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PARP-1 Modulates HIF-2α Accumulation and EPO Expression in Immortalized MEFs Under Hypoxia

This study investigates the role of PARP-1 in the regulation of HIF-2α accumulation and EPO mRNA expression under hypoxic conditions in immortalized mouse embryonic fibroblasts (MEFs). Immunofluorescence analysis reveals that hypoxia-induced nuclear HIF-2α accumulation is diminished in PARP-1 knockout MEFs compared to wild-type cells. Additionally, the silencing efficacy of siRNAs targeting PARP-1, HIF-1α, and HIF-2α was validated using western blotting. EPO mRNA levels were quantified via Q-PCR, demonstrating significant differences between genotypes under hypoxia, with statistical significance noted.

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PARP-1 Modulates HIF-2α Accumulation and EPO Expression in Immortalized MEFs Under Hypoxia

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  1. Figure S1 parp-1 +/+ parp-1 -/- H N H N HIF-2 DAPI Fig S1. Nuclear HIF-2 accumulation by hypoxia is reduced in parp-1 -/- immortalizedMEFs.Immunofluorescence staining of parp-1 +/+ and parp-1 -/- immortalized MEFs incubated in normoxia (N) or hypoxia (H, 16 hours at 1 % O2). Representative images from several independent experiments are shown.

  2. Figure S2 H H PARP-1 HIF-1α Control HIF-2α Control Control siRNA: siRNA: siRNA: HIF-1α HIF-2α PARP-1 β-Actin β-Actin β-Actin Figure S2. Validation of silencing efficacy: siRNA of PARP-1, HIF-2 or HIF-1. (Cells were transfected with the correspondig siRNAs and 48h later, the expression levels of the targeted protein was evaluated by WB. HIF expression was analyzed after 16 hours of exposure to hypoxia (1% O2).

  3. Figure S3 parp-1+/+ 75 15 15 Liver Kidney Brain parp-1-/- 50 10 10 Epo mRNA expression (fold induction) N N N H H H * 25 5 5 ** 0 0 0 Figure S3. EPO mRNA levels were determined by Q-PCR from liver, kidney and brain . The level of mRNA in each sample was expressed as fold of the value in control wild-type mice. Numeric value represents mean of hypoxic induction in each genotype at 8 hours, respectively. (*) and (**) indicate p< 0.05 and p<0.001, with respect to wild-type mice.

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