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. 2020 Mar 16;9:e52241. doi: 10.7554/eLife.52241

Figure 1. Activation of PACT-PKR axis promotes proinflammatory gene expression in response to high-intensity hyperosmotic stress.

(A) As stress intensity increases, signaling shifts from c-Rel-mediated adaptation to PACT/PKR-mediated inflammation. (B) MEFs selected for shRNA-mediated knockdown of PACT and control vector were treated with 500 or 600 mOsm sucrose for the indicated durations. Lysates were analyzed via western blot for the indicated proteins. (C) PKR KO MEFs reconstituted with FLAG-tagged human PKR were treated with sucrose or DSS for 3 hr. Co-immunoprecipitation with FLAG antibody was analyzed by western blot. Inputs are 5% of immunoprecipitated sample. (D) Control and shPACT MEFs were treated with 500 or 600 mOsm sucrose. Lysates were assayed for caspase-3 enzymatic activity. (E) Control and shPACT MEFs were treated with 500 or 600 mOsm sucrose. RNA was isolated, and transcripts were analyzed via RT-qPCR for the indicated mRNAs.

Figure 1—source data 1. Graph values for caspase-3 activity assays and RT-qPCR experiments in Figure 1.

Figure 1.

Figure 1—figure supplement 1. PACT Ser-246 and Ser-287 are necessary for PKR interaction and downstream inflammatory signaling.

Figure 1—figure supplement 1.

(A) PACT KO MEFs were reconstituted with FLAG-WT PACT or FLAG-S246A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. Proteins from total cell extracts or co-immunoprecipitated with the FLAG antibody were analyzed via western blot. (B) PACT KO MEFs were reconstituted with FLAG-WT PACT, FLAG-S246A, or FLAG-S287A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. Total lysates were analyzed via western blot. (C) PACT KO MEFs were reconstituted with FLAG-WT PACT or FLAG-S246A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. RNA was isolated, and Nos2 transcript levels were analyzed via qPCR. (D) PKR KO MEFs reconstituted with wild type PKR, PKR mutated at the RNA-binding residues (K64R/K154R), or kinase activity-deficient PKR (K296R) were treated with 500 or 600 mOsm sucrose for the indicated durations. RNA was isolated, and Nos2 transcript levels were analyzed via qPCR.

Figure 1—figure supplement 2. PACT influences the TonEBP-dependent adaptive transcription program.

Figure 1—figure supplement 2.

(A) MEFs deficient in PACT were treated with 500 mOsm hyperosmotic media for the indicated durations. Total mRNA was isolated, and target mRNA levels analyzed via RT-qPCR. (B) MEFs deficient in PACT were treated with 500 mOsm hyperosmotic media for 3 hr. Actinomycin D was added, and total mRNA was harvested after the indicated duration. mRNA half-life was calculated using linear regression analysis.