Additional file 1 of Photodynamic and nitric oxide therapy-based synergistic antimicrobial nanoplatform: an advanced root canal irrigation system for endodontic bacterial infections

Additional file 1: Table S1. Ce6 loading capacity and encapsulation efficiency of the CGP. Fig. S1. 1H nuclear magnetic resonance spectra of G-PEG-PCL. Fig. S2. The stability of CGP during 14 days. Fig. S3. Ultraviolet-visible absorption spectra of free Ce6, CPP, and CGP. Fig. S4. Fluorescence emission spectra of free Ce6, CP, and CGP. Fig. S5. Total ROS generation profiles of different groups over various durations. Data are presented as mean ± SEM, n = 3, **** p ≤ 0.0001. Comparison between CGP+Laser versus other groups. Fig. S6. Representative images of plate samples of Enterococcus faecalis after various treatments. Fig. S7. bacterial viability of Enterococcus faecalis after various treatments. Fig. S8. HE staining of heart, lung, liver, spleen, and kidney in healthy group and CGP+Laser treated AP group

IAV infection promotes YTHDC1 expression.

(A) A549 cells were infected with the PR8 virus at an MOI of 5 (top) or at an MOI of 0.01 (below). Cell lysates were collected at 3, 6, 9, 12, 24, and 36 hpi and subjected to western blotting analysis. (B) A549 cells were infected with the PR8 virus at MOI 0.01 for 24 h, mock-infected cells as a control, and YTHDC1 mRNA was determined by RT-qPCR which normalized to GAPDH mRNA. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (Student t-test; ns, not significant). (C) A549 cells were infected with the PR8 virus at an MOI of 0.01 for 24 h. Viral NP and host protein YTHDC1 were detected by the confocal assay. Diamidino-2-phenylindole shows the nuclei of cells. Data are presented as the average of 30 sights of the fluorescence which was calculated by ImageJ, and error bars indicate the standard error of the mean (SEM) (Student t-test, *** (TIF)</p

NS1 associates with YTHDC1.

(A) HEK293T cells were infected with PR8 virus at a multiplicity of infection (MOI) of 0.01 or transfected with Flag-NS1 and vector for 24 hours, cell lysates were immunoprecipitated with an anti-Flag or anti-NS1 antibody and then analyzed by silver stain. (B-C) HEK293T cells were transfected with the indicated plasmids for 24 h. The cell lysates were treated with or without 100U RNase A/T at 37°C for 30 min. CoIP assay was performed using an anti-HA (B) and anti-Flag (C) antibody and analyzed by western blotting. (D) A549 cells were infected with the PR8 virus at an MOI of 0.01 for 24 hours. The cell lysates were treated with or without 100U RNase A/T at 37°C for 30 min. CoIP assay was performed using an anti-YTHDC1 antibody. Input and IP complexes (NS1 and YTHDC1) were analyzed by western blotting. (E-G) A549 cells were infected with the influenza virus at MOI of 5. Cells were fixed and analyzed for the colocalization of YTHDC1 with NS1 after 3 and 6 hours post infection. Scale bar, 20 μm. (F and G) The normalized fluorescence of YTHDC1 and NS1 along the white arrowheads shows overlapping peaks.</p

NSA530C attenuated on MAVS knockout MDCK cells.

MAVS knockout MDCK cells were infected with WT and NSA530C virus for 12, 24, and 36 h. The virus titer was determined by TCID50. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) (two-way ANOVA test; ns, not significant; *, P (TIF)</p

Analysis of GGAC motif NS segment of IAV.

The ratio of ISS in the NS segment (with GGAC number/without GGAC number/total of numbers analyzed) in each analyzed subtype of IAVs isolated from human and avian species. Both human H5 (1821/0/1821), H7 (1035/0/1035), and H9 (15/0/15), and avian H5 (3930/28/3958), H7 (1438/132/1570), and H9 (2617/8/2625) viruses. (TIF)</p

Proteins pulled down by NS1 and Flag-NS1.

N6-methyladenosine (m6A) modification on viral RNAs has a profound impact on infectivity. m6A is also a highly pervasive modification for influenza viral RNAs. However, its role in virus mRNA splicing is largely unknown. Here, we identify the m6A reader protein YTHDC1 as a host factor that associates with influenza A virus NS1 protein and modulates viral mRNA splicing. YTHDC1 levels are enhanced by IAV infection. We demonstrate that YTHDC1 inhibits NS splicing by binding to an NS 3′ splicing site and promotes IAV replication and pathogenicity in vitro and in vivo. Our results provide a mechanistic understanding of IAV-host interactions, a potential therapeutic target for blocking influenza virus infection, and a new avenue for the development of attenuated vaccines.</div

Analysis of GGAC motif in NS segment of H5, H7 and H9 IAVs.

Analysis of GGAC motif in NS segment of H5, H7 and H9 IAVs.</p

Identification proteins interact with NS1.

(A) Schematic strategy for purification and identification of NS1 binding proteins via IP assay. Plasmid expressing Flag-tagged NS1 was transient transfection into HEK293T cells. HEK293T cells were infected or mock-infected with the PR8 virus. Cell lysates were performed for affinity purification by immunoprecipitation with protein A/G beads connected with Flag or NS1 antibody. The purified elutes were boiled in the SDS-PAGE loading buffer and then analyzed by MS. (B) Venn diagram showing the overlaps of differentially candidate NS1 binding proteins either in transfected or infected HEK293T cells. (C) A549 cells were transfected with indicated plasmids. Twenty-four hours after transfection, cells were fixed, permeabilized, and probed with anti-Flag/HA antibodies. FITC and Cy3 were used to visualize the indicated proteins. Diamidino-2-phenylindole shows the nuclei of cells. (D) The normalized fluorescence of YTHDC1 and NS1 along the white arrowheads shows overlapping peaks. (TIF)</p

Primers and probes.

N6-methyladenosine (m6A) modification on viral RNAs has a profound impact on infectivity. m6A is also a highly pervasive modification for influenza viral RNAs. However, its role in virus mRNA splicing is largely unknown. Here, we identify the m6A reader protein YTHDC1 as a host factor that associates with influenza A virus NS1 protein and modulates viral mRNA splicing. YTHDC1 levels are enhanced by IAV infection. We demonstrate that YTHDC1 inhibits NS splicing by binding to an NS 3′ splicing site and promotes IAV replication and pathogenicity in vitro and in vivo. Our results provide a mechanistic understanding of IAV-host interactions, a potential therapeutic target for blocking influenza virus infection, and a new avenue for the development of attenuated vaccines.</div

Working model for the regulation of NS mRNA splicing.

NEP mRNA is a spliced form of NS1 mRNA. The splicing complex binds (scissors) to the 3’ splicing site of NS1 mRNA and regulates alternative splicing. YTHDC1 binds to the m6A modification site on the 3’ splice site and inhibits the splicing process. The A530C mutation results in reduced binding of NS mRNA by YTHDC1, which promotes NS1 mRNA splicing and produces more NEP, thereby inhibiting viral polymerase activity.</p