-CoV-2-enhanced osteoclastogenesis through RANKL independent mechanism. (A) Quantifying the number of TRAP-positive osteoclasts (expressed as an absolute count) at 12 dpi in the absence of RANKL. (B) Representative images of (A) (x200). Ni, non-infected control. Wh, ancestral variant. BA.5, Omicron variant. Scale bar: 200 μm. *p < 0.05. n: 2-3 donors were used.