(A) mESCs were transfected with empty vector control or MEK5DD for (48 h) and treated with either DMSO or 10 µM AX15836 (24 h prior to lysis). ERK5 activation was assessed by band-shift following ERK5 immunoblotting (P = phosphorylated active ERK5, UnP = unphosphorylated inactive ERK5) and by KLF2 induction. Expression levels of ZSCAN4 were determined by immunoblotting. ERK1/2 was used as a loading control. (B) mESCs were transfected with either empty vector or MEK5DD. mRNA levels of Klf2, Zscan4, Tdpoz3 and Zfp352 were determined by qRT-PCR. Klf2 induction is used as a positive control for ERK5 activity. Each data point represents one biological replicate calculated as an average of two technical replicates (n = 3). Error bars represent mean ± SEM. Statistical significance was determined by student t-test. (C) Erk5/Mapk7−/− mESCs were transfected with MEK5DD and either empty vector, wild-type ERK5 or kinase inactive (D200A) ERK5. mRNA levels of Klf2, Zscan4, Tdpoz3 and Zfp352 were determined by qRT-PCR. Klf2 induction is used as a positive control. Each data point represents one biological replicate calculated as an average of two technical replicates (n = 3). Error bars represent mean ± SEM. Statistical significance was determined by student t-test. (D) Genomic DNA from mESCs maintained in LIF/FBS or 2i media was subjected to qPCR using primers against the telomeric repeats (T) and a single locus control region (S). T/S ratio was calculated to give the average relative telomere length. Each data point represents one biological replicate calculated as the average of three technical replicates (n = 7). A power calculation was used to determine sample size, which was randomly selected from biological replicates. Error bars represent mean ± SEM. Statistical significance was determined by student t-test. (E) Genomic DNA from mESCs transfected with either empty vector or MEK5DD (48 h) and treated with either DMSO or 10 µM AX15836 (24 h prior to lysis) was collected and subjected to qPCR using primers against the telomeric repeats (T) and a single locus control region (S). T/S ratio was calculated to give the average relative telomere length. Each data point represents one biological replicate calculated as the average of three technical replicates (n = 22). A power calculation was used to determine sample size, which was randomly selected from biological replicates. Error bars represent mean ± SEM. Statistical significance was determined by student t-test. (F) Genomic DNA from mESCs transfected with either empty vector or ZSCAN4 (48 h) was collected and subjected to qPCR using primers against the telomeric repeats (T) and a single locus control region (S). T/S ratio was calculated to give the average relative telomere length. Each data point represents one biological replicate calculated as the average of three technical replicates (n = 7). A power calculation was used to determine sample size, which was randomly selected from biological replicates. Error bars represent mean ± SEM. Statistical significance was determined by student t-test.