Journal: bioRxiv

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1101/2025.09.11.675615

Figure Lengend Snippet: (A) A549 cells were treated with DMSO, KIRA6 (10 μM), or Ceapin-A7 (6 μM) for 24 hours. Alamar blue assay was used to assess cell viability based on cellular metabolic activity. (B-C) A549 cells were primed with DMSO or Tg (0.05 μM) in the absence or presence of KIRA6 (10 μM) or Ceapin-A7 (6 μM). Cell lysates were collected at 24 hours post-treatment. Xbp1s (B) or HERPUD1 (C) protein expression was assessed by western blot. (D-F) A549 cells were primed with DMSO or Tg (0.05 μM) in the presence or absence of KIRA6 (10 μM), prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or KIRA6 (10 μM). (D-F) Cell lysates were collected at 24 hpi to assess XBP1s and N gene expression by RT-qPCR (D, F) , or Xbp1s and HCoV-229E N protein expression by western blot (E) . (G-I) A549 cells were primed with DMSO or Tg (0.05 μM) in the presence or absence of Ceapin-A7 (6 μM) prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or Ceapin-A7 (6 μM). (G-I) Cell lysates were collected at 24 hpi to assess HERPUD1 and N by RT-qPCR (G, I) , or HERPUD1 and HCoV-229E N protein expression by western blot (H) . RT-qPCR data are normalized to actin and set relative to DMSO. (J) A549 cells were primed with DMSO or Tg (0.05 μM) in the presence or absence of KIRA6 (10 μM) or Ceapin-A7 (6 μM), alone or in combination, prior to infection with HCoV-229E (MOI 0.05). Inoculum was removed 2 hours later and replaced with media containing the respective inhibitor(s). Viral supernatants were collected 24 hpi and titrated by plaque assay on Huh7 cells. Graphs show mean +/− SEM from three independent experiments performed in triplicate (*p<0.05, ****p<0.0001). Representative western blots are shown.

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1:1000), ATF6 (CST 8089, dilution 1:1000), PERK (CST 5683, dilution 1:1000), XBP1s (CST 12782T, dilution 1:1000), HERPUD1 (Abcam ab150424, dilution 1:1000), eIF2α-P (Ser-51) (CST 3398S, dilution 1:1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1:10,000), beta-actin (Abcam ab8226, dilution 1:5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1:5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1:1000) were used for western blotting.

Techniques: Alamar Blue Assay, Activity Assay, Expressing, Western Blot, Infection, Gene Expression, Quantitative RT-PCR, Plaque Assay