DBR1 regulates the activation of PKR and integrated stress response rather than IFN response. (A) RT-qPCR detection of DBR1 mRNA in BJ cells transduced with scrambled and shDBR1. Data representative of three independent experiments. Graphs depict mean with SD and points represent biological replicates. Statistical analysis was performed with t tests. ***, P < 0.001. (B) BJ cells with scrambled shRNA or shDBR1 were stimulated with polyI:C, followed by RT-qPCR analysis of the indicated IFN or ISGs. Data representative of three independent experiments. Graphs depict points that represent biological replicates. (C) RT-qPCR detection of DBR1 mRNA in BJ cells transduced with overexpressing DBR1 relative to control. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. ***, P < 0.001. (D) BJ cells with EV or DBR1 overexpression were stimulated with IFNβ, followed by RT-qPCR analysis of EV and DBR1 BJ cells for expression of the indicated ISGs. Data representative of three independent experiments. Graphs depict points that represent biological replicates. (E) RT-qPCR detection of DBR1 mRNA in HEK293T cells transduced with scrambled and shDBR1. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with unpaired t tests. ****, P < 0.0001. (F) RT-qPCR detection of DBR1 mRNA in HEK293T cells transduced with DBR1 relative to control. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with t tests. ****, P < 0.0001. (G) RT-qPCR determination of ID1, DKK1 RNA lariat levels in HEK293T scrambled control and shDBR1 cells. Data representative of three independent experiments. Graphs depict mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (H and I) HEK293T scrambled control or shDBR1 cells were infected with VSV at an MOI of 0.1 (H) or HSV-1 at an MOI of 0.5 (I) for 24 h, and viral replication was then detected by RT-qPCR. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (J) HEK293T cells transfected with the scramble shRNA or shDBR1 were stimulated with polyI:C (1 µg/ml), followed by WB for indicated proteins. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (K and L) HEK293T cells transduced with the scrambled shRNA, shDBR1, or shDBR1 with DBR1 for rescue were analyzed by WB with the indicated antibodies (K). GAPDH was used as a loading control. Data shown are representative of three independent experiments. RT-qPCR for RNA lariat detection (L). Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ***, P < 0.001. (M) HEK293T cells transduced with shDBR1 or shDBR1 cells transduced with DBR1 for rescue were infected with HSV-1 (left) or VSV (right), and viral mRNA was then quantified by RT-qPCR. Statistical analysis was performed with unpaired t tests. **, P < 0.01. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (N) RT-qPCR analysis of control, shDBR1-transduced and DBR1-overexpressing HEK293T cells for assessment of the expression of the indicated ISR genes. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01. (O) Stimulation with polyI:C (1 µg/ml) in THP-1 cells transduced with the scramble shRNA or shDBR1, followed by WB for indicated proteins. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (P) THP1 cells transduced with EV or DBR1 were stimulated with polyI:C (1 µg/ml) and WB was then performed with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (Q) HEK293T cells transduced with the scrambled shRNA or shDBR1 were subjected to HSV-1 infection (MOI: 0.5) for various time points. WB was then performed with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (R and S) THP-1 cells transduced with the scrambled shRNA or shDBR1 were subjected to HSV-1 infection (MOI: 0.5) (R) or VSV infection (MOI: 0.1) (S) for various time points, followed by WB for the indicated proteins. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (T) RT-qPCR quantification of ID1 and DKK1 RNA lariat levels in fibroblasts from the control and patients. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. (U–W) Cells from the control and the patient were infected with VSV at a MOI of 0.1, and RT-qPCR was then performed for detection of the expression of viral genes (U), IFN and ISG genes (V and W). Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (X) Patient’s cells were rescued by transfection with DBR1, and RT-qPCR was then performed to assess ID1 and DKK1 lariat levels. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01; ***, P < 0.001. (Y) Control and patient’s cells were infected with HSV-1 (MOI: 0.5), and viral mRNA was then quantified by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01. Source data are available for this figure: SourceData FS1.
Figure S1.

DBR1 regulates the activation of PKR and integrated stress response rather than IFN response. (A) RT-qPCR detection of DBR1 mRNA in BJ cells transduced with scrambled and shDBR1. Data representative of three independent experiments. Graphs depict mean with SD and points represent biological replicates. Statistical analysis was performed with t tests. ***, P < 0.001. (B) BJ cells with scrambled shRNA or shDBR1 were stimulated with polyI:C, followed by RT-qPCR analysis of the indicated IFN or ISGs. Data representative of three independent experiments. Graphs depict points that represent biological replicates. (C) RT-qPCR detection of DBR1 mRNA in BJ cells transduced with overexpressing DBR1 relative to control. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. ***, P < 0.001. (D) BJ cells with EV or DBR1 overexpression were stimulated with IFNβ, followed by RT-qPCR analysis of EV and DBR1 BJ cells for expression of the indicated ISGs. Data representative of three independent experiments. Graphs depict points that represent biological replicates. (E) RT-qPCR detection of DBR1 mRNA in HEK293T cells transduced with scrambled and shDBR1. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with unpaired t tests. ****, P < 0.0001. (F) RT-qPCR detection of DBR1 mRNA in HEK293T cells transduced with DBR1 relative to control. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with t tests. ****, P < 0.0001. (G) RT-qPCR determination of ID1, DKK1 RNA lariat levels in HEK293T scrambled control and shDBR1 cells. Data representative of three independent experiments. Graphs depict mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (H and I) HEK293T scrambled control or shDBR1 cells were infected with VSV at an MOI of 0.1 (H) or HSV-1 at an MOI of 0.5 (I) for 24 h, and viral replication was then detected by RT-qPCR. Data representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001. (J) HEK293T cells transfected with the scramble shRNA or shDBR1 were stimulated with polyI:C (1 µg/ml), followed by WB for indicated proteins. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (K and L) HEK293T cells transduced with the scrambled shRNA, shDBR1, or shDBR1 with DBR1 for rescue were analyzed by WB with the indicated antibodies (K). GAPDH was used as a loading control. Data shown are representative of three independent experiments. RT-qPCR for RNA lariat detection (L). Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ***, P < 0.001. (M) HEK293T cells transduced with shDBR1 or shDBR1 cells transduced with DBR1 for rescue were infected with HSV-1 (left) or VSV (right), and viral mRNA was then quantified by RT-qPCR. Statistical analysis was performed with unpaired t tests. **, P < 0.01. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. (N) RT-qPCR analysis of control, shDBR1-transduced and DBR1-overexpressing HEK293T cells for assessment of the expression of the indicated ISR genes. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01. (O) Stimulation with polyI:C (1 µg/ml) in THP-1 cells transduced with the scramble shRNA or shDBR1, followed by WB for indicated proteins. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (P) THP1 cells transduced with EV or DBR1 were stimulated with polyI:C (1 µg/ml) and WB was then performed with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (Q) HEK293T cells transduced with the scrambled shRNA or shDBR1 were subjected to HSV-1 infection (MOI: 0.5) for various time points. WB was then performed with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (R and S) THP-1 cells transduced with the scrambled shRNA or shDBR1 were subjected to HSV-1 infection (MOI: 0.5) (R) or VSV infection (MOI: 0.1) (S) for various time points, followed by WB for the indicated proteins. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (T) RT-qPCR quantification of ID1 and DKK1 RNA lariat levels in fibroblasts from the control and patients. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. (U–W) Cells from the control and the patient were infected with VSV at a MOI of 0.1, and RT-qPCR was then performed for detection of the expression of viral genes (U), IFN and ISG genes (V and W). Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (X) Patient’s cells were rescued by transfection with DBR1, and RT-qPCR was then performed to assess ID1 and DKK1 lariat levels. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01; ***, P < 0.001. (Y) Control and patient’s cells were infected with HSV-1 (MOI: 0.5), and viral mRNA was then quantified by RT-qPCR. Data representative of three independent experiments. Graphs depict mean with SD, and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01. Source data are available for this figure: SourceData FS1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal