N^G-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, attenuated hypoxia-inducible Tβ4 expression and cell migration.

<p><b>A:</b> HeLa cells were transfected with Tβ4-GLuc for 6 h and incubated under normoxic or hypoxic conditions for 4 h in the presence or absence of L-NMMA. GLuc activity in culture medium was measured using a luminometer with a GLuc substrate. Data in the bar graph are means ± SED. **<i>p</i><0.01, statistical significance <i>vs</i>. Tβ4 promoter activity in the normoxia control group. ^##<i>p</i><0.01, statistical significance <i>vs</i>. Tβ4 promoter activity in L-NMMA-untreated control at each normoxic or hypoxic condition. <b>B–C:</b> HeLa cells were incubated under normoxic or hypoxic conditions for 4 h in the presence or absence of L-NMMA. RNA was purified with TRIzol reagent and cell lysates were prepared as described in the Materials and Methods. The Tβ4 level was measured by RT-PCR (<b>B, left</b>) and Western blot analysis (<b>B, right</b>). HIF-1α was detected using Western blot analysis (<b>C</b>). <b>D:</b> NO production was detected with Griess reagents as described in the Materials and Methods. Data in the bar graph represent mean ± SED. **<i>p</i><0.01, statistical significance <i>vs</i>. NO production in the normoxia control group. ^#<i>p</i><0.05; ^##<i>p</i><0.01, statistical significance <i>vs</i>. NO production in L-NMMA-untreated control at each normoxic or hypoxic condition. <b>E–G:</b> HeLa cells were scratched and incubated under normoxic conditions for 15 h in the presence or absence of L-NMMA after 1 h-incubation under normoxic or hypoxic condition. Cell migration into the space left by the scratch was photographed with a phase-contrast microscope. Photographs were taken at the same magnification; 200×. Data represent four experiments (<b>E</b>). The empty area was quantified using NIH image analysis software (version 1.62) and compared with the initiation of cell migration. Percentage of cell migration is presented as a bar graph. Data in the bar graph are means ± SED. **<i>p</i><0.01, statistical significance <i>vs</i>. cell migration in the normoxia control group. ^##<i>p</i><0.01, statistical significance <i>vs</i>. cell migration in the L-NMMA-untreated control under each normoxic or hypoxic condition (<b>F</b>). NO production was detected with Griess reagents as described in the Materials and Methods. Data in the bar graph represent mean ± SED. *<i>p</i><0.05, statistical significance <i>vs</i>. NO production in the normoxia control group at each time point (<b>G</b>).