Journal: Molecular Therapy

Article Title: Erythropoietin Gene-enhanced Marrow Mesenchymal Stromal Cells Decrease Cisplatin-induced Kidney Injury and Improve Survival of Allogeneic Mice

doi: 10.1038/mt.2011.162

Figure Lengend Snippet: Effect of conditioned media (CM) from mesenchymal stromal cells (MSCs) and from Epo-MSCs on P-Akt expression in kidney cells. MM55.K mouse kidney epithelial cells were exposed to cisplatin, with/without MSCs CM or Epo-MSCs CM (a) at increasing concentrations or (b) at highest concentrations with/without a neutralizing anti-IGF-1 antibody (IGF1 Ab), and/or anti-VEGF antibody (VEGF Ab), or anti-Epo antibody (Epo Ab), as detailed in the Materials and Methods. Cells were collected 42 hours later and lysates used for western blot analysis of phosphorylated Akt (Ser473) (P-Akt) expression, as well as of loading controls GAPDH or total Akt expression. (b) The bar graph represents the mean ± SEM of seven independent experiments.

Article Snippet: The Epo-MSCs CM was similarly tested alone or in combination with neutralizing antibodies against IGF-1, VEGF or Epo (MAB959) (R&D Systems), or against both IGF-1and VEGF.

Techniques: Expressing, Western Blot

Journal: Toxics

Article Title: Mercury Chloride Impacts on the Development of Erythrocytes and Megakaryocytes in Mice

doi: 10.3390/toxics9100252

Figure Lengend Snippet: HgCl 2 does not impact the production of EPO, but increases the expression of EPOR in EMPs in B10.S mice. EPO protein in the serum and BM, EPO mRNA in the kidney, and EPOR in EMPs in the BM in B10.S mice treated with 50 μM HgCl 2 or the control for 4 w were measured. FACS-purified EMPs from the BM of B10.S mice were treated with HgCl 2 or vehicle or EPO in vitro, and the Jak2/STAT5 signaling pathway and differentiation of EMPs were measured thereafter. ( A ) EPO protein (IU/L) in the serum of B10.S mice treated with 50 μM HgCl 2 or water for 4 w. ( B ) EPO protein (IU/g protein) in the BM of B10.S mice treated with 50 μM HgCl 2 or water for 4 w. ( C ) EPO mRNA (fold change) in the kidney of B10.S mice treated with 50 μM HgCl 2 or water for 4 w. ( D ) The expression of EPOR (MFI) in EMPs in the BM of B10.S mice treated with 50 μM HgCl 2 or water for 4 w. ( E ) The expression of EPOR mRNA (fold change) in FACS-purified EMPs from the BM of B10.S mice treated with 50 μM HgCl 2 or water for 4 w. ( F ) EMPs were purified from the BM of control B10.S mice, and the FACS-purified EMPs were treated thereafter with EPO or vehicle in vitro for 16 h. After that, the expression of p-Jak2 (MFI) in EMPs was measured. ( G ) EMPs were purified from the BM of control B10.S mice, and the FACS-purified EMPs were treated thereafter with EPO or vehicle in vitro for 16 h. After that, the expression of p-STAT5 (MFI) in EMPs was measured. ( H ) EMPs were purified from the BM of B10.S mice treated with 50μM HgCl 2 or water for 4 w, and the EMPs were treated thereafter with EPO or vehicle in the presence or absence of the Jak2 inhibitor fedratinib for 16 h in vitro. After that, the expression of p-STAT5 (MFI) in the EMPs was measured. ( I ) EMPs were purified from the BM of control B10.S mice, and the FACS-purified EMPs were treated thereafter with HgCl 2 and/or EPO or vehicle in vitro for 16 h. After that, the expression of p-Jak2 (MFI) in EMPs was measured. Each dot represents one mouse or one sample, and a total of 5 to 12 mice or samples were used for each group. Asterisk indicates a significant difference compared to the counterpart control group. p < 0.05 was considered as the level of significant difference.

Article Snippet: Ab (clone) and fluorescein used to stain progenitors, erythroblasts and leukocytes included lineage cocktail (Lin, CD3 (145-2C11), CD11b (M1/70), B220 (RA3-6B2), Gr-1 (RB6-8C5) and Ter119 (TER-119))-FITC or biotin, c-Kit-APC (2B8), Scal-1-PerCP-Cy5.5 (D7), CD150-PE-Cy7 (TC15-12F12.2), CD150-APC (TC15-12F12.2), CD41-FITC (MWReg30), CD41-PE (MWReg30), CD105-PB (MJ7/18); CD105-PE-Cy7 (MJ7/18), CD71-PE-Cy7 (RI7217), CD45-PE-Cy7 (30-F11), CD11b-PerCP-Cy5.5 (M1/70), Ly6G-PE (1/A8), F4/80-PE-Cy7 (BM8), Ki67-PE (16A8), p-STAT1-PE (pY701); p-STAT3-PE (Tyr705), streptavidin (SA)-PB, SA-APC-Cy7, anti-rabbit Ab-PE, anti-rabbit Ab-FITC and anti-rabbit Ab-APC (Biolegend, San Diego, CA, USA), and rabbit anti-mouse p-Jak1 (Tyr1022) (Nanjing Jiancheng, China), rabbit anti-mouse p-Jak2 (Tyr1007/1008), rabbit anti-mouse p-STAT5 (Tyr694), and rabbit anti-mouse EPO receptor (EPOR) (Beyotime Biotechnology, China), and rat anti-mouse CD16/32 (Fc block, 2.4G2) (BD Biosciences, San Diego, CA), and 4’6-diamidino-2-phenylindole (DAPI) (Sigma, St Louis, MO, USA).

Techniques: Expressing, Purification, In Vitro

Journal: Toxics

Article Title: Mercury Chloride Impacts on the Development of Erythrocytes and Megakaryocytes in Mice

doi: 10.3390/toxics9100252

Figure Lengend Snippet: A schematic model for the impact of Hg 2+ on erythro-megakaryopoiesis in mice. In hosts carrying certain H-2 haplotypes sensitive to Hg-induced autoimmunity (e.g., B10.S mice, H-2 s ), Hg 2+ induces an autoimmune environment, which leads to the increased expression of EPOR in EMPs and the resultant hypersensitivity of EMPs in response to EPO in the BM. Consequently, the Jak2/STAT5 signaling pathway is overactivated by EPO interaction with EPOR in EMPs, thus resulting in the increased production of RBCs and platelets in the BM. Hg 2+ does not impact erythro-megakaryopoiesis in mice carrying H-2 haplotypes resistant to Hg-induced autoimmunity (e.g., DBA/2 mice, H-2 d ).

Article Snippet: Ab (clone) and fluorescein used to stain progenitors, erythroblasts and leukocytes included lineage cocktail (Lin, CD3 (145-2C11), CD11b (M1/70), B220 (RA3-6B2), Gr-1 (RB6-8C5) and Ter119 (TER-119))-FITC or biotin, c-Kit-APC (2B8), Scal-1-PerCP-Cy5.5 (D7), CD150-PE-Cy7 (TC15-12F12.2), CD150-APC (TC15-12F12.2), CD41-FITC (MWReg30), CD41-PE (MWReg30), CD105-PB (MJ7/18); CD105-PE-Cy7 (MJ7/18), CD71-PE-Cy7 (RI7217), CD45-PE-Cy7 (30-F11), CD11b-PerCP-Cy5.5 (M1/70), Ly6G-PE (1/A8), F4/80-PE-Cy7 (BM8), Ki67-PE (16A8), p-STAT1-PE (pY701); p-STAT3-PE (Tyr705), streptavidin (SA)-PB, SA-APC-Cy7, anti-rabbit Ab-PE, anti-rabbit Ab-FITC and anti-rabbit Ab-APC (Biolegend, San Diego, CA, USA), and rabbit anti-mouse p-Jak1 (Tyr1022) (Nanjing Jiancheng, China), rabbit anti-mouse p-Jak2 (Tyr1007/1008), rabbit anti-mouse p-STAT5 (Tyr694), and rabbit anti-mouse EPO receptor (EPOR) (Beyotime Biotechnology, China), and rat anti-mouse CD16/32 (Fc block, 2.4G2) (BD Biosciences, San Diego, CA), and 4’6-diamidino-2-phenylindole (DAPI) (Sigma, St Louis, MO, USA).

Techniques: Expressing