Figure 5 | Scientific Reports

Figure 5

From: Downregulation of PARP1 transcription by promoter-associated E2F4-RBL2-HDAC1-BRM complex contributes to repression of pluripotency stem cell factors in human monocytes

Figure 5

PARP1 protein recovery enhances transcription of pluripotency stem cell factors POU5F1, NANOG and SOX2. In order to recover PARP1 transcription human monocytes were transfected with siRBL2, siPARP1 and siRBL2/siPARP1 or incubated with iHDAC for 48 h and additionally transfected with siPARP1. The PARP1 and RBL2 protein was determined with western blot (a). The expression of embryonic stem cell relevant transcription factors and RUNX1, GATA2 and PAX5 was analyzed with real-time PCR (bh). THP-1 cell were transfected with pCMV3-EMPTY vector (negative control) and pCMV3-PARP1 expression vector and differentiated with PMA (10 ng/ml) for 48 h. PARP1 level was monitored with western blot (i) and real-time PCR (j). POU5F1, NANOG and ZFP42 transcription was quantified in genomic DNA-free samples using primers designed to span exon-exon boundary (Supplem. Table 1: POU5F1 ex-ex, NANOG ex-ex, ZFP42 ex-ex) by real-time PCR (k,l,n). The same method was employed to determine mRNA level of the intronless SOX2 (m). For normalization of POU5F1, NANOG, SOX2 and ZFP42 transcription (kn) three housekeeping genes (HSKG) were used (ACTB, GAPDH, B2M).

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