Journal: International Journal of Molecular Sciences

Article Title: SARS-CoV-2 Spike Protein Induces Time-Dependent and Brain-Region-Specific Alterations in Ferroptosis Markers: A Preliminary Study in K18-hACE2 Mice

doi: 10.3390/ijms27031526

Figure Lengend Snippet: Ferroptosis machinery is composed of three hallmarks: iron dysregulation, antioxidant failure, and lipid peroxidation. Iron dysregulation, a driver of ferroptosis, occurs through increased labile iron pool (LIP). This can happen by release of iron from its storage sites in ferritin by ferritinophagy, increased iron import through transferrin receptor 1 (TFR1) and divalent metal transporter 1 (DMT1), and decreased iron export from the cell through ferroportin 1 (FPN1). Antioxidant failure occurs through increased ROS production that is fostered by increased LIP (ferrous iron: Fe +2 ) through a Fenton-like reaction. On the other hand, depletion of antioxidant defenses occurs by decreased glutathione (GSH), glutathione peroxidase 4 (GPx4), the main brakes of ferroptosis, and a decrease in the antioxidant transcription factor Nuclear Factor Erythroid 2-Related Factor 2 (NrF2). Decreased NrF2 leads to a reduction in GSH production and inhibits the cystine/glutamate antiporter SLC7A11/xCT. Slc7a11 is needed for exporting glutamate and importing cystine that is needed to form GSH along with glycine and glutamate after conversion to cysteine. Acyl-CoA synthetase long-chain family member 4 (ACSL4) incorporates long polyunsaturated fatty acids (PUFAs) into the cell membrane and converts them to PUFA-COA, which is crucial for the process of lipid peroxidation. Both iron dysregulation (through a Fenton-like reaction) and the production of hydrogen peroxide (H 2 O 2 ) propagate the process of lipid peroxidation and the formation of more lipid peroxides (PUFAOOH . ), which execute ferroptosis that leads to membrane disruption. “Created in BioRender. Hatem, A. (2026) https://BioRender.com/qzdemai , accessed on 28 December 2025”.

Article Snippet: We used primary antibodies for transferrin receptor 1 (TFR1) (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 46222) (1:1000), ferroportin 1 (FPN1) (Proteintech, Chicago, IL, USA, Cat. no. 26601-1-AP) (1:2000), divalent metal transporter-1 (DMT-1) (Abcam, Washington, DC, USA, Cat. no. ab55735) (1:1000), MDA-conjugated proteins (Abcam, Washington, DC, USA, Cat. no. ab243066) (1:1000), Nuclear Factor Erythroid 2-Related Factor 2 (NrF2) (Abcam, Washington, DC, USA, Cat. no. ab62352) (1:2000), GPx4 (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 52455) (1:1000), and beta-actin (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 3700T) (1:1000).

Techniques: Membrane, Disruption

Journal: International Journal of Molecular Sciences

Article Title: SARS-CoV-2 Spike Protein Induces Time-Dependent and Brain-Region-Specific Alterations in Ferroptosis Markers: A Preliminary Study in K18-hACE2 Mice

doi: 10.3390/ijms27031526

Figure Lengend Snippet: Changes in expression of ferroptotic markers in the hippocampus. Representative Western blot bands of measured ferroptosis markers and beta-actin are shown in ( A ). Two-way ANOVA identified a significant time effect # on TFR1 (F 1.584,15.05 = 4.972, p = 0.028), but no statistically significant differences were observed in post hoc pairwise comparisons ( B ) or in FPN1 (time effect #: F 2,12 = 6.879, p = 0.0102). Šídák post hoc comparisons showed a significant increase in FPN1 in the spike group at 2 weeks (adjusted p = 0.0126) ( D ); NRF2 ( C ), DMT1 ( E ), MDA-conjugated proteins ( F ), and GPx4 ( G ) showed no significant main effects or interactions. Data are represented as mean ± SEM; * p < 0.05. ns: non-significant.

Article Snippet: We used primary antibodies for transferrin receptor 1 (TFR1) (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 46222) (1:1000), ferroportin 1 (FPN1) (Proteintech, Chicago, IL, USA, Cat. no. 26601-1-AP) (1:2000), divalent metal transporter-1 (DMT-1) (Abcam, Washington, DC, USA, Cat. no. ab55735) (1:1000), MDA-conjugated proteins (Abcam, Washington, DC, USA, Cat. no. ab243066) (1:1000), Nuclear Factor Erythroid 2-Related Factor 2 (NrF2) (Abcam, Washington, DC, USA, Cat. no. ab62352) (1:2000), GPx4 (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 52455) (1:1000), and beta-actin (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 3700T) (1:1000).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SARS-CoV-2 Spike Protein Induces Time-Dependent and Brain-Region-Specific Alterations in Ferroptosis Markers: A Preliminary Study in K18-hACE2 Mice

doi: 10.3390/ijms27031526

Figure Lengend Snippet: Changes in expression of ferroptotic markers in the prefrontal cortex. All Western blot representative bands of measured ferroptosis markers and beta-actin are shown in ( A ). Two-way ANOVA showed no significant main effects or interactions for TFR1 ( B ), NRF2 ( C ), or FPN1 ( D ). For DMT1, two-way ANOVA identified significant main effects of time # (F 1.541,15.41 = 20.12, p = 0.0001) and treatment ## (F 1,20 = 12.61, p = 0.002). Šídák post hoc testing demonstrated a significant increase in the spike group at 2 weeks (adjusted p = 0.002), with no differences at 6 or 12 weeks ( E ). For MDA-conjugated proteins, two-way ANOVA revealed a significant time effect # (F 1.858,11.15 = 6.411, p = 0.015) ( F ). For GPx4, two-way ANOVA identified a significant main effect of treatment ## (F 1,20 = 13.60, p = 0.001), with no statistically significant difference observed at any time point ( G ). Šídák post hoc comparisons were not significant (at 6 weeks, adjusted p = 0.052). Data are represented as mean ± SEM; ** p < 0.01, ns = non-significant.

Article Snippet: We used primary antibodies for transferrin receptor 1 (TFR1) (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 46222) (1:1000), ferroportin 1 (FPN1) (Proteintech, Chicago, IL, USA, Cat. no. 26601-1-AP) (1:2000), divalent metal transporter-1 (DMT-1) (Abcam, Washington, DC, USA, Cat. no. ab55735) (1:1000), MDA-conjugated proteins (Abcam, Washington, DC, USA, Cat. no. ab243066) (1:1000), Nuclear Factor Erythroid 2-Related Factor 2 (NrF2) (Abcam, Washington, DC, USA, Cat. no. ab62352) (1:2000), GPx4 (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 52455) (1:1000), and beta-actin (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 3700T) (1:1000).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SARS-CoV-2 Spike Protein Induces Time-Dependent and Brain-Region-Specific Alterations in Ferroptosis Markers: A Preliminary Study in K18-hACE2 Mice

doi: 10.3390/ijms27031526

Figure Lengend Snippet: Changes in expression of ferroptotic markers in cerebellum. All Western blot representative bands of measured ferroptosis markers and beta-actin are shown in ( A ). Two-way ANOVA identified significant main effects of time # (F 1.654,16.54 = 8.343, p = 0.0045) and treatment ## (F 1,20 = 11.44, p = 0.003) for TFR1. Šídák post hoc comparisons were not significant at individual time points (at 2 weeks, adjusted p = 0.054) ( B ). NRF2 showed no significant main effects or interaction ( C ). For FPN1, two-way ANOVA showed significant main effects of time # (F 1.829,18.29 = 4.339, p = 0.0315) and treatment ## (F 1,20 = 6.266, p = 0.0211). Šídák post hoc testing demonstrated a significant increase in the spike group at 2 weeks (adjusted p = 0.0219), with no differences at 6 or 12 weeks ( D ). For DMT1, a significant main effect of treatment ## was detected (F 1,8 = 6.296, p = 0.0364), with no significant time or interaction effects. Šídák comparisons were not significant (at 12 weeks, adjusted p = 0.0684) ( E ). For MDA-conjugated proteins, two-way ANOVA revealed a significant time effect # (F 1.830,10.98 = 9.883, p = 0.004. Šídák post hoc testing identified a significant increase in the spike group at 12 weeks (adjusted p = 0.043) ( F ). For GPx4, two-way ANOVA showed significant main effects of time # (F 1.842,11.05 = 15.17, p = 0.0008) and treatment ## (F 1,8 = 12.47, p = 0.0077). Šídák post hoc comparisons demonstrated a significant increase in the spike group at 2 weeks (adjusted p = 0.0033), with no differences at later time points. ( G ). Data are represented as mean ± SEM; * p < 0.05, ** p < 0.01, ns = non-significant.

Article Snippet: We used primary antibodies for transferrin receptor 1 (TFR1) (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 46222) (1:1000), ferroportin 1 (FPN1) (Proteintech, Chicago, IL, USA, Cat. no. 26601-1-AP) (1:2000), divalent metal transporter-1 (DMT-1) (Abcam, Washington, DC, USA, Cat. no. ab55735) (1:1000), MDA-conjugated proteins (Abcam, Washington, DC, USA, Cat. no. ab243066) (1:1000), Nuclear Factor Erythroid 2-Related Factor 2 (NrF2) (Abcam, Washington, DC, USA, Cat. no. ab62352) (1:2000), GPx4 (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 52455) (1:1000), and beta-actin (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 3700T) (1:1000).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SARS-CoV-2 Spike Protein Induces Time-Dependent and Brain-Region-Specific Alterations in Ferroptosis Markers: A Preliminary Study in K18-hACE2 Mice

doi: 10.3390/ijms27031526

Figure Lengend Snippet: Changes in expression of ferroptotic markers in the olfactory bulb. All Western blot representative bands of measured ferroptosis markers and beta-actin are shown in ( A ). Two-way ANOVA identified a significant main effect of time # for TFR1 (F 1.998,11.99 = 11.93, p = 0.001), with no significant main effect of treatment. Šídák post hoc comparisons between saline and spike groups were not significant at 2, 6, or 12 weeks ( B ). For FPN1, two-way ANOVA showed a significant main effect of treatment ## (F 1,8 = 8.329, p = 0.02) with no significant time or interaction effects. Šídák post hoc testing identified a significant increase in the spike group at 12 weeks (adjusted p = 0.0006) ( D ). For DMT1, a significant main effect of treatment ## was detected (F 1,20 = 5.962, p = 0.024). Šídák comparisons were not significant at individual time points ( E ). NRF2 ( C ) and MDA-conjugated proteins ( F ) showed no significant main effects or interactions. For GPx4, two-way ANOVA revealed a significant main effect of treatment ## (F 1,8 = 8.051, p = 0.021) with no significant time or interaction effects. Šídák post hoc comparisons were not significant (at 2 weeks, adjusted p = 0.0799) ( G ). Data are represented as mean ± SEM; *** p < 0.001, ns = non-significant.

Article Snippet: We used primary antibodies for transferrin receptor 1 (TFR1) (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 46222) (1:1000), ferroportin 1 (FPN1) (Proteintech, Chicago, IL, USA, Cat. no. 26601-1-AP) (1:2000), divalent metal transporter-1 (DMT-1) (Abcam, Washington, DC, USA, Cat. no. ab55735) (1:1000), MDA-conjugated proteins (Abcam, Washington, DC, USA, Cat. no. ab243066) (1:1000), Nuclear Factor Erythroid 2-Related Factor 2 (NrF2) (Abcam, Washington, DC, USA, Cat. no. ab62352) (1:2000), GPx4 (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 52455) (1:1000), and beta-actin (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 3700T) (1:1000).

Techniques: Expressing, Olfactory, Western Blot, Saline