Figure 2. Loss of NRF2 function produced severe splenomegaly and inflammation in SCD mice. (A) The weight (left) and size (right) of spleens from NRF2 wild-type (+/+), heterozygote (+/−), and knockout (−/−) SCD mice were determined. (B) Histologic section of spleen tissue stained with hematoxylin and eosin is shown; scale bars: (i and ii), 100 µm; (iii and iv), 40 µm. (C) Using 2'-7'-dichlorodihydrofluorescein diacetate staining and flow cytometry, ROS levels in RBCs were determined (n ≥ 6); RBC gating was performed as described in supplemental Methods (supplemental Figure 2). (D) Western blot was completed to determine the expression of antioxidant proteins in spleen whole-tissue extracts for adult SCD/NRF2+/+ mice (red bars) and SCD/NRF2−/− mice (blue bars) (n = 3 per group) (top), and quantitative data were generated (bottom); β-actin was used as a loading control. (E) Western blot was completed as in panel D for the following: intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), and vascular endothelial growth factor (VEGF-A) (top), and quantitative data analyzed (bottom). (F) The mRNA levels of the various genes shown were measured by qRT-PCR in spleen cells isolated from 2- to 3-month-old mice (n = 6 per group). Red bars, C57BL/6 mice; blue bars, SCD/NRF2+/+ mice; light green bars, SCD/NRF2−/− mice. The level of mouse 18s ribosomal RNA was used as an internal control. **P < .01; *P < .05. CAT, catalase; GCLC, γ-glutamylcysteine ligase catalytic subunit; HMOX1, heme oxygenase 1; NQO1, NAD(P)H quinone dehydrogenase 1; qRT-PCR, quantitative reverse transcription polymerase chain reaction.
Figure 2.

Loss of NRF2 function produced severe splenomegaly and inflammation in SCD mice. (A) The weight (left) and size (right) of spleens from NRF2 wild-type (+/+), heterozygote (+/−), and knockout (−/−) SCD mice were determined. (B) Histologic section of spleen tissue stained with hematoxylin and eosin is shown; scale bars: (i and ii), 100 µm; (iii and iv), 40 µm. (C) Using 2'-7'-dichlorodihydrofluorescein diacetate staining and flow cytometry, ROS levels in RBCs were determined (n ≥ 6); RBC gating was performed as described in supplemental Methods (supplemental Figure 2). (D) Western blot was completed to determine the expression of antioxidant proteins in spleen whole-tissue extracts for adult SCD/NRF2+/+ mice (red bars) and SCD/NRF2−/− mice (blue bars) (n = 3 per group) (top), and quantitative data were generated (bottom); β-actin was used as a loading control. (E) Western blot was completed as in panel D for the following: intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), and vascular endothelial growth factor (VEGF-A) (top), and quantitative data analyzed (bottom). (F) The mRNA levels of the various genes shown were measured by qRT-PCR in spleen cells isolated from 2- to 3-month-old mice (n = 6 per group). Red bars, C57BL/6 mice; blue bars, SCD/NRF2+/+ mice; light green bars, SCD/NRF2−/− mice. The level of mouse 18s ribosomal RNA was used as an internal control. **P < .01; *P < .05. CAT, catalase; GCLC, γ-glutamylcysteine ligase catalytic subunit; HMOX1, heme oxygenase 1; NQO1, NAD(P)H quinone dehydrogenase 1; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

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