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. Author manuscript; available in PMC: 2021 Jul 21.
Published in final edited form as: Cancer Res. 2020 Apr 7;80(11):2125–2137. doi: 10.1158/0008-5472.CAN-19-3018

Figure 4. Compartment-specific regulation of stem cell transcription factors by PADI4.

Figure 4.

(A) Schematic of the SORE6 cancer stem cell reporter (for more details, see text). (B) Flow cytometry of MCF10Ca1h cells transduced with control vector (minCMV>GFP) or CSC reporter (SORE6>GFP) and treated with PADI4 inhibitor (GSK484) or control (GSK106) for 3 days. (C) Quantification of SORE6+ CSCs in MCF10Ca1h and MDA-MB-231 LM2 cells. Results are mean +/− SD for n=3; unpaired t-test. (D) Experimental schematic for analysis of cell-compartment specific effects of PADI4. (E) Ingenuity Pathway Analysis of Biofunction enrichment in RNA-Seq generated genelists following PADI4 inhibition in unsorted cells, sorted CSCs and non-CSCs. Enriched biofunctions with an absolute z-score>2 in any cell compartment are shown. A positive z-score reflects activation on PADI4 inhibition. (F,G) Fold change in NANOG (F) and POU5F1 (G) mRNA in CSCs and non-CSCs following PADI4 inhibition in MCF10Ca1h and MDA-MB-231 LM2 cells as assessed by RT-qPCR. (H,I) H3R17me2a ChIP of NANOG (H) and POU5F1 (I) promoters in CSCs and non-CSCs in MCF10Ca1h and MDA-MB-231 LM2 cells. (J,K) H3K4me3 ChIP of NANOG (J) and POU5F1 (K) promoters. Results are percent enrichment compared to input and normalized to H3; mean +/− SD, n=3; unpaired t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.