Fig 1: CLS promotes M2-like macrophage polarization in RSV-infected mice. (A) Flow cytometry analysis of BALF cells for F4/80+iNOS+ M1 cells and F4/80+CD206+ M2 cells. (B) Immunofluorescence staining of iNOS (green) and CD206 (red) in lung sections with DAPI nuclei (blue). Scale bar: 30 µm. (C) Relative mRNA expression of M1 marker (iNOS, IL-1β, IL-6, and TNF-α) and M2 marker (Arg-1, CD206, IL-10, and TGF-β). Data are shown as mean ± SD; n=3 per group. *P<0.05, **P<0.01, ***P<0.001, ns P>0.05 compared to the RSV group (one-way ANOVA with Dunnett’s post hoc).
Fig 2: CLS combination synergistically reduces RSV infection and enhances macrophage survival compared to cordycepin monotherapy in RSV-infected cells. (A) Immunofluorescence staining of RSV-F (red, with blue DAPI nuclei) in alveolar macrophages (AMs) across experimental groups: AM controls, RSV-infected, RSV+CLS, and RSV+Cordycepin (CD). Scale bar: 20 µm. (B) RSV mRNA expression levels. (C) Dual immunofluorescence for M1 marker iNOS (red) and M2 marker CD206 (green) with DAPI nuclei (blue). Scale bar: 20 µm. (D) Levels of LDH in culture supernatants indicate cytotoxicity. (E) CCK-8 analysis of macrophage proliferation. (F) EdU staining of macrophages. Scale bar: 200 µm. Data are presented as mean ± SD; n=6 per group. *P<0.05, **P<0.01, ***P<0.001, ns P>0.05 (one-way ANOVA with Tukey’s post hoc).
Fig 3: FUS‐assisted hydrogen‐gene therapy attenuated neuroinflammation in the hippocampus. a) Immunofluorescence of CD206 (M2 marker, green) and inos (M1 marker, green) in hippocampus. Iba1 (microglia marker) were stained red and nuclei were stained blue (Con, A+S, PBS, S, HPP, HPPS) (n = 3 biologically independent experiments). b) Western blot of lba‐1, inos, CD206, NF‐κB p65, NF‐κB p‐p65 of different groups (Con, A+S, PBS, S, HPP, HPPS) (n = 3 biologically independent experiments). c,d) The number of platform crossings and the duration spent in the third quadrant during the MWM test for the different experimental groups (Con, A+S, HPPS+FUS) (n = 6 biologically independent rat). e) Representative swimming tracks and time heatmap visualizations in the MWM test of various groups (Con, A+S, HPPS+FUS). f,g) The duration of interaction with both the novel and familiar objects during the NOR test across different groups (Con, A+S, HPPS+FUS) (n = 6 biologically independent rat). h) Representative movement tracks and time heatmap visualizations in the testing phase of NOR (Con, A+S, HPPS+FUS). Data are presented as Mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns: no significance.
Fig 4: CLS synergy amplifies M2-like polarization in RSV-infected alveolar macrophages beyond cordycepin alone. (A) Flow cytometry analysis of F4/80+iNOS+ M1 cells and F4/80+CD206+ M2 cells. (B) RT-qPCR results of M1 marker mRNA (iNOS, IL-1β, IL-6, and TNF-α) and M2 marker mRNA (Arg-1, CD206, IL-10, and TGF-β) expression. Data are shown as mean ± SD; n=6 per group. *P<0.05, **P<0.01, ***P<0.001, ns P>0.05 (one-way ANOVA with Tukey’s post hoc).
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