Fig 1: Traf6 transcripts recognized by YTHDF1 depends on DDX60. (A) Volcano plot depicting the Log10 (P-value) versus the Log2 fold change (YTHDF1-IP/IgG-IP) in MS-identified proteins obtained from two biological replicates. Intensities were obtained from a MaxQuant database search. (B) Pathway analyses (with Gene Ontology direct terms) of proteins measured using co-IP of YTHDF1 in LPS-stimulated IPEC-J2 cells. (C) Summary of the interaction between the DDX60, THRAP3, and BCLAF1 proteins and the Traf6 transcript measured using RIP q-PCR, normalized to the input levels. (D) DDX60 immunofluorescence (green) and FISH analysis of Traf6 mRNA (red) in an IPEC-J2 cell 6 h after LPS stimulation. The nucleus was counterstained with DAPI (blue). (E) Co-IP experiment using anti-YTHDF1 (upper) or DDX60 (lower) and control IgG. After immunoprecipitation, the samples were washed and incubated with RNase where indicated. YTHDF1 and DDX60 proteins were then detected using western blot analysis. (F) RIP-qPCR analysis of the interaction between YTHDF1 and the Traf6 transcript in IPEC-J2 cells expressing a scrambled shRNA or the Ddx60 shRNA. (G) RIP-qPCR analysis of the interaction between DDX60 and the Traf6 transcript in IPEC-J2 cells expressing a scrambled shRNA or the Ythdf1 shRNA. (H, I) Western blot analysis and summary of TRAF6 in IPEC-J2 cells co-expressing the Ythdf1 shRNA, the FLAG-Ythdf1 plasmid or empty vector, and either a scrambled siRNA or Ddx60 siRNA (H). The reciprocal experiment is shown in (I). (J) Luciferase assay to measure TRAF6 and NF-?B expression in IPEC-J2 cells expressing the Ythdf1 shRNA, the FLAG-Ythdf1 plasmid or empty vector, and a scrambled siRNA or the Ddx60 siRNA. Luciferase activity is expressed relative to Renilla luciferase activity.
Fig 2: The interaction between DDX60 and YTHDF1 is required for initiating translation of the Traf6 transcript. (A) Western blot analysis and summary of TRAF6 protein in IPEC-J2 cells expressing the scrambled shRNA or theDdx60 shRNA and stimulated with LPS for the indicated times. (B) Total RNA was extracted from the same samples in (A), and Tnf-α and Il-6 mRNA was measured using qPCR and expressed relative to Gapdh mRNA. (C) Western blot analysis and summary of TRAF6 protein in LPS-stimulated Caco2 cells expressing a scrambled siRNA or Ddx60 siRNA. (D) Total RNA was extracted from the same samples in (C), and Tnf-α and Il-6 mRNA was measured and expressed relative to Gapdh mRNA. (E) Western blot analysis and summary of TRAF6 protein in LPS-stimulated Traf6 KO IPEC-J2 cells expressing the FLAG-Traf6 plasmid or an empty vector together with either a control siRNA or Ddx60 siRNA. (F) Total RNA was extracted from the same samples in (E), and Tnf-α and Il-6 mRNA was measured and expressed relative to Gapdh mRNA. (G-H) Total RNA was extracted from the same samples in (A), and Traf6 mRNA was measured and expressed relative to Gapdh mRNA. (I) FISH analysis of Traf6 (red) transcripts in LPS-stimulated IPEC-J2 cells expressing the scrambled shRNA or Ddx60 shRNA; the nuclei were counterstained with DAPI (blue). Scale bars, 10 μm. (J) The fold enrichment of Traf6 and Gapdh mRNA was measured using RIP-qPCR in IPEC-J2 cells expressing the indicated plasmids. The structure of the full-length and truncated DDX60 proteins is shown schematically at the left; the DEADc and HELICc domains are indicated. (K) Total RNA was extracted from IPEC-J2 expressing the Ddx60 shRNA and the indicated Myc-Ddx60 plasmids, and Traf6 mRNA was measured and expressed relative to Gapdh mRNA. (L) Western blot analysis and summary of TRAF6 protein measured in the same samples in (K). (M) Total RNA was extracted from the same samples in (K), and Tnf-α and Il-6 mRNA was measured and expressed relative Gapdh mRNA. (N, O) Co-IP experiments with LPS-stimulated IPEC-J2 cells co-expressing the indicated FLAG-Ythdf1 plasmids together with Myc-Ddx60 or an empty vector (N) or co-expressing the indicated Myc-Ddx60 plasmids together with FLAG-Ythdf1 or an empty vector (O). For reference, western blot analyses using whole-cell lysate (WCL) samples are shown at the bottom.
Fig 3: Levels of IFNß and DDX-60 mRNA and protein after pDNA electrotransfer in B16.F10 mouse melanoma cells. (a) IFNß mRNA levels as measured by real-time RT-PCR, n = 5–9. (b) IFNß promotor activity as measured by luciferase expression, n = 4. (c) IFNß protein levels as measured by ELISA, n = 3. (d) Fold cells positive for IFNß as measured by flow cytometry normalized to the control, n = 6–8. (e) Median IFNß fluorescent intensity as measured by flow cytometry normalized to the control, n = 6–8. (f) Fold cells positive for DDX60 as measured by flow cytometry normalized to the control, n = 7. (g) Median DDX60 fluorescent intensity as measured by flow cytometry normalized to the control, n = 7. (h) Western blot of cell lysate demonstrating an increased expression of DDX60 9 hours after pDNA electrotransfer. Control, 40 µl of cells and 10 µl of saline; EP, 40 µl of cells and 10 µl of saline were electroporated by the delivery of eight 5 ms pulses with a voltage-to-distance ratio of 600 V/cm with a plate electrode; pDNA, 40 µl of cells and 10 µl of 2 mg/ml gWiz Blank plasmid; EP+pDNA, 40 µl of cells and 10 µl of 2 mg/ml gWiz Blank plasmid were lectroporated by the delivery of eight 5 ms pulses with a voltage-to-distance ratio of 600 V/cm with a plate electrode.
Fig 4: Correlation between helicases expression and cancer stages, and between their expression and MyD88/MAVS/TRIF expression. The expression data of helicases were retrieved from the TCGA microarray database in Oncomine® platform, and were grouped by stages or patient IDs for further analysis. (A-C) Correlation between DHX9, DHX36, and DDX60 and cancer stages were shown. (D-G) Expression correlation between DHX9 and MAVS/MyD88, DHX36 and MyD88/TRIF. n=22 for health normal control, 44 for stage I, 78 for stage II, 52 for stage III, and 23 for stage IV. Data were expressed as mean ± SEM, Log2 median-centered ratio expression. *p < 0.05; ***p < 0.001.
Fig 5: The expression profile of helicases in human and mouse CRC tissues and TCGA database. RNA was extracted from cancer and matched peri-carcinomatous tissues of CRC patients, as well as tissues from AOM/DSS treated mice, and reverse-transcribed into cDNA. Then the gene expression levels of helicases were determined by quantitative PCR. The data from the same patient were connected by straight lines. The data of the expression of helicases were retrieved from the TCGA microarray database in Oncomine® platform. Expression of DHX9 (A, B, C), DHX36 (D, E, F), and DDX60 (G, H, I), in human CRC tissues, tissues from AOM/DSS treated mice, and TCGA database. Control: matched peri-carcinomatous tissues, n=44 for clinical samples, data represent two independent experiments; n=7 for PBS-treated mice and n=5 for AOM/DSS- treated mice. *p < 0.05, **p < 0.01, ***p < 0.001; NS: Not significantly different.
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