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. 2021 Sep 30;17(9):e1009950. doi: 10.1371/journal.ppat.1009950

Fig 5. Pre induction of miR-24 by MAPK activator promotes viral replication and effect of miR-24 is regulated through 3’UTR of STING mRNA.

Fig 5

(A) HT1080 cells were treated by PMA (10μM) and miR-24 expression was determined. (B) HT1080 cells or HT1080 STING KD cells were treated with PMA and then HSV1 infection was given 2 hours post treatment. HSV1 virus titer was quantified in cells and culture supernatants 8 hours post infection. Control group was treated with DMSO. (C) HT1080 cells and HT1080 STING KD cells reconstituted with STING-ORF were transfected by Scramble or miR-24 and cells were harvested after 24 hours to determine STING protein level by western blotting. (D) HT1080 cells and HT1080 STING KD cells reconstituted with STING-ORF were infected with HSV1 and cells were harvested after 6 and 12 hours to determine STING protein level by western blotting. (E) HT1080 cells and HT1080 STING KD cells reconstituted with STING-ORF were infected with HSV1, cells and culture media were harvested after 12 hours to determine viral titer. (F) HT1080 cells (WT), HT1080 STING KD cells reconstituted with ORF-UTR (containing STING ORF+3’UTR) and HT1080 STING KD cells reconstituted with ORF- mut UTR (containing STING ORF + mutated 3’ UTR where the binding site for miR-24 has been mutated in 3’UTR) were transfected with Scramble RNA or miR-24 and cells were harvested after 24 hours to determine STING protein level by western blotting. (G) HT1080 cells, HT1080 STING KD cells reconstituted with ORF-UTR (containing STING ORF+3’UTR) and HT1080 STING KD cells reconstituted with ORF- mut UTR (containing STING ORF + mutated 3’ UTR where the binding site for miR-24 has been mutated in 3’UTR) were infected with HSV1 and cells were harvested after 12 hours to determine STING protein level by western blotting. (Numbers above the blot represent densitometric quantification of STING normalized against actin) (H) HT1080 cells, HT1080 STING KD cells reconstituted with ORF-UTR and HT1080 STING KD cells reconstituted with ORF-mut UTR were infected with HSV1 and cells and culture media were harvested after 16 hours to determine viral titer. (A, B, E, H—Mean ± SEM, N = 3). *P<0.05, **P<0.01, ***P<0.001.