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. 2020 Sep 18;9:e58825. doi: 10.7554/eLife.58825

Figure 1. IFN-α pretreatments with different durations lead to opposite effects to the second stimulation.

(A) Schematic of HeLa reporter cell line engineered using CRISPR/Cas9 (top). STAT1 was tagged with mCherry at C-terminus to monitor the translocation and expression. The coding sequence for P2A-YFP was inserted at the C-terminus of IRF9 coding sequence to generate a transcription reporter (PIRF9). A simplified diagram of the IFN-α pathway components that can be monitored using the reporter cell line (bottom). (B) Time lapse images of a representative cell treated with 100 ng/ml IFN-α for 48 hrs. Scale bar: 20 μm. The time traces of nuclear/cytoplasmic STAT1-mCherry and PIRF9-YFP signals of the cell are shown on the right. Vertical dashed lines represent cell divisions. (C) Averaged time traces of nuclear/cytoplasmic STAT1-mCherry, PIRF9-YFP, and the time derivative of PIRF9-YFP (dYFP/dt) (n = 257 cells). Data are represented as the mean (solid lines) and + standard deviation (SD) (shaded areas). (D) Schematic of IFN-α pretreatment experiments (top). Cells were pretreated with 100 ng/ml IFN-α for 0, 2, 10 and 24 hrs followed by 8 hrs of break time and re-stimulated with 100 ng/ml IFN-α for an additional 10 hrs. Bottom: A diagram of the microfluidic set-up. Two syringes filled with culture medium with or without IFN-α were connected to programmable Arduino-controlled valves that control the duration of IFN-α treatments. Images were captured every 5 min throughout the entire experiment that lasted for a total of 52 hr. (E) Averaged time traces of PIRF9-driven YFP induction (left) and the rate of induction (dYFP/dt, right) in response to the second IFN-α treatment under different pretreatment conditions. For PIRF9-YFP induction, the baselines at the beginning of the second stimulation were normalized to the same level for the comparison of induction levels under different pretreatment conditions. Results were from at least three independent experiments. (F) Amounts of PIRF9-YFP induction by the second IFN-α stimulation under different pretreatment conditions were shown in bar graph (left) and violin plot (right). The bar showed the averages from three independent experiments, represented as mean + standard error of the mean (SEM). The differences between the pretreatment conditions and the control are all statistically significant (p<0.001). The violin plots showed the distributions of single-cell responses under different pretreatment conditions. The violin plots cover single-cell data from three independent experiments.

Figure 1.

Figure 1—figure supplement 1. Cell line construction and validation.

Figure 1—figure supplement 1.

(A) Illustration of cell line construction steps. Full detail was described in Materials and methods. Fluorescent reporters introduced and the targeted genes are shown. In each step, homogenous clones grew from single cells were carefully validated with PCR and sequencing to determine correct integration and homozygosity. Only one positive clone was selected to proceed with the next step. (B) Schematics of the ACTB (top), STAT1 (middle) and IRF9 (bottom) tagged loci. Sequences of the gRNA along with the recognition direction and the synonymous substitutions to avoid Cas9 recognition are shown. Targeted integration loci and the design of the donor DNA with indicated homology arms along with the inserts are also shown. (C) Representative images of the reporter cell line in response to IFN-α. Cells were treated without or with 100 ng/ml IFN-α for 48 hrs. Scale bar: 20 μm. (D) Time course western blots showing the dynamics of phosphorylation (pY701) and expression of STAT1, and the dynamics of IRF9 expression in the reporter cell line. Cells were treated with 100 ng/ml IFN-α for indicated times, harvested and lysed for immunoblotting with indicated antibodies. The dynamics of the endogenous protein phosphorylation and expression, measured using immunoblotting, are similar to those measured using fluorescence microscopy (compare with Figure 1C).
Figure 1—figure supplement 2. Dose-dependent responses to IFN-α treatment.

Figure 1—figure supplement 2.

Time traces of nuclear to cytoplasmic ratio for STAT1-mCherry, STAT1-mCherry fluorescence, and PIRF9-YFP fluorescence in response to different concentrations (ng/ml) of IFN-α, as indicated. Averages of single-cell traces were shown.
Figure 1—figure supplement 3. Dose dependence of desensitization to IFN-α treatment.

Figure 1—figure supplement 3.

Bar graphs showing the amounts of PIRF9-YFP induction to the second IFN input (100 ng/ml) in WT (A - C) and USP18-KD (D – F), pretreated with different concentrations of IFN-α for different durations, as indicated. The results were normalized to the non-pretreatment condition (control). Error bars represent standard deviations of single cells.
Figure 1—figure supplement 4. Dependence of desensitization effects on the break time.

Figure 1—figure supplement 4.

Bar graphs showing the amounts of PIRF9-YFP induction to the second IFN input followed by different break time durations. Cells were pretreated with 100 ng/ml IFN-α for 24 hrs or not pretreated (control), washed with PBS three times and stimulated with 100 ng/ml IFN-α. The average PIRF9-YFP induction was normalized to the non-pretreatment control. Error bars represent standard deviations of single cells.