Fig 2: Proinflammatory activation and M1 polarization of HMC3 by SARS-CoV-2 infection. (A and B) Heat maps of significantly upregulated genes during SARS-CoV-2 infection enriched in (A) immune response and (B) microglial M1 polarization. (C) The graphs show the measurements of secreted proinflammatory cytokines, including IL-1β, IL-6, and TNF-α, and anti-inflammatory cytokine IL-10 by enzyme-linked immunosorbent assay (ELISA). (D) Confocal immunofluorescence images of mock or SARS-CoV-2-infected HMC3 with anti-CD68 antibody. (E) Quantitative analysis of microglial activation markers, including CD68, CX3CL1, and CX3CR1 using Western blotting. Actin served as the loading control. (F) RT-qPCR analysis of the representative M1 (NOS2; left) and M2 markers (Arginase-1; right). (G) Assessment of proteins of M1 markers, including CD16, phopho-Stat1, and Stat1 by Western blotting. Actin served as the loading control. Statistically significant differences between the groups were determined using ANOVA; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Symbols represent mean ± SEM.

Fig 3: Sox2 overexpression suppressed noise-induced upregulation of IL6 and TNF-α. A IL6 immunofluorescence in cochlear cross-section after noise treatment. The IL6 expression level was elevated after traumatic noise exposure in HCs, while it was inhibited in the Sox2 overexpression group. Myosin7a is used as a marker of cochlear HCs. B Western blot analysis of sensory epithelia shows increased expression levels of TNF-α and IL6 in control mice after noise injury, but it was significantly inhibited in Prestin-Sox2OE mice. β-Actin served as the sample loading control. Data are presented as means ± SEM.; n = 3. *P < 0.05, **P < 0.01. Scale bar = 20 μm

Fig 4: Sox2 overexpression suppressed noise-induced upregulation of inflammatory cytokines. A The mRNA expression levels of apoptosis-related genes (Aif, Bax, EndoG, Casp3, Casp8, and Casp9) were elevated 6 h after the completion of noise exposure. B The mRNA expression levels of necrosis-related genes (Prkaα1, Ripk1, and Ripk3) and pro-inflammatory cytokines (Tnf, Il-1β, and Il-6) were elevated 6 h after the completion of noise exposure. C TNF-α immunofluorescence in HCs after noise exposure. The TNF-α expression level was elevated after traumatic noise exposure in HCs, while it was inhibited in the Sox2 overexpression group. Parvalbumin is used as a marker of cochlear HCs. Data are presented as means ± SEM.; n = 6. *P < 0.05, **P < 0.01. Scale bar = 20 μm

Fig 5: Sox2 overexpression suppressed LPS-induced upregulation of IL6 and TNF-α. A IL6 immunofluorescence in cochlear cross-section after LPS treatment. Myosin7a is used as a marker of cochlear HCs. (B) Western blot analysis of sensory epithelia shows increased expression levels of TNF-α and IL6 in control mice after LPS treatment, but the LPS-induced increase of the TNF-α and IL6 protein was significantly inhibited in Prestin-Sox2OE mice. β-Actin served as the sample loading control. Data are presented as means ± SEM.; n = 3. *P < 0.05, **P < 0.01. Scale bar = 20 μm