Fig 2: Assessment of CRT, PERK, eIF2a, and p-eIF2a protein content by Western blot analysis. The protein content of CRT, PERK, eIF2a, and p-eIF2a in 12 pairs of DOX-resistant endometrial cancer patients and DOX-sensitive endometrial cancer patients was assessed. In the DOX-resistant endometrial cancer samples, the CRT, PERK, eIF2a, and p-eIF2a protein content was not significantly different between the pre-NAC and post-NAC. (A) In the DOX-sensitive endometrial cancer samples, the CRT, PERK, and p-eIF2a protein content was significantly increased in the post-NAC group compared with the pre-NAC group. (C) There was not a significant change in the eIF2a protein content in the DOX-sensitive endometrial cancer samples. The expression levels were normalized to the actin/pan-cadherin expression values. (B and D) The data are expressed as the means ± standard deviations, which were calculated from three parallel experiments. *P<0.05. CRT; calreticulin; PERK, PKR-like endoplasmic reticulum kinase.

Fig 3: Compounds induce PERK downstream signaling.(A) Wild type and PERK knock out MEF cells were treated with 1 µM Tg or 20 µM compounds for 1 h. (B) NHBE cells were transfected with PERK siRNA or non-targeting (NT) siRNA for 48 h, then treated with compounds at 3 µM for 24 h. (C) NHBE cells were treated with compounds at 10 µM for 1 h or 3 µM for 24 h. Tg (0.5 µM) and hemin (3 µM) served as controls. Treatment was all at 37°C and 5% CO2. Cells were lysed and protein expression was determined by Western Blotting. Data was representative of at least 2 separate experiments.

Fig 4: Dose curves and structures of 3 compounds from focus screening.For the activity analysis, GFP-eIF2a BacMam transduced U-2 OS cells were incubated with DMSO or 0.5 µM PERK inhibitor for 30 min at 37°C and 5% CO2, then plated into 384 well plates and treated with compounds at different doses for 2.5 h at 37°C and 5% CO2. Cells treated with 2 µM Tg only were used as the control for 100% activity. LanthaScreen was performed for detection of phospho-GFP-eIF2a. Data was the average of 2 repeats.

Fig 5: Model for potential crosstalk between PERK and NRF2 signaling pathways.