Figure 6.
Figure 6. Neutrophils with decreased TG2 activity express lower levels of GP91PHOX mRNA and protein, and generate less superoxide. (A-B) Northern blot and real-time quantitative PCR (Q-PCR) analysis of GP91PHOX mRNA expression in NB4 cells. (A) NB4 cells were treated with 1 μM ATRA or 1 μM ATRA plus 15 μM MDC for 3 days. RNA was prepared and subjected to Northern blot analysis as described in “Materials and methods.” (B) NB4 cells were incubated with MDC from day 1 in the presence of ATRA and harvested from following days. Relative expression of GP91PHOX was normalized to the expression of human cyclophilin. (C) NBT positivity of TG2–/– and TG2+/+ mouse neutrophils. Wild-type and TG2–/– mice were injected intraperitoneally with 1 mL 10% yeast extract. Four hours after the injection, the peritoneal lavage fluid was withdrawn and washed. Granulocytes were allowed to adhere for 0.5 hours, and were then analyzed for NBT reduction. Inset images are NBT-stained neutrophils in a 96-well microplate. Visualization was done using an inverted microscope (Axiovert 135; Zeiss, Oberkochen, Germany), an Achrostigmat 20×/0.45 Ph2 objective, adapter ring VAD-S70, and a DSC-S70 digital still camera (Sony, Tokyo, Japan). Film was imaged in AlphaImager 2200 (Alpha Innotech, San Leandro, CA). (D) Superoxide generation and gp91phox protein level of TG2–/– and TG2+/+ mouse neutrophils. Wild-type and TG2–/– mouse neutrophils were collected and separated as previously described. The reaction volume of 500 μL contained 1 × 105 cells and 5.0 μL L-012 (100 μM). ROSs were generated by adding 50 nM PMA. Chemiluminescence was detected as described in “Materials and methods.” Inset: Proteins of wild-type and TG2–/– mouse neutrophils were analyzed by Western blotting using an antibody against the gp91phox and beta-actin. (E) Q-PCR analysis of gp91phox mRNA expression of TG2–/– and TG2+/+ mouse neutrophils; gp91phox mRNA levels were normalized to mouse cyclophilin. Data in panels B-D are representative of 2 independent experiments; error bars represent SD.

Neutrophils with decreased TG2 activity express lower levels of GP91PHOX mRNA and protein, and generate less superoxide. (A-B) Northern blot and real-time quantitative PCR (Q-PCR) analysis of GP91PHOX mRNA expression in NB4 cells. (A) NB4 cells were treated with 1 μM ATRA or 1 μM ATRA plus 15 μM MDC for 3 days. RNA was prepared and subjected to Northern blot analysis as described in “Materials and methods.” (B) NB4 cells were incubated with MDC from day 1 in the presence of ATRA and harvested from following days. Relative expression of GP91PHOX was normalized to the expression of human cyclophilin. (C) NBT positivity of TG2–/– and TG2+/+ mouse neutrophils. Wild-type and TG2–/– mice were injected intraperitoneally with 1 mL 10% yeast extract. Four hours after the injection, the peritoneal lavage fluid was withdrawn and washed. Granulocytes were allowed to adhere for 0.5 hours, and were then analyzed for NBT reduction. Inset images are NBT-stained neutrophils in a 96-well microplate. Visualization was done using an inverted microscope (Axiovert 135; Zeiss, Oberkochen, Germany), an Achrostigmat 20×/0.45 Ph2 objective, adapter ring VAD-S70, and a DSC-S70 digital still camera (Sony, Tokyo, Japan). Film was imaged in AlphaImager 2200 (Alpha Innotech, San Leandro, CA). (D) Superoxide generation and gp91phox protein level of TG2–/– and TG2+/+ mouse neutrophils. Wild-type and TG2–/– mouse neutrophils were collected and separated as previously described. The reaction volume of 500 μL contained 1 × 105 cells and 5.0 μL L-012 (100 μM). ROSs were generated by adding 50 nM PMA. Chemiluminescence was detected as described in “Materials and methods.” Inset: Proteins of wild-type and TG2–/– mouse neutrophils were analyzed by Western blotting using an antibody against the gp91phox and beta-actin. (E) Q-PCR analysis of gp91phox mRNA expression of TG2–/– and TG2+/+ mouse neutrophils; gp91phox mRNA levels were normalized to mouse cyclophilin. Data in panels B-D are representative of 2 independent experiments; error bars represent SD.

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