Fig 1: APE1 regulates VEGF gene expression in HUVECs. HUVECs were transfected with adenovirus Ad5 or F35-shAPE1 for 48 h to overexpress or knock down APE1 expression, respectively. Cells were then cultured in hypoxic conditions (1% O2). VEGF mRNA levels were measured by qPCR after 8 h of hypoxic exposure (A). VEGF and APE1 protein levels in whole cell extracts were assessed by Western blot after 24 h of hypoxic exposure (B). VEGF secretion in the culture medium was assessed by ELISA after 48 h of hypoxic exposure (C). * P < 0.01, treated group vs control group by Student's t-test.
Fig 2: Both mono-functional and bi-functional DNA glycosylases depend on APE1 for DNA backbone cleavage in cell lysates(A) Purified methylpurine DNA glycosylase (MPG) and APE1 probed for lesion repair activity using DRMB-Hx and DRMB-Con2 assays. (B) Analysis of APE1 activity: LN429/SCR (LN429 cells expressing a scrambled control shRNA) and LN429/APE1-KD cell lysates probed for lesion repair activity using DRMB-THF2 assays, as compared to a control beacon (DRMB-Con2) containing no DNA lesion. (C) Analysis of uracil glycosylase activity: LN429/SCR (LN429 cells expressing a scrambled control shRNA) and LN429/APE1-KD cell lysates probed for lesion repair activity using DRMB-dU/A assays, as compared to a control beacon (DRMB-Con2) containing no DNA lesion. (D) Simplified schematic detailing the lesion removal and strand cleavage activities of mono-functional and bi-functional DNA glycosylases and APE1 in BER. Mono-functional DNA glycosylase activity (Left) leaves an abasic site as a substrate for APE1, resulting in DNA strand cleavage. Bifunctional DNA glycosylases excise the base and then catalyze either (Center) β-elimination, resulting in a 3’phospho, unsaturated aldehyde (PUA) and a 5’phosphate (P) in the gap that is then further processed by APE1. (Right) β,δ-elimination resulting in a 3’phosphate (P) and a 5’phosphate (P) in the gap that can then be further processed by polynucleotide kinase 3′-phosphatase (PNKP). (E) Analysis of APE1 activity: U2OS/SCR (U2OS cells expressing a scrambled control shRNA) and U2OS/APE1-KD cell lysates probed for lesion repair activity using DRMB-THF2 assays, as compared to a control beacon (DRMB-Con2) containing no DNA lesion. (F) Analysis of glycosylase activity for removal of thymine glycol: U2OS/SCR (U2OS cells expressing a scrambled control shRNA) and U2OS/APE1-KD cell lysates probed for lesion repair activity using DRMB-Tg assays, as compared to a control beacon (DRMB-Con2) containing no DNA lesion. Plot data show normalized fluorescence values and are the mean of three independent experiments with error bars representing SEM. Statistical values are listed in Supplementary Tables 1 and 2.
Fig 3: Evaluation of APE1 activity using a DRMB-Bead assay via flow cytometry analysis(A) Diagram of the DRMB-Biotin-THF with a T at the loop region of the DRMB-THF2 assay replaced with a biotin labeled T to allow conjugation to streptavidin beads. Further, the CC-FAM at the 5′ end of the DRMB-THF2 was replaced with GG-Iowa Black® Dark quencher and the GG-Dabcyl at the 3′ end was replaced with a CC-FAM. Thus, after cleavage of beacon and release of the quencher, the fluorescent signal (FAM) will remain on the beads for signal measurement by flow cytometry. (B) Purified APE1 protein probed for abasic site cleavage activity using DRMB-THF2 and DRMB-Biotin-THF assays in solution.Plot data are the mean of three independent experiments, with error bars representing SEM. (C) Mixing fully cleaved and un-cleaved DRMB-Biotin-THF formed different ratios of fully cleaved and un-cleaved DRMB-Biotin-THF beacons. After those beacon mixtures were captured by streptavidin beads and analyzed by flow cytometry, the ratio of fully cleaved and un-cleaved DRMB-Biotin-THF were then differentiated by the Geo mean value. Data is represented as MFI (mean fluorescence intensity-geometric mean) of each mixture of beacons, R2 value = 0.9958. (D) Flow cytometry scan of the DRMB-Biotin-THF bound to beads and after incubation in buffer (1: without APE1) or with added APE1 (2: with APE1). Data is represented as MFI (mean fluorescence intensity-geometric mean). (E) Immunoblots showing the depletion of APE1 protein in U2OS/APE1-KD cell lysate compared to that of the U2OS/SCR control. (F) Flow cytometry scan of the DRMB-Biotin-THF bound to beads and after incubation in cell lysate (1: U2OS/APE1-KD or 2: U2OS/SCR). Data is represented as MFI (mean fluorescence intensity-geometric mean).
Fig 4: Apex1-deficiency leads to neural crest cell differentiation defects.(A) qPCR expression analysis of pluripotency (Pou5f1), endoderm (Gata6), mesoderm (Eomes), neuroectoderm (Pax6) and neural crest (Pax3) marker genes of control and Apex1-deficient mESCs, EBs and EBs + RA. Expression of marker genes was normalized to Tbp and is relative to control mESCs (s.d., n = 3 technical replicates). (B) Quantification of differentially expressed genes in Apex1-deficient mESCs, EBs and EBs + RA. (C) Overlap of differentially expressed genes from Neil1, Neil2 and Apex1 single-deficient EBs + RA. (D) Pathway enrichment analysis of downregulated genes from Apex1-deficient EBs + RA. Dashed line indicates the significance threshold FDR = 0.05.
Fig 5: DRMB assay detected functional changes of APE1 with single amino acid substitutions(A) The position of the nine single amino acid substitutions of APE1 is indicated in the diagram. The red boxes indicate mutants with increased initial rate activity as determined by the DRMB-THF2 assay. The blue boxes indicate mutants with decreased initial rate activity as determined by the DRMB-THF2 assay. The black boxes indicate a mutant with a loss of activity as determined by the DRMB-THF2 assay. (B) APE1 mutants and WT protein (0.5 µg each) were analyzed for abasic site cleavage activity using the DRMB-THF2 assay. The activity of each protein was normalized to the maximum fluorescent signal within each well, Fl(Tmax), as described in the Materials and Methods and previously [34] and then normalized to each APE1 protein concentration detected by immunoblotting analysis using an APE1 antibody. Plots show the mean of three independent experiments, with error bars representing SEM. Statistical values are listed in Supplementary Tables 1 and 2.
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