Fig 1: piR-36741 bound to METTL3 to regulate the m6A activity of METTL3. (A) RNA pull-down assay was performed using bio-NC or bio-piR-36741 to verify the binding of piR-36741 and METTL3. (B–F) RIP assay was carried out to confirm the binding of piR-36741 with METTL3, METTL14, WTAP, FTO and ALKBH5. (G) Biotin-based RNA pull-down assay was used to detect the specific binding of piR-36741 with PIWIL4. (H) RIP assay was performed to verify the binding piR-36741 with PIWIL4. (I) BMSCs were transfected with METTL3 and PIWIL4 overexpression vectors, and co-IP was carried out using specific antibodies, and the immunocomplex were purified and subjected to Western blotting analysis using METTL3 or PIWIL4 antibodies. (J) RNA pull-down assay was used to confirm the binding ability of purified PIWIL4 or METTL3 proteins with biotin-labelled piR-36741. (K, L) BMSCs were transfected with mimic-piR-36741, antagomir-piR-36741 or respective controls for 24 h, and the mRNA and protein levels of METTL3 were determined. (M, N) m6A levels of BMSCs with or without piR-36741/PIWIL4 overexpression were detected by using the EpiQuik™ m6A RNA methylation quantification kit. N = 5 in each group. *P < 0.05, **P < 0.01. Each test was independently repeated at least three times.
Fig 2: Detection and differential expression of piRNAs in PIWIL4-silenced iSAE cells following RSV infection.iSAE cells were transfected with either non-targeting or PIWIL4-targeting siRNA and total RNA was extracted from uninfected cells (Ctrl) or infected with RSV for 15 hours. Samples from one experiment done in triplicate were pooled and the number of differentially expressed genes (DEGs) were identified by piRNA array analysis. (A) Venn diagrams illustrating the distribution of piRNAs detected among the four experimental groups: non-targeting control (NTarg Ctrl), non-targeting RSV-infected (NTarg RSV), PIWIL4 targeting control (Targ Ctrl), and PIWIL4 targeting RSV-infected (Targ RSV) groups. Numbers within each section indicate piRNAs detected exclusively in one condition or shared between conditions. For NTarg cells, 975 piRNAs were detected only in RSV-infected samples, 247 only in controls, and 9,948 for both conditions. For Targ cells, 650 piRNAs were unique to RSV-infected samples, 566 unique to controls, and 9,908 were detected in both. (B) Bar graphs display the numbers of differentially upregulated (open bars) or downregulated (black bars) expressed piRNAs comparing PIWIL4 targeting versus non-targeting conditions within either the control or RSV-infected condition. Differential expressions were determined using a fold-change cutoff of FC ≥ 1.5.
Fig 3: PIWIL4 mRNA and protein expression in primary small airway epithelial (SAE) cells following RSV infection and Poly I:C treatment.SAE cells control (Ctrl), infected with RSV or treated with Poly I:C were harvested at 6, 15, and 24 hours to either extract total RNA or prepare total cell lysates. (A) PIWIL4 mRNA levels measured by RT-qPCR. Data are means ± SEM (n=3, from three independent experiments run in triplicate); ***p<0.001, ****p<0.0001 infected/treated vs. control by one-way ANOVA followed by Tukey’s multiple comparisons test. (B) PIWIL4 protein levels in SAE cells following RSV infection or (C) Poly I:C treatment were measured by western blot analysis using an antibody anti-PIWIL4. Membranes were stripped and re-probed with anti-β-actin for loading control. Images are representative of two independent experiments. Graphs show relative protein fold change determined by quantification of bands and normalization to the loading control (β-actin) expressed as mean ± SEM.
Fig 4: Effect of PIWIL4 silencing on gene expression in iSAE cells.(A) iSAE cells were transfected with either non-targeting or PIWIL4-targeting siRNA and total RNA was extracted from cells uninfected (Ctrl) or infected with RSV for 15 hours. Samples from one experiment done in triplicate were pooled and the number of differentially expressed genes (DEGs) were identified by gene array analysis. Number of upregulated (open bars) and downregulated (black bars) DEG in PIWIL4-targeting (Targ) versus non-targeting (NTarg) iSAE cells under Ctrl and RSV infection conditions. Differential expressions were determined using a fold-change cutoff of FC ≥ 1.5. (B) Heatmaps showing the expression profiles of significantly differentially expressed genes from the selected IPA-enriched canonical pathways in Tables 1 and 2. Left panels: DEGs under Ctrl conditions; right panels: DEGs under RSV infection. Upper panels: upregulated genes; lower panels: downregulated genes. Red indicates higher relative expression and green indicates lower relative expression (mean-centered log fold-change values).
Fig 5: Validation of selected upregulated (A) and downregulated (B) piRNAs in PIWIL4-targeting iSAE cells following RSV infection.Radar plots depicting the expression profiles of twenty selected piRNAs in PIWIL4-targeting (Targ) versus non-targeting (NTarg) iSAE cells either infected with RSV for 15 hours (red shaded area) or uninfected (control) (blue shaded area). piRNA expression levels were measured by RT-qPCR and calculated as fold changes relative to NTarg control cells using the 2^−ΔΔCT method. Data are normalized to U6 small nuclear RNA and presented as mean ± SEM (n=2, from two independent experiments run in triplicate).
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