Fig. 5.

Amino acid limitation affects HSF1 binding affinity and HSP mRNA levels. a HEK293 cells were cultured in medium containing limiting amounts of leucine for 24 h. The concentrations are indicated relative to the standard leucine concentration in medium (+), which is 450 μM. EMSA was performed with a double-stranded oligonucleotide with the HSE sequence. The arrow indicates the primary complexes formed. b HEK293 cells were cultured for the indicated times in medium containing 45 μM leucine. As a control, cells were cultured in parallel in standard medium containing 450 μM leucine (+). Medium was changed every day. Nuclear extracts were used in EMSA with a double-stranded oligonucleotide with the HSE or NSRU sequence. The closed arrow indicates the HSE complex formed. Open arrows indicate the NSRU complexes formed. c ASNS, DNAJB1, and HSPA1A mRNA levels were determined by QPCR analysis and are shown relative to GAPDH mRNA levels. Error bars represent SD; *P < 0.05; **P < 0.01; ***P < 0.001, relative to 0 days