Fig. 5: PAM mRNA increase under hypoxia is HIF1α dependent.

a Levels of PAM mRNA in different glioblastoma cell lines under hypoxia for 24 h. Data are normalized to housekeeping gene EEF2 and the respective normoxic conditions, and are represented as the mean of three independent experiments ± SEM (t test: p value < 0.05* and <0.01**). b PHM activity in different glioblastoma cell lines under 24 h of hypoxia. The experiments were performed in three independent biological replicates ± SEM (t test: p value < 0.05* and <0.001***). N normoxia, H hypoxia. c Relative mRNA (mean of three independent replicates ± SEM; t test with p value < 0.05*) and protein d levels of PAM and PAM sfCD under nontarget or HIF1α knockdown in LN308 cells. EEF2 was used as housekeeping gene for RT-PCR in (c). e HIF1α and PAM sfCD protein levels in glioblastoma cells upon PERK silencing when cultivated under hypoxia for 24 h. EEF2 was used as a loading control. The same protein extracts were also used in Fig. 2a. f Relative PAM mRNA level in the presence of AP-1i (SR11302) in LN308 cells under 24 h of hypoxia. The data are represented as a mean of two independent biological replicates. RPS13 was used as housekeeper gene. g PAM protein level under hypoxia with PERK kinase inhibition (PERKi; GSK2606414 (500 nM)) and AP-1i (SR11302; 2 µM). EEF2 was used as a loading control.