To knock down Hsp70 and Hsc70 expression, and prevent induction during treatments, RNA interference experiments were performed using ON-TARGET plus SMARTpool siRNAs (GE Healthcare/Dharmacon, Freiburg, Germany). Cells are cultured in antibiotic-free medium. Then, 24h after seeding, cells were transfected with siRNA and DharmaFECT 3 Transfection Reagent (GE Healthcare/Dharmacon, Freiburg, Germany) according to the manufacturer's instructions. For Hsp70 knockdown 12.5 nM Hspa1a siRNA and 12.5 nM Hspa1b siRNA were used. Hsc70 knockdown was performed using 6.25 nM Hspa8 siRNA. Furthermore, non-targeting scrambled siRNA was used as negative control. Appropriate siRNA concentrations were tested before on cell viability using MTT assay (Fig. 3B ).
To express an Hsp70-GFP fusion protein, the cells were transfected using TurboFect Transfection Reagent (Thermo Fisher Scientific, Schwerte, Germany) 24h after seeding. The vector was purchased from Origene (Rockville, USA): pCMV6-AC-GFP with Insert HSPA1A (human heat shock 70 kDa protein 1A) (RG200270, Origene, Rockville, USA). A pCMV6-AC-GFP without Insert (PS100010, Origene, Rockville, USA) transfected with the same conditions was used as control.
To express an Hsp70-GFP fusion protein, the cells were transfected using TurboFect Transfection Reagent (Thermo Fisher Scientific, Schwerte, Germany) 24h after seeding. The vector was purchased from Origene (Rockville, USA): pCMV6-AC-GFP with Insert HSPA1A (human heat shock 70 kDa protein 1A) (RG200270, Origene, Rockville, USA). A pCMV6-AC-GFP without Insert (PS100010, Origene, Rockville, USA) transfected with the same conditions was used as control.