(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 …
(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 …
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 …
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 …
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 …
(A) Representative time-lapse images of STAT1 nuclear translocation in response to the second IFN-α treatment under different pretreatment conditions in WT cells (top) and USP18-KD (bottom). (B) …
(A) Western blots of USP18 expression in WT and USP18-KD cells. Cells were treated with 100 ng/ml IFN-α for indicated times, harvested and lysed for immunoblotting with the USP18 antibody. Time …
(A) Averaged time traces of nuclear/cytoplasmic STAT1-mCherry in WT in response to the second IFN-α treatment under different pretreatment conditions (depicted in Figure 1D). (B) Violin plot shows …
(A) Quantitative PCR results comparing SOCS1 expression in WT (back) and SOCS1-KD (red). The data was normalized to WT without IFN-α treatment. The knockdown efficiency (73.71%) was calculated as …
Cells with or without a 24-hr pretreatment of 100 ng/ml IFN-α and an 8-hr break time were infected with 2500 PFU (plaque forming units) of VSV. After 18 hrs, viral supernatant was collected and …
(A) A diagram for the simple kinetic model of the IFN-driven gene regulatory network. (B) Fitting errors between simulations and the data with different assigned values of the delay time in USP18 …
(A) A diagram for the simple kinetic model of the IFN-driven gene regulatory network with reaction parameters labeled. (B) Fitting results with different delay times. The model fitting results …
(A) The violin plots showing single-cell distributions of PIRF9-YFP induction upon 5 × 8-hr pulsatile (blue) or 40-hr sustained (red) IFN-α treatments in WT (top) and USP18-KD (bottom) cells. The …
(A) Schematic of the dual reporter cell line. A coding sequence for NLS-CFP-P2A was inserted endogenously into the N-terminus of USP18 coding sequence of the previous cell line. IFN-α induces …
(A) Illustration showing the introduction of PUSP18-CFP into the dual reporter cell line in Figure 1A. NLS-CFP-P2A coding sequence was inserted between the promoter and coding sequence of the USP18 …
(A) Scatterplots showing the relationship of PUSP18-CFP expression upon IFN-α treatment with (i) the basal level of PIRF9-YFP, (ii) PIRF9-YFP expression upon IFN-α treatment, (iii) the basal level …
(A) Illustration of measurement of cell mitosis times from time-lapse images. (Top) Representative time-lapse images of cells over multiple cell divisions. (Bottom) The size of nucleus (left) and …
(A) Distributions of delay times in cells treated with different cell cycle perturbations. Cells were serum-starved or treated with lovastatin (5 μM), or with roscovitine (5 μM) for 36 hrs prior to …
Cells were serum-starved or treated with lovastatin (5 μM), or with roscovitine (5 μM) for 36 hrs prior to IFN-α treatment. The mean activation times (with 95% confidence interval) of IRF9 were …
(A) Illustration of how the FUCCI reporter works. The fluorescent signals of chromatin licensing and DNA replication factor 1(Cdt1) and Geminin (Gem) proteins oscillate throughout a cell cycle to …
(A) Histogram showing the numbers of CpG sites at ISG promoters. The promoter region is defined as 1000 bp upstream of the transcription start site. The data are collected from ENCODE database and …
Cells were cultured with medium in the absence (control) or presence of 100 μM decitabine for 48 hrs prior to 100 ng/ml IFN-α treatment. The mean activation times (with 95% confidence interval) of IR…
(A) Representative single-cell time traces from decitabine-treated cells. Cells were treated with 100 μM decitabine for 36 hrs prior to being imaged and kept in decitabine throughout the experiment. …
(A) A diagram for the simple model of the IFN-driven gene regulatory network that incorporates the cell cycle gating of USP18 upregulation. (B) Cell-cycle-dependent delay times in the model …
The image sequence was acquired for 50 hrs in which the first 2 hr were before the addition of IFN-α. The NLS-2xiRFP nuclear marker is shown in red and is merged with phase images of the cells. …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB | This paper | NHM003 | Available upon request from Hao lab |
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry | This paper | NHM008 | Available upon request from Hao lab |
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine | This paper | NHM025 | Available upon request from Hao lab |
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine, shRNA USP18 | This paper | NHM026 | Available upon request from Hao lab |
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine, shRNA USP18 negative control | This paper | NHM027 | Available upon request from Hao lab |
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine, shRNA SOCS1 | This paper | NHM031 | Available upon request from Hao lab |
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine, PUSP18-NLS-mCerulean-USP18 | This paper | NHM032 | Available upon request from Hao lab |
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PUSP18-NLS-mCerulean-USP18, PCMV-DHB-mCherry | This paper | NHM035 | Available upon request from Hao lab |
Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PUSP18-NLS-mCerulean-USP18, PCMV-mCh-Gem1-P2A-CFP-Cdt1 | This paper | NHM036 | Available upon request from Hao lab |
Primers used in this study.
Plasmids constructed in this study.
Best-fit parameter values of the model.