Fig 2: PACT is not required for spontaneous activation of Ser6- and Ser97-Ala PKR mutants. PACT-PKR DKO cells and PKR-KO cells were transfected with plasmids expressing either wt PKR or indicated PKR mutants. After 48 h, PKR activation (phospho-Thr446) was analyzed by western blot (a) and stress granule formation was quantified in transfected cells using eIF3 detection by immunofluorescence (b). (a) Detection of phospho-Thr446 PKR (PKR-pT446), PKR and PACT by western blot. Note the similar activation of PKR in PACT-positive and PACT-deficient cells. ß-actin was detected as a loading control. Acquisition was made with a CCD camera (Fusion Solo-S, Vilber) using the Bio1D software (version 15.08). (b) Graph (generated with Microsoft Excell 2011) showing the percentage of stress granule-positive cells counted among PKR+ cells, for PACT-PKR DKO and PKR-KO cell lines (mean ± SEM) (n = 4). 2-way ANOVA (Graphpad Prism 7) was used to compare mutant and wt PKR in each cell line.

Fig 3: In vitro interaction of PKR and PACT by HTRF. (A) Schematic representation of full length PKR, PKR 1–265 fragment in fusion with GST at its N-terminus, PACT and PACT in fusion with His tag at its N-terminus. The position of the two Double Stranded RNA Binding Domains are indicated (respectively K1 and K2 for PKR and A1 and A2 for PACT) as well as the M3 domain of PACT. The PKR-Nter fragment ends at aa 265, located in the 234–275 basic domain of PKR (grey box). PRI peptide corresponds to the amino-acids 60 to 80 of first DRBD of PKR. (B) Analysis of the GST-PKR-Nter and His-PACT proteins by SDS-PAGE after purification on glutathione-sepharose and on Ni-charged His-bind resin, respectively. Gel analysis shows the profile of GST-PKR-Nter and His-PACT after purification. The purified fractions were pooled and processed for resuspension as soluble proteins as described in Materials and Methods. (C) schematic representation of the HTRF assay (Cisbio) showing transfer of energy from the donor fluorophore (Lumi4Tb) to the acceptor (XL665), each coupled to anti-6His or anti-GST antibodies, when GST-PKR and His-PACT are interacting. (D) GST-PKR and His-PACT (100 nM each) were mixed and incubated for 30 min at 30 °C before addition of 4 nM of anti-6His Lumi4Tb antibodies and either 15 or 30 nM of anti-GST XL665 antibodies. (E) GST-PKR (100 nM) was first incubated for 30 min with different concentrations of PRI as indicated before addition of His-PACT (100 nM). After another 30 min of incubation, the anti-6His Lumi4Tb (4 nM) and anti-GST XL665 (15 nM) antibodies were added. (F) GST-PKR and His-PACT (100 nM each) were mixed and incubated for 30 min at 30 °C before addition of DMSO at the indicated concentrations. After another 30 min, the anti-6His Lumi4Tb (4 nM) and anti-GST XL665 (15 nM) antibodies were added. Incubation and reading of fluorescence were performed for the indicated times. The percentage of Delta F was calculated as described in Materials and Methods.

Fig 4: Effect of compounds on the PKR/PACT interaction monitored by NPCA. (A) HEK 293 T cells (30,000 cells per well in 96-well plates) were transfected with 100 ng of PKR-Nter or PACT plasmids bearing the Nanoluc moiety 1 or 2 at their N- or C-terminus, as indicated. At 24 h post-transfection, the medium of the cells was discarded and the cells were incubated in the presence of 50 µl of Nano-Glo reagent. Luciferase enzymatic activity was measured using a Berthold Centro XS LB960 luminometer and counting luminescence for 5 s. The values represent the mean of triplicate experiments. Saturation of luciferase signal was found to occur frequently in the HEK293T cells and the subsequent NPCA experiments were then performed in the Huh7.5/CD81 cells, which proved to give less intense and still satisfactory luciferase signal. (B,C) Huh7.25/CD81 cells in 96-well plates were transfected with 100 ng each of PKR-N2 and PACT-N1. They were then either untreated or incubated with different concentrations of the Lyon compounds (referred to as #36, #39 and #40) (B), or luteolin, quercetin and myricetin (C) as indicated. Treatment was done either at the time of transfection or up to 6 hrs post transfection with identical effect on the luciferase activity. Luciferase enzymatic activity was measured after 24 hrs. The values represent the mean of triplicate experiments. (D,F) The DRBDs of PKR or PACT were cloned in fusion with the moeity 1 or 2 of luciferase at their N-terminus. The Huh7.25/CD81 cells in 96-well plates were transfected with 100 ng of the resulting plasmids expressing the first or second DRBD of PKR (K1 or K2; (D), or PACT (A1 or A2; (E) in homodimerization (D,E) or heterodimerization (F) configuration. The cells were either untreated or incubated with different concentrations of luteolin. Luciferase enzymatic activity was measured after 24 hrs. The values represent the mean of triplicate experiments.

Fig 5: The PKR/PACT inhibitor Luteolin triggers activation of the inflammasome. The THP1 cells were differentiated by PMA treatment for 72 hrs as described in Materials and Methods. The cells were either untreated (NT) or treated with 50 µM of luteolin (Lut) or 10 µM of Salubrinal (salub) alone. After 30 min, incubation was continued for 8 hrs as such or in the presence of 5 µM of thapsigargin and 100 ng/ml KLA (Tg + KLA). Where indicated, the cells were treated with luteolin in the presence of each of the other drugs. After 8 hrs, activation of Caspase-1 (A) and secretion of IL-1ß (B) in the supernatants were determined by ELISA and the values were normalised to the amount of total protein for each condition. Data represents means ± SD from three independent supernatants.