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letter
. 2021 Sep 26;11(9):e391. doi: 10.1002/ctm2.391

FIGURE 2.

FIGURE 2

DDX5 N terminal domain interacts with S2 region of S protein which mediates MSR1 expression inhibition and DDX5 cooperates with METTL3 to regulating MSR1 mRNA transcription through modifying R‐loop resolution. (A) Western blot detection of DDX5 protein in the cytoplasmic or the nuclear fractions of macrophages incubated with HIS‐tagged recombinant viral S protein (GAPDH as cytoplasmic maker; Histone‐H3 as nuclear maker). (B) Fluorescence analysis of the subcellular distribution of DDX5 (red) in macrophages incubated with recombinant viral S protein (green) or PBS. Nucleus, blue. (C) Domain mapping analysis of the interaction between MYC‐tagged DDX5 protein and HIS‐tagged viral S protein S, S1 region or S2 region by Co‐IP. (D‐E) Western blot analysis of MSR1 expression in macrophages incubated with HIS‐tagged recombinant viral S1 protein (D) or S2 protein (E). (F) Western blot analysis of MSR1 expression in macrophages transfected with MYC‐tagged DDX5 plasmids and incubated with HIS‐tagged recombinant viral S2 protein. (G) Domain mapping analysis of the interaction between MYC‐tagged DDX5, N‐terminal domain (N), p68HR domain (HR), HELIC domain (HC), DEXD domain (DE), and HIS‐tagged recombinant viral S2 protein in 293T cells. (H) MeRIP‐qRTPCR analysis of MSR1 mRNA expression in macrophages transfected with MYC, MYC‐DDX5, or MYC‐METTL3 plasmids. (I) DRIP‐qRTPCR analysis of MSR1 mRNA expression in macrophages transfected with MYC, MYC‐DDX5, or MYC‐METTL3 plasmids. (J) DRIP‐qRTPCR analysis of MSR1 mRNA expression in macrophages transfected with control siRNA (NC), siRNA‐DDX5, or siRNA‐METTL3. (K) Fluorescence analysis of the subcellular distribution of R‐loop foci in macrophages transfected with NC, si‐DDX5, or si‐METTL3. (L) DRIP‐qRTPCR analysis of MSR1 mRNA in macrophages transfected with NC or si‐DDX5 with RNaseH1 treatment or not. (M) Fluorescence analysis of the subcellular distribution of R‐loop foci in macrophages transfected with NC or si‐DDX5 with RNaseH1 treatment or not. (N) DRIP‐qRTPCR analysis of MSR1 mRNA in macrophages transfected with MYC, or MYC‐METTL3 plasmid with RNaseH1 treatment or not. (O) qRTPCR analysis of MSR1 new transcript in macrophages transfected with NC, si‐DDX5, si‐METTL3, or si‐FTO siRNAs. (P) qRTPCR analysis of MSR1 new transcript in macrophages treated with RNaseH1. *P < .05