Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction (2020)
Figure 4

Figure 4, Model analysis of the dynamics of pSTAT1 complex formation and application to Roferon and HepG2-hNTCP cells

Model analysis reveals impact of different prestimulation doses on the dynamics of pSTAT1-containing nuclear complexes. The time-resolved amounts of nuclear pSTAT1 homodimers, pSTAT1:pSTAT2 heterodimers and pSTAT1:pSTAT2:IRF9 trimers were calculated by the mathematical model. Simulations were performed for Huh7.5 cells stimulated with 1400 pM IFNα that were either untreatead or prestimulated with 28 pM IFNα or 280 pM IFNα for 24 hours. Different STAT1 comprising transcription factor complexes are indicated.

Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) (left panel), of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers (middle panel) and of the pSTAT1:pSTAT2:IRF9 trimers (right panel) in untreated Huh7.5 cells and in Huh7.5 cells prestimulated for 24 hours with 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Lines with shading represent model predictions with 68% confidence intervals using the prediction profile likelihood method. For experimental validation of the dynamics of OccGASbs, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were incubated with radioactively-labeled oligonucleotides probes harboring the GAS-binding region of the human IRF1 promoter. Samples were resolved on a native polyacrylamide gel and radioactivity was visualized and quantified from three independent experiments (left panel). For experimental validation of the dynamics of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations (IP) were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation with antibodies recognizing STAT2 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (middle panel). For experimental validation of the dynamics of the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation (IP) with antibodies recognizing IRF9 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (right panel). Antibodies and the corresponding proteins in the complexes are underlined. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3) using a combined scaling and error model.

Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) and of occupied ISRE binding sites (OccISREbs) in Huh7.5 cells without prestimulation and in cells prestimulated for 24 hours with 28 pM and 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Model predictions were performed using the prediction profile likelihood method. Lines with shading represent model predictions with 68% confidence intervals. For experimental validation, growth factor-depleted Huh7.5 cells were prestimulated with 0 pM, 28 pM and 280 pM IFNα. After 24 hours cells were stimulated with 1400 pM IFNα and IFNα-induced expression of target genes was measured by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP, averaged and displayed as fold change, represented by filled circles with errors representing standard error of the mean calculated from biological replicates (N=3). Except for gene-specific parameters (mRNA synthesis and degradation rates, time-delay parameter and Hill coefficient), qRT-PCR data were used for model validation but not for model calibration.

Dose-dependent sensitization of signal transduction induced by the therapeutic interferon Roferon. Growth factor-depleted Huh7.5 were prestimulated with 0, with 1.2 or 608 pM Roferon and stimulated with 608 pM Roferon 24 hours later. Concentrations of Roferon correspond to equipotent concentrations of IFNα. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescense utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=8) using a combined scaling and error model. Model trajectories are represented by lines.

Dose-dependent sensitization of signal transduction induced by Roferon in HepG2-hNTCP. Growth factor-depleted HepG2-hNTCP were prestimulated with 0, 1.8 or 887 pM Roferon and stimulated with 887 pM Roferon 24 hours later. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescence utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3) using a combined scaling and error model. Model trajectories are represented by lines.