Fig 2: IFIT3 interplays with NF-κB pathway during chemotherapy. (A) Western blot showed IFIT3 was up-regulated after gemcitabine treatment. Protein samples were collected from L3.6pl and TBO368 cells with or without gemcitabine treatment (3 ng/ml for L3.6pl and 400 ng/ml for TBO368) for indicated time. Membrane was stripped after IFIT3 detection and re-probed with α-Tubulin. (B) IFIT3 and NF-kB pathway was activated after gemcitabine treatment in L3.6pl and TBO368. NFKB1, NFKB3, IL6 and XIAP were selected as indicators for activation of NF-κB pathway. RNA samples were collected from L3.6pl and TBO368 cells with or without gemcitabine treatment for indicated time. (C) Three putative p65 binding sites on IFIT3 gene promoter are depicted. Chromatin immunoprecipitation indicates direct binding of p65 to the promoter region of IFIT3 (TSS +122 to +342, black bar). Chromatin was extracted from 1% PFA fixed L3.6pl and TBO368 cells pre-treated with gemcitabine and immunoprecipitated with anti-p65 antibody or normal IgG. (D) Expression of IFIT3 was diminished by NF-κB inhibitor Bay 11-7082 with or without gemcitabine treatment. Bay 11-7082, 10 µM. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: IFIT3 renders chemotherapy resistance in PDAC cells. (A) IFIT3 knockdown was confirmed with western blot in both L3.6pl and TBO368 cells. Membrane was stripped after IFIT3 detection and re-probed with α-Tubulin. (B) IFN pathway and NF-κB pathway targeted genes, IFIT1, IFIT2, RIG-I, IL6, and XIAP were significantly down-regulated after knockdown of IFIT3. (C-D) Knockdown of IFIT3 increased sensitivity of L3.6pl (C) and TBO368 (D) to chemotherapy. Cells were treated with gemcitabine (3 ng/ml for L3.6pl and 400 ng/ml for TBO368), paclitaxel (10 nM) or FOLFIRINOX (Oxaliplatin: Irinotecan: Folinic acid Calcium: 5-Fluorouracil=1:2:4:25 µM as 1X, used as 0.025X for L3.6pl and 0.5X for TBO368) for indicated time. Apoptosis was determined by flow cytometry analysis of Annexin V/DAPI staining. Representative FACS dot plots are shown on the left. Bar graphs are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant, p > 0.05.

Fig 4: IFIT3 regulates mitochondria-associated apoptosis. (A) Confocal immunofluorescence labeled with anti-FLAG (green), anti-Tom20 (red), and counterstained with DAPI (blue), showed co-localization of FLAG-tagged IFIT3 and Tom20 in mitochondria. L3.6pl and TBO368 cells stably express FLAG-tagged IFIT3 were used here. Scale was shown in the lower-right corner. (B) Western blot showed localization of IFIT3 in cytosol and mitochondria of L3.6pl and TBO368. Mitochondria were isolated as indicated in methods section. Cyto represent cytosol and Mito represent mitochondria. We probed α-Tubulin as cytosolic marker, BAK and VDAC2 as mitochondrial marker. (C) IFIT3 knockdown in L3.6pl altered mitochondrial membrane potential (ΔΨm) with or without gemcitabine treatment. TMRE was used to determine the mitochondrial membrane potential (ΔΨm). MFI: mean fluorescence intensity. (D) IFIT3 knockdown in L3.6pl showed more MitoSOX positive cells when treated with gemcitabine. Representative FACS dot plot and bar graph are shown. (E) Mass spectrometry results of immunoprecipitated samples by anti-IFIT3 antibody are shown in Venn diagram. Cells were treated with gemcitabine for indicated time before harvest. Proteins with p < 0.01 and log2 difference > 1 are considered significant. Proteins identified in the intersection of L3.6pl and TBO368 are listed beside the diagram. (F) Interaction between IFIT3 and VDAC2 was confirmed with western blot in L3.6pl. Data are presented as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001.

Fig 5: XRN2 and IFIT3 expression during RSV infection. HEp2 cells were infected with RSV (+) or left uninfected (-) and harvested at indicated time points post infection. Cells were lysed and proteins analyzed by SDS-PAGE and subsequent immunoblotting using polyclonal antibody raised against IFIT3 (A) or either XRN2a or XRN2a/b (B). Western blot analysis of indicated offgel fractions of a whole cell lysate of RSV infected cells 24 hpi using a polyclonal antibody raised against XRN2a (C).