Journal: International Journal of Molecular Sciences
Article Title: ER Stress-Activated HSF1 Governs Cancer Cell Resistance to USP7 Inhibitor-Based Chemotherapy through the PERK Pathway
doi: 10.3390/ijms25052768
Figure Lengend Snippet: ER stress PERK pathway involves HSF1 phosphorylation and activation. ( A ) Phosphorylated-eIF2α (p-eIF2α), as an ER stress marker, was measured in P22077 (10 µM)- or P5091 (10 µM)-treated cells. ( B ) HEK293 cells transfected with Flag-HSF1-CT were pre-incubated with 2 mM of 4-PBA for 1 h, and the cells were then further incubated with 10 µM of P22077 for 2 h. ( C ) Schematic diagram of signal transduction pathway and key kinases regulating ER stress response. ( D ) Flag-HSF1-CT transfected HEK293 cells were pre-incubated with inhibitors targeting PERK (PERKi, GSK2606414, 100 nM), JNK (JNKi, SP600125, 10 µM), and ASK1 (ASK1i, GS-4997, 20 µM) for 1 h, and the cells were then further incubated with 10 µM of P22077 for 2 h. ( E ) HeLa cells expressing Flag-HSF1-CT were incubated with P22077 (10 µM) and PERKi (100 nM) for 2 h. ( F ) HeLa cells expressing Flag-HSF1-CT were incubated in the absence or presence of P22077 (10 µM), PERKi (100 nM), and 4-PBA (2 mM) for 2 h, as indicated. Phosphorylated HSF1-CT was measured using immunoprecipitation and Western blotting with indicated antibodies. ( G ) HeLa cells expressing Flag-HSF1-CT were incubated in the absence or presence of P22077 (10 µM), MG132 (20 µM), and H 2 O 2 (50 µM) for 2 h. ( H ) Various concentrations of HA-ubiquitin (Ub) and Flag-HSF1-FL were co-transfected in HEK293 cells. ( I , J ) HeLa ( I ) and HEK293 ( J ) cells were pre-incubated with PERKi (100 nM) or 4-PBA (2 mM) for 1 h, and the cells were then further incubated with P22077 (10 µM) for 2 h. ( K ) A shift in the molecular weight of HSF1-CT in control or PERK-silenced HeLa cells. The values represent the mean ± SD (n = 3); * p < 0.05 and ** p < 0.01. One-way ANOVA Tukey’s post hoc test was performed for statistical analysis.
Article Snippet: P22077 (Cat# S7133), P5091 (Cat# S7132), ASK1 inhibitor (GS-4997, Cat# S8292), PYR41 (Cat# S7129), TAK981 (Cat# S8829), and NGI1 (Cat# S8750) were purchased from Selleckchem (Houston, TX, USA).
Techniques: Phospho-proteomics, Activation Assay, Marker, Transfection, Incubation, Transduction, Expressing, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Molecular Weight, Control
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