(a) Transcript levels for Padi1, Padi2 and Padi3 in ES, NS and iPS cells, as assessed by qRT-PCR. Padi6 was undetectable in all three cell types. Expression normalized to endogenous levels of Ubiquitin (UbC). Error bars represent the standard error of the mean of three biological replicates.
(b) Transcript levels of Padi1, Padi2 and Padi3 in ES cells upon switch to 2i containing medium for one passage, as assessed by qRT-PCR. Padi6 was undetectable in both conditions. Expression normalized to UbC. Error bars represent the standard error of the mean of three biological replicates.
(c) Immunoblot analysis of H3Cit levels in ES, NS and iPS cells. Total H3 is presented as loading control.
(d) Immunoblot analysis of total citrullination the levels in ES, NS and iPS cells, using an antibody against Modified Citrulline (ModCit) which recognizes peptidylcitrulline irrespective of amino acid sequence. Total H3 is presented as loading control.
(e) ZHBTc4.1 and 2TS22C ES cell lines were treated with 1µg/ml doxycycline for 48 hours, resulting in depletion of Oct4 or Sox2 (data not shown). Padi4 mRNA was significantly reduced upon Oct4, but not Sox2 knockdown, as assessed by qPCR. Error bars represent standard error of the mean of four biological replicates.
(f) ChIP-qPCR for Oct4, Sox2, Klf4, RNA polymerase II (PolII), H3K4me3 and H2A on the promoter of Padi4 in mES and NS cells. For each cell condition, the signal is presented as fold enrichment over Input and after subtracting background signal from the beads. Error bars represent the standard deviation of three technical qPCR replicates.
Asterisks denote difference with ES cells (a) or media (b), and 0h time point (e); - not significant, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 by ANOVA (a) or t-test (b,e).