Journal: PLoS ONE
Article Title: Oxygen Availability for Porphyrin Biosynthesis Enzymes Determines the Production of Protoporphyrin IX (PpIX) during Hypoxia
Figure Lengend Snippet: Transcriptional regulation is not associated with the alteration of porphyrin biosynthesis during hypoxia. (A) MKN45, KatoIII, and MKN74 cells were incubated for 24 h under atmospheres containing 21% O 2 or 1% O 2 . mRNA expression levels of ALAD , HMBS , UROS , UROD , CPOX , PPOX , FECH , PEPT1 , ABCB6 , and ABCG2 in each cell line were analyzed by quantitative PCR. Data are expressed as the means ± S.D. from multi-replicated (n = 2) experiments. ALAD , aminolevulinic acid dehydrogenase; HMBS , hydroxymethylbilane synthase; UROS , uroporphyrinogen III synthase; UROD , uroporphyrinogen III decarboxylase; CPOX , coproporphyrinogen III oxidase; PPOX , protoporphyrinogen oxidase; FECH , ferrochelatase; PEPT1 , Peptide transporter 1 (B) KatoIII cells were incubated with 100 μM CoCl 2 , 100 μM deferoxamine (DFO), or 1 mM dimethyloxaloglycine (DMOG) for 24 h under atmospheres containing 21% O 2 or 1% O 2 . Immunoblots were carried out using antibodies against HIF-1α and actin. (C-F) KatoIII cells were incubated with 1 mM ALA and 100 μM CoCl 2 , 100 μM DFO, or 1 mM DMOG for 24 h under atmospheres containing 21% O 2 or 1% O 2 . (C) Intracellular PpIX, (D) intracellular CPIII, (E) extracellular PpIX and (F) extracellular CPIII were measured using HPLC. (G-J) KatoIII cells were incubated with 1 mM ALA and 10 μg/mL cyclohexamide. (G) Intracellular PpIX, (H) intracellular CPIII, (I) extracellular PpIX and (J) extracellular CPIII were measured using HPLC. Data are expressed as the means ± S.D. from multi-replicated (n = 3) experiments.
Article Snippet: Briefly, PpIX and CPIII were separated using an HPLC system (Prominence, Shimadzu, Kyoto, Japan) equipped with a reversed-phase C18 Column (CAPCELL PAK, C18, SG300, 5 μm, 4.6 mm × 250 mm, Shiseido Co., Ltd., Tokyo, Japan).
Techniques: Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, High Performance Liquid Chromatography