Journal: Antioxidants (Basel, Switzerland)

Article Title: Nrf2 Plays a Key Role in Erythropoiesis during Aging.

doi: 10.3390/antiox13040454

Figure Lengend Snippet: Figure 4. Sorted Nrf2−/−mouse erythroblasts display overactivation of the system and impaired autophagy with caspase-3 pro-apoptotic pathway activation. (a) Wb analysis with specific anti- phospho-NF-kB (p-NF-kB) and NF-kB in sorted erythroid precursors from bone marrow of 12-month- old wild-type (WT) and Nrf2−/−mice. Actin was used as protein loading control. One representative gel of the other four is shown (upper panel). Densitometric analysis of immunoblots is shown in the lower panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (b) Wb analysis with specific anti-HSP70, ATF6 and GADD34 in sorted erythroid precursors as in a. Densitometric analysis of immunoblots is shown in the lower panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (c) Caspase 3 activity determined by cleavage of a fluorescent substrate in sorted erythroid precursors from bone marrow of WT and Nrf2−/−mice. Data are presented as means ± SD * p < 0.05 compared to WT. (d) Wb analysis with specific anti-LC3 I/II, Atg4, Atg5, Rab5 and p62 in sorted erythroid precursors from bone marrow of 12-month-old WT and Nrf2−/−mice. Actin was used as protein loading control. Densitometric analysis of immunoblots is shown in the right panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (e) Rab5 immunostaining of sorted erythroid precursors from bone marrow of 12-month-old WT and Nrf2−/−mice. DAPI was used to stain nuclei. Large clusters of positive cells were measured using ImageJ. At least 40 cells were analyzed in 8 different fields of acquisition. Data are presented as median and minimum/maximum, with boxes indicating 25–75th percentiles; * p < 0.05 compared to WT mice.

Article Snippet: The following specific antibodies were used: anti-NFkB-phospho-S536 (3033 Cell Signaling Technology, Leiden, NL, USA); anti-NFkB p65 and anti-Atg5 (8242, 12994 Cell Signaling Technology, Leiden, NL, USA); anti-Nrf2-phospho-S40 (ab76026 Clone EP1809Y, Abcam, Cambridge, UK); anti-Nrf2 (ab62352 Abcam, Cambridge, UK); anti Gadd34, anti-Lamp-1, anti-SQSTM1/P62, anti-Rab5 and anti-LC3A/B (ab9869, ab24170, ab109012, ab109534, ab62721 Abcam, Cambridge, UK); anti-APG7 (Atg7) (3615 ProSci, Poway, CA, USA); anti-ATF6 (70B1413.1 Novus Biologicals, Centennial, CO, USA); antiCHOP (ma1-250 Thermo Fisher Scientific, Waltham, MA, USA); anti-Actin (SAB4301137 clone BIII-136; Sigma-Aldrich, Saint Louis, MO, USA) and anti-GAPDH (HPA040067 SigmaAldrich, Saint Louis, MO, USA) as loading controls.

Techniques: Activation Assay, Control, Western Blot, Activity Assay, Immunostaining, Staining

Journal: Antioxidants (Basel, Switzerland)

Article Title: Nrf2 Plays a Key Role in Erythropoiesis during Aging.

doi: 10.3390/antiox13040454

Figure Lengend Snippet: Figure 7. Schematic diagram of the role of Nrf2 in erythropoiesis during aging and the protective effects of astaxanthin PLGA nanoparticles. Aging is associated with increased ROS production, which is limited by the activation of Nrf2. This results in the upregulation of ARE genes encoding for antioxidants and cytoprotective systems as well as by the activation of adaptative mechanisms such as the UPR system to face ER stress and autophagy to clear damaged proteins. The absence of Nrf2 (Nrf2−/−mice) negatively affects the antioxidant cell machinery, resulting in severe and sustained oxidation. Nrf2−/−mouse red cells display severe membrane oxidation, exposition of phosphatidylserine, membrane binding of hemichromes and reduced expression of antioxidants and cytoprotective systems such as Prdx2. Red cell membrane protein oxidation favors band 3 protein clusterization, which is recognized by the naturally occurring anti-band 3 IgG antibodies. Both mechanisms drive Nrf2−/−mouse red cells towards erythrophagocytosis by splenic macrophages. In erythroblasts lacking Nrf2, the prolonged and severe oxidation due to the downregulation of antioxidants and cytoprotective systems promotes intense ER stress with overactivation of the UPR system and autophagy. Although the persistence of oxidative stress promotes compensatory activation of NF-kB, this is insufficient to prevent the overwhelming of proteostasis with impairment autophagy and accumulation of Rab5. This drives Nrf2−/−erythroblasts towards apoptosis via the caspase-3 pathway, resulting in ineffective erythropoiesis. ATS-NPs act as efficient antioxidants preventing the deleterious effects of the absence of Nrf2 on erythropoiesis and red cells during aging. PS: phosphatidylserine; ER: endoplasmic reticulum; UPR: unfolded protein response; ARE-: antioxidant-related element; ROS: reactive oxygen species; Prdx2: peroxiredoxin-2; Atg: autophagy- related protein; GADD34: growth arrest and DNA damage-inducible protein 34; PLGA: poly(lactic- co-glycolic acid).

Article Snippet: The following specific antibodies were used: anti-NFkB-phospho-S536 (3033 Cell Signaling Technology, Leiden, NL, USA); anti-NFkB p65 and anti-Atg5 (8242, 12994 Cell Signaling Technology, Leiden, NL, USA); anti-Nrf2-phospho-S40 (ab76026 Clone EP1809Y, Abcam, Cambridge, UK); anti-Nrf2 (ab62352 Abcam, Cambridge, UK); anti Gadd34, anti-Lamp-1, anti-SQSTM1/P62, anti-Rab5 and anti-LC3A/B (ab9869, ab24170, ab109012, ab109534, ab62721 Abcam, Cambridge, UK); anti-APG7 (Atg7) (3615 ProSci, Poway, CA, USA); anti-ATF6 (70B1413.1 Novus Biologicals, Centennial, CO, USA); antiCHOP (ma1-250 Thermo Fisher Scientific, Waltham, MA, USA); anti-Actin (SAB4301137 clone BIII-136; Sigma-Aldrich, Saint Louis, MO, USA) and anti-GAPDH (HPA040067 SigmaAldrich, Saint Louis, MO, USA) as loading controls.

Techniques: Activation Assay, Membrane, Binding Assay, Expressing