Fig 1: IFN induction capability of D279.LAB genomes were introduced into HT-29 cells using lipofection. After 24 h, the medium was collected and subjected to an enzyme-linked immunosorbent assay. Cultures were performed in triplicate to quantify IFNs in each experiment. Values are presented as mean ± S.E. (n = 3). N.D. indicates not detected. Asterisks indicate a statistically significant difference compared with D279 analyzed using Student’s t-test (**P < 0.01). (A) All IFNa subtypes. (B) All IFN? subtypes.
Fig 2: CICD triggers mtRNA-dependent Type I IFN production in cGAS/STING pathway deficient tumor cells.a Western blot showing C32, A375, Colo205, and SK-MEL-28 baseline expression of indicated proteins. Data are representative of three independent experiments (n = 3). b Indicated cell lines were treated with 0.5 µg/ml Poly(dG:dC)/LyoVec (cytosolic dsDNA agonist) or Poly(I:C) (LMW)/LyoVec (cytosolic dsRNA agonist) for 16 h. RT-qPCR analysis was performed to measure IFIT3 expression. Each dot represents a biological replicate, and A375 and Colo205 experiments were performed in biological triplicate (n = 3). SKMEL28 dsDNA treatment was performed in duplicate (n = 2), and dsRNA treatment was performed in triplicate (n = 3). c Colo205 and SK-MEL-28 cells were subjected to 5 days of EtBr (100 ng/ml) or control-media pre-treatment. The pre-treated EtBr and control cells were exposed to the designated combinations of 2 µM PLX-4720 and 2 µM S63845 (PLX + S6) and 10 µM Emricasan (E) for 48 h. ISG15 and IFIT3 transcripts were measured with RT-qPCR. d SK-MEL-28 cells were treated with 2 µM PLX-4720, 2 µM S63845, and 10 µM Emricasan in the presence or absence of 2.5 µM IMT1B for 36 h. RT-qPCR was performed to measure expression levels of ISG15 and IFIT3. e, f A375, Colo205, and SK-MEL-28 cells were conditioned with EtBr or control media as described above. e EtBr and control media-treated A375 and SK-MEL-28 cells were subjected to 30 nM and 10 nM paclitaxel (Pacl), respectively, with or without Emricasan (E) for 48 h. RT-qPCR was performed to measure expression levels of ISG15 and IFIT3. f EtBr and control-treated A375, Colo205, and SK-MEL-28 were treated with 300 nM, 200 nM, and 200 nM doxorubicin (Dox), respectively, with or without 10 µM Emricasan (E) for 48 h. RT-qPCR was performed to measure expression levels of ISG15 and IFIT3. c–f, Each dot represents a biological replicate, and the data are the results of three independent experiments (n = 3). b–f mRNA levels are normalized to DMSO-treated control cells. One-way ANOVA with Tukey’s multiple-comparison test, p-values are included in the figure; ns = not significant. Data are presented as mean ± SEM.
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