Figure 6.

Mn treatment enhances RNA-mediated or RNA-virus-infection-mediated innate immune responses

(A) Human macrophages were treated with or without Mn²⁺ at 25 μM, then transfected with 5′ppp-dsRNA (1 μg/mL) or infected with SeV at 40 HA units/mL. Total RNA was extracted 24 h after RNA stimulation. Real-time RT-PCR was performed to measure the gene expression level of IFNL1, IFNB, and IFNA. One representative experiment of at least three independent experiments is shown. And each was done as triplicate. Data are shown as mean ± SD (n = 3). Not significant (ns) p> 0.05, ∗p< 0.05, ∗∗∗∗p< 0.0001 (One-way ANOVA)) were indicated in the figures where the IFN mRNA expression level was compared between RNA-transfected/infected and Mn²⁺/RNA-treated cells.

(B) The effect of Mn²⁺ pretreatment on morphological changes of SeV-infected human primary macrophages. Human macrophages were pretreated with Mn²⁺, then mock-infected (uninfected) or infected with SeV at 40 HA units/mL. Cells were observed under a phase-contrast microscope at 40× and 100× magnification 48 h after SeV infection. Scale bar = 100 μM. One representative experiment of at least three independent experiments is shown.

(C) The total cell lysate was collected at 24 h after infection, and Western blotting was performed using anti-SeV and anti-GAPDH antibodies. One representative experiment of at least three independent experiments is shown. The band intensity of SeV proteins was normalized by the band intensity of GAPDH (Image J), the values are indicated in the image.

(D) Macrophages were treated with or without Mn²⁺, then infected with SeV at 40 HA units/mL. The total cell lysate was collected at 24 h after infection, and WB was performed using anti-p-TBK1, anti-TBK1 and anti-GAPDH antibodies. One representative experiment of at least three independent experiments is shown.

(E) Cell culture supernatants were collected at 24, 48, and 72 h after SeV virus infection. The protein expression of IFN-α, IFN-β, and IFN-λ1 was detected by ELISA. One representative experiment of at least three independent experiments is shown. Each was done as duplicate. Data are shown as mean ± SD (n = 2). ∗∗∗p < 0.001 and ∗∗∗p< 0.0001 (One-way ANOVA)) were indicated in the figures where the IFN protein expression level was compared between SeV-infected and Mn²⁺/SeV-infected cells.