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. Author manuscript; available in PMC: 2020 Nov 16.
Published in final edited form as: Mol Cancer Res. 2020 Mar 19;18(5):735–747. doi: 10.1158/1541-7786.MCR-19-0018

Figure 3. Formation of CECN is dependent on PAD4.

Figure 3.

(A) Treatment of 4T1 cells with the PAD inhibitor, YW3–56, reduced both hypercitrullination and the formation of CECN. Cells were treated for 15 min with YW3–56 followed by 8 h with A23187. Upper panels: arrow denotes inhibition of CECN formation. H3Cit (red), nuclei (blue). Scale Bar: 50 μm. Lower panels: Quantification of percentage of cells with citrullinated nuclei and/or decondensed chromatin. Data are shown as mean ± SD (n = 8–10 fields from three independent experiments). **p<0.01, ***p<0.001. (B) Chromatograms from Sanger sequencing of the region encompassing mutations on exon 1 (left) and exon 2 (right). Green arrows denote mutations. (C) Knockout of the Padi4 gene in 4T1 cells via CRISPR/Cas9 was verified at protein level by Western blot. (D) Transient transfection of hPAD4 into Padi4 CRISPR cells before 8 h 4 μM A23187 treatment rescued the formation of CECN in 4T1 cells. H3Cit (red), nuclei (blue). Scale Bars: 25 μm. Data shown are representative results from 3 independent transfection experiments.