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 IFNα subtypes. (B) All IFNλ 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.
Fig 3: cGAS–STING pathway coordinates antiviral defense in M‐ChPO. (A) Schematic for testing cGAS–STING in sensing HSV‐1 DNA viruses in microglia in M‐ChPO. (B) Quantification of IFN‐α level measured by ELISA from organoid supernatants at 3 dpi. The ChPO and M‐ChPO were pretreated with cGAS inhibitor RU.512 (1 μM) or STING inhibitor H‐151 (1 μM) for 24 h and then the HSV‐1 virus was added to the culture medium. Mean ± SD; n = 5; ***p < 0.001, one‐way ANOVA with Bonferroni's post hoc test. (C) Quantification of virus titers from organoid supernatants. M‐ChPO and ChPO were pretreated with cGAS inhibitor RU.512 or STING inhibitor H‐151 before HSV‐1 infection. Mean ± SD; n = 5; ***p < 0.001, one‐way ANOVA with Bonferroni's post hoc test. (D) Costaining for HSV‐1, DAPI, and Claudin5 in ChP epithelium layer in ChPO and M‐ChPO incubated with RU.512 or H‐151 before HSV‐1 infection. (E) Representative confocal images of fluorescent immunostaining for HSV‐1, TTR, DAPI, and ZO‐1 expression in HSV‐1‐infected ChPO and M‐ChPO with RU.512 or H‐151 treatment. (F) Quantification of percentages of HSV‐1+ cells among ChP cells in both ChPO and M‐ChPO with RU.512 or H‐151 before HSV‐1 infection. Mean ± SD; n = 6; ***p < 0.001, one‐way ANOVA with Bonferroni's post hoc test. (G–I) Quantification of TTR, Claudin5, and ZO‐1 expressions in M‐ChPO and ChPO. Mean ± SD; n = 6; ***p < 0.001, one‐way ANOVA with Bonferroni's post hoc test. ANOVA, analysis of variance; cGAS, cyclic GMP‐AMP synthase; ChPO, choroid plexus organoid; DAPI, 4′,6‐diamidino‐2‐phenylindole; ELISA, enzyme‐linked immunosorbent assay; HSV‐1, herpes simplex virus type 1; IFN‐α, interferon‐α; M‐ChPO, microglia‐containing ChPO; TTR, transthyretin; ZO‐1, zonula occludens‐1.
Fig 4: Microglia limit HSV‐1 infection and protect choroid plexus (ChP) barrier. (A) Schematic for studying the different effects of HSV‐1‐induced physiology in the presence or absence of microglia using ChPO and M‐ChPO. (B) Quantification of virus titers from culture supernatants. Mean ± SD; n = 5; ***p < 0.001. (C) qRT‐PCR analysis of TK mRNA expression using ChPO and M‐ChPO with HSV‐1 infection. Mean ± SD; n = 3; ***p < 0.001. (D) Quantification of IFN‐α level measured by ELISA using ChPO and M‐ChPO after HSV‐1 (3000 pfu/organoid) or vehicle treatment at 3 dpi. (E) Costaining for HSV‐1, IBA1, and Claudin5 in ChPO and M‐ChPO infected with HSV‐1 or mock at 3 dpi. (F) Quantification of percentages of HSV‐1+ cells in the ChP epithelium layer in ChPO and M‐ChPO after HSV‐1 treatment at 3 dpi. Mean ± SD; n = 7; ***p < 0.001, Student's t‐test. (G) Quantification of Claudin5 expression in M‐ChPO and ChPO infected with HSV‐1 or mock shown in (E). Mean ± SD; n = 6; ***p < 0.001, Student's t‐test. (H) Representative confocal images of fluorescent immunostaining for the HSV‐1, ZO‐1, and TTR in the ChP epithelium layer in ChPO and M‐ChPO. (I, J) ZO‐1 and TTR expressions in M‐ChPO and ChPO infected with HSV‐1 or mock shown in (E) and (F). Mean ± SD; n = 6; **p < 0.01; ***p < 0.001, one‐way ANOVA with Bonferroni's post hoc test. (K) Costaining for C‐CASP3, HSV‐1, and TTR in ChPO and M‐ChPO infected with HSV‐1 or mock at 3 dpi. (L) Quantification of percentages of C‐CASP3+ cells among TTR+ cells in ChP epithelium layer in ChPO and M‐ChPO with HSV‐1 or mock at 3 dpi shown in (K). Mean ± SD; n = 6; ***p < 0.001, Student's t‐test. ANOVA, analysis of variance; ChPO, choroid plexus organoid; dpi, days postinfection; ELISA, enzyme‐linked immunosorbent assay; HSV‐1, herpes simplex virus type 1; IFN‐α, interferon‐α; M‐ChPO, microglia‐containing ChPO; pfu, pore‐forming unit; TK, thymidine kinase; TTR, transthyretin; ZO‐1, zonula occludens‐1.
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