Figure 3: Oridonin activates HSF1 transcription factor and promotes the expression of HSP70 and ubiquitin.

(a) qPCR showing changes of chaperons and ubiquitins in K562 cells after oridonin (20 μM) treatment. (b) HSE reporter gene assay. Upper, schematic diagrams showing the HSE elements (red boxes) in the promoter regions of hsp70, ubb and ubc genes. Mutations introduced for the luciferase reporter assays were shown in red. Lower, K562 cells were transfected with luciferase reporter plasmids and treated with DMSO or oridonin (10 μM). The activity of firefly luciferase was analyzed relative to that of renilla luciferase. *P < 0.05; **P < 0.01. (c) Immunoblotting showing the increase of phosphorylated HSF1 by oridonin (K562: 20 μM for 2 hours; KU812 and SUP-B15: 10 μM for 2 hours). (d) Detection of HSF1 trimer and monomer. Cell lysates from K562 cells treated with DMSO or oridonin (ori, 20 μM) for 2 hours, or heat shock (HS) at 42 °C for 30 minutes, were cross-linked with 1 mM EGS at room temperature for 20 minutes before subjected to immunoblotting. The position of HSF1 monomers and trimers are indicated on the right. (e) Immunofluorescence detecting HSF1 (Red) in Ph⁺ leukemia cells. Cells were treated with DMSO or oridonin (K562: 20 μM; KU812 and SUP-B15: 10 μM) for 24 hours, immuno-stained and analyzed by confocal microscopy. (f) Knockdown of HSF1 prevented oridonin-induced degradation of BCR-ABL and MYC. Forty-eight hours after transfection, K562 cells were treated with 20 μM oridonin for 2 hours and subjected to immunoblotting.