DBR1 regulates the PKR-mediated stress response during viral infection. (A) RT-qPCR determination of ID1, DKK1 RNA lariat levels in BJ scrambled control and shDBR1 cells. Data are representative of three independent experiments. Graphs depict the mean with standard deviation (SD), and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01. (B) BJ cells stably expressing Flag-DBR1 and an shRNA against DBR1 together with a scrambled/EV control were transfected and stimulated with polyI:C (1 µg/ml), and were then subjected to WB analysis with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (C) shDBR1 BJ cells transfected with EV or DBR1 were transfected with polyI:C (1 µg/ml) for 0, 2, or 4 h. Cell lysates were then prepared and subjected to WB with the indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (D) Following stimulation with polyI:C (1 µg/ml) in scrambled control and shDBR1 BJ cells, RT-qPCR was performed to quantify CHOP and ATF4 mRNA levels. Data are 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. (E) BJ scrambled control or shDBR1 cells were infected with VSV at an MOI of 0.1 for 24 h, and viral replication was then detected by RT-qPCR. Data are 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 < 0.01. (F) BJ cells transduced with scrambled shRNA or shDBR1 were infected with VSV at an MOI of 0.1 for various times. The cells were then lysed and subjected to WB with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (G) BJ cells transduced with scrambled shRNA or shDBR1 were infected with HSV-1 at an MOI of 0.5 for 24 h and then quantified for viral mRNA 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 < 0.001. (H) BJ cells transduced with scrambled shRNA or shDBR1 were infected with HSV-1 at an MOI of 0.5 for various times. The cells were then lysed and subjected to WB with the indicated antibodies, followed by the p-PKR/PKR WB grayscale analysis of three independent experiments. GAPDH was used as a loading control. (I and J) BJ cells were infected with HSV-1 (MOI: 0.5) for 24 h and then quantified for the expression of the indicated ISR genes (I), IFNB and the indicated ISGs (J) by RT-qPCR. Data are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01; ***, P < 0.001. Source data are available for this figure: SourceData F1.
Figure 1.

DBR1 regulates the PKR-mediated stress response during viral infection. (A) RT-qPCR determination of ID1, DKK1 RNA lariat levels in BJ scrambled control and shDBR1 cells. Data are representative of three independent experiments. Graphs depict the mean with standard deviation (SD), and points represent biological replicates. Statistical analysis was performed with one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01. (B) BJ cells stably expressing Flag-DBR1 and an shRNA against DBR1 together with a scrambled/EV control were transfected and stimulated with polyI:C (1 µg/ml), and were then subjected to WB analysis with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (C) shDBR1 BJ cells transfected with EV or DBR1 were transfected with polyI:C (1 µg/ml) for 0, 2, or 4 h. Cell lysates were then prepared and subjected to WB with the indicated antibodies. Tubulin was used as a loading control. Data shown are representative of three independent experiments. (D) Following stimulation with polyI:C (1 µg/ml) in scrambled control and shDBR1 BJ cells, RT-qPCR was performed to quantify CHOP and ATF4 mRNA levels. Data are 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. (E) BJ scrambled control or shDBR1 cells were infected with VSV at an MOI of 0.1 for 24 h, and viral replication was then detected by RT-qPCR. Data are 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 < 0.01. (F) BJ cells transduced with scrambled shRNA or shDBR1 were infected with VSV at an MOI of 0.1 for various times. The cells were then lysed and subjected to WB with the indicated antibodies. GAPDH was used as a loading control. Data shown are representative of three independent experiments. (G) BJ cells transduced with scrambled shRNA or shDBR1 were infected with HSV-1 at an MOI of 0.5 for 24 h and then quantified for viral mRNA 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 < 0.001. (H) BJ cells transduced with scrambled shRNA or shDBR1 were infected with HSV-1 at an MOI of 0.5 for various times. The cells were then lysed and subjected to WB with the indicated antibodies, followed by the p-PKR/PKR WB grayscale analysis of three independent experiments. GAPDH was used as a loading control. (I and J) BJ cells were infected with HSV-1 (MOI: 0.5) for 24 h and then quantified for the expression of the indicated ISR genes (I), IFNB and the indicated ISGs (J) by RT-qPCR. Data are representative of three independent experiments. Graphs depict the mean with SD and points represent biological replicates. Statistical analysis was performed with t tests. **, P < 0.01; ***, P < 0.001. Source data are available for this figure: SourceData F1.

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