www.sciencesignaling.org/cgi/content/full/8/406/ra126/dc1 Supplementary Materials for The microrna mir-485 targets host and influenza virus transcripts to regulate antiviral immunity and restrict viral replication Harshad Ingle, Sushil Kumar, Ashwin Ashok Raut, Anamika Mishra, Diwakar Dattatraya Kulkarni, Takeshi Kameyama, Akinori Takaoka, Shizuo Akira, Himanshu Kumar* This PDF file includes: *Corresponding author. E-mail: hkumar@iiserb.ac.in Published 8 December 2015, Sci. Signal. 8, ra126 (2015) DOI: 10.1126/scisignal.aab3183 Fig. S1. Kinetics of viral infections of human cells. Fig. S2. Kinetics of the effects of transfection of various human cells with poly(i:c). Fig. S3. mir-485 is increased in abundance in HEK 293T cells after viral infection. Fig. S4. mir-485 suppresses the expression of RIG-I. Fig. S5. Analysis of the mir-485 mediated regulation of MDA5 expression. Fig. S6. mir-485 suppresses the virus-induced expression of IFN-encoding genes and promotes NDV replication. Fig. S7. An mir-485 inhibitor differentially regulates the replication of H5N1 and NDV. Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/8/406/ra126/dc1) Table S1 (Microsoft Excel format). mirna expression profile in HEK 293T cells infected with NDV.
Fig. S1. Kinetics of viral infections of human cells. (A to L) Quantification of the abundances of NP (A, D, and G), RIG-I (B, E, H, and J), and IFNβ (C, F, I, K, and L) mrnas in human SAECs (A to C), A549 cells (D to F), HEK 293T cells (G to K), and human PBMCs (L) that were left uninfected (Ctrl) or were infected for the indicated times with H5N1 (A to I) and NDV (J to L) at an MOI of 1.0. Data are means SEM of triplicate samples from a single experiment and are representative of three independent experiments.
Fig. S2. Kinetics of the effects of transfection of various human cells with poly(i:c). (A to D) Quantification of the abundances of IFNβ (A and C) and RIG-I (B and D) mrnas in HEK 293T cells (A and B) and MCF7 cells (C and D) that were left untransfected (Ctrl) or were transfected with poly(i:c) (5 μg/ml) for the indicated times. Data are means SEM of triplicate samples from a single experiment and are representative of three independent experiments.
Fig. S3. mir-485 is increased in abundance in HEK 293T cells after viral infection. (A to C) Quantification of the abundance (A) and copy number (B and C) of mir-485 in HEK 293T cells infected for the indicated times with NDV at an MOI of 1. The abundance was calculated with the absolute quantification method. The copy number was calculated using mir-485 mimic as a standard. The percentage of mir-485 in small RNA was determined by Bioanalyser 2000. Data are means SEM of triplicate samples from a single experiment and are representative of three independent experiments.
Fig. S4. mir-485 suppresses the expression of RIG-I. (A) Conserved mir-485 binding sites at positions 213 and 1784 in the 3 UTR of RIG-I. Hsa, human; Mmu, mouse; Ptr, chimpanzee; Mml, rhesus macaque. (B) Information on the human Ago2-binding site in the 3 UTR of RIG-I as predicted by the iclip database. (C to E) Quantification of the abundance of RIG-I mrna in HEK 293T cells (C to E) and MCF7 cells (E) transfected with the mir-485 mimic (m-485) (C and D), with anti-mir485, anti-mir-125a, anti-mir-205a, or anti-mir-146a (C and D), or with p485 (E) and then infected for the indicated times with NDV at an MOI of 1 (C and D) or transfected with poly(i:c) (5 μg/ml) for the indicated times (E). (F to H) Quantification of the abundance of mir-485 (F, left and H) or RIG-I mrna (F, right and G) in human SAECs (F), human PBMCs (G), and HEK 293T cells (H) transfected with mir-485 mimic (F to H), mir-1 (F), anti-mir-485 (G), or Ago2 Flag (H) and infected for the indicated times with H5N1 (F and G) or NDV (H) at an MOI of 1. Data are means SEM of triplicate samples from a single experiment and are representative of three independent experiments.
Fig. S5. Analysis of the mir-485 mediated regulation of MDA5 expression. (A to D) Quantification of the abundance of MDA5 mrna in HEK 293T cells (A to C) or BMDCs (D) transfected with p485 (A and B) or mir-485 mimic (C and D) and then infected for the indicated times with H5N1 (A) or NDV (B and D) or transfected for the indicated times with poly (I:C) (C). Data are means SEM of triplicate samples from a single experiment and are representative of three independent experiments.
Fig. S6. mir-485 suppresses the virus-induced expression of IFN-encoding genes and promotes NDV replication. (A) Transcriptional activity of the IFNα-4 promoter in HEK 293T cells that were untransfected or were transfected with mir-485 and then infected for 24 hours with NDV at an MOI of 1. (B to F) Quantification of the abundances of IFNα (B and C), IFNβ (D to F), IP10 (D and E), and IL-29 (D) mrnas in BMDCs (C), HEK 293T cells (B and D to F), and H485 cells (E) transfected with p485 (B to D), mir-485 (C and F), or am-125a, am-146a, am-205a, or am-485 (F) and followed by transfection with poly(i:c) (E) or infection for the indicated times with H5N1 at an MOI of 1 (B) or NDV at an MOI of 1, 5, or 10 (B, C, D, and F). Data are means SEM of triplicate samples from a single experiment and are representative of three independent experiments.
Fig. S7. An mir-485 inhibitor differentially regulates the replication of H5N1 and NDV. (A and C) Quantification of the abundances of NDV RNA (A) and NP mrna (C) in HEK 293T cells (A) and human PBMCs (C) transfected with m-485, am-485, am-125a, am-205a, or am- 146a and then infected for the indicated times with NDV or H5N1 at an MOI of 1. (B) Densitometric analysis of the relative abundance of RIG-I protein in the indicated samples from three independent Western blots from experiments shown in Fig. 5E. (D and E) Quantification of mir-485 abundance after Ago2 pulldown in HEK 293T cells transfected with plasmid encoding Ago2 Flag with or without the mir-485 mimic and infected with H5N1 virus at an MOI of 1 or 5. Data are means SEM of triplicate samples from a single experiment and are representative of three independent experiments.