Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Functional significance of erythropoietin receptor expression in breast cancer.

doi: 10.1097/01.lab.0000020415.72863.40

Figure Lengend Snippet: Figure 1. Results of immunohistochemical studies. Contiguous tissue sections from breast tumors were immunostained to detect EPO, EpoR, or presence of pimonidazole adducts as a marker for hypoxia. Panels A, B, and C, Immunostaining of contiguous sections from the same tumor showing reactivity for EpoR (A), EPO (B), and pimonidazole adducts (C). Panels D and E, Immunostaining of contiguous sections for EpoR (D) and EPO (E), demonstrating EpoR immunoreactivity and no EPO expression. Panel F, High magnification of tissue section showing strong EpoR immunoreactivity in breast cancer cells. Panel G, Z-chamber consists of plexiglass rings (1), with an access port on the side (2), and covered with 180 m nylon mesh on both sides allowing the in-growth of vessels (3). Panels H and I, Representative section of tumor removed from Z-chamber stained with hematoxylin and eosin (H & E). Panel H, Histology of typical tumor formation at Day 7 after implantation of the T-ZCs. The maximal tumor depth (green arrow) is representative of negative control chambers containing rat mammary adenocarcinoma R3230 Ac cells and vehicle. Panel I, Reduction in maximal tumor depth (green arrow) observed in a T-ZC containing recombinant soluble EpoR (compare panels H and I). A to F, bar indicates 100 m; H and I, bar indicates 500 m.

Article Snippet: In these experiments, recombinant human soluble EPO receptor (R and D Systems, Minneapolis, Minnesota) or neutralizing anti-EPO monoclonal antibody (MAB-287; R and D Systems) were reconstituted in PBS (cat#14190–144, Life Technologies) and added to the chambers at the indicated final concentrations.

Techniques: Immunohistochemical staining, Marker, Immunostaining, Expressing, Staining, Negative Control, Recombinant

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Functional significance of erythropoietin receptor expression in breast cancer.

doi: 10.1097/01.lab.0000020415.72863.40

Figure Lengend Snippet: Figure 3. Inhibition of tumor growth by EPO-EpoR antagonists. Rat R3230Ac mammary adenocarcinoma cells were injected into tumor Z-chambers as described in “Materials and Methods.” The chambers contained one-time doses each of recombinant sEpoR, MAB-287 anti-EPO neutralizing antibody, AG-490 Jak2 tyrosine kinase inhibitor, or vehicle (Control). Treatment groups are indicated on top of the chart and final concentrations of each compound are indicated on the x axis. At Day 7, the tumors in the Z-chambers were removed and H&E staining was performed. Quantitative analysis of maximal tumor depth was carried out in each treatment group and is expressed as a percentage of control, as indicated in the y axis. The data is expressed as mean standard error of the mean. To determine significant effects of treatment, the mean of each treatment group was compared with control using the Students t test (two-tail) and p values for each comparison are indicated above each bar. * p 0.05. NS, not significant.

Article Snippet: In these experiments, recombinant human soluble EPO receptor (R and D Systems, Minneapolis, Minnesota) or neutralizing anti-EPO monoclonal antibody (MAB-287; R and D Systems) were reconstituted in PBS (cat#14190–144, Life Technologies) and added to the chambers at the indicated final concentrations.

Techniques: Inhibition, Injection, Recombinant, Control, Staining, Comparison

Journal: Molecular Medicine Reports

Article Title: Gaseous signalling molecule SO 2 via Hippo-MST pathway to improve myocardial fibrosis of diabetic rats

doi: 10.3892/mmr.2017.7714

Figure Lengend Snippet: SO 2 improves myocardial fibrosis in diabetic rats. (A) Morphological changes in myocardium assessed by Masson staining. Images were acquired at ×400 magnification. Expression levels of (B) MMP9, (C) MMP24 and (D) TIMP1 in each group. Date are expressed as mean ± standard deviation (n=3). *P<0.05 vs. control group; # P<0.05 vs. STZ group. SO 2 , sulfur dioxide; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; STZ, streptozotocin; HDX, L-Aspartic acid β-hydroxamate.

Article Snippet: The antibodies for matrix metalloproteinase (MMP)9, MMP24, tissue inhibitor of metalloproteinase (TIMP)1 and GAPDH were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Staining, Expressing, Standard Deviation, Control

Journal: PLoS ONE

Article Title: Melanocortins Contribute to Sequential Differentiation and Enucleation of Human Erythroblasts via Melanocortin Receptors 1, 2 and 5

doi: 10.1371/journal.pone.0123232

Figure Lengend Snippet: ( A ) Schematic diagram of an in vitro differentiation protocol for deriving erythroblasts from human HPCs. Human CD34 + HPCs were expanded for 7 days (E0–E7) and stocks were frozen in liquid nitrogen (LN 2 ). The stock cells were differentiated for 3 days (D0–D3) before undergoing maturation (M0–M7). The number of enucleated erythrocytes increased from M5 to M7. ACTH, adrenocorticotropic hormone; EPO, erythropoietin; FL, flt-3 ligand; IL-3, interleukin-3; IL-6, interleukin-6; α-MSH, α-melanocyte stimulating hormone; SCF, stem cell factor; TPO, thrombopoietin. ( B ) May-Grunwald-Giemsa staining of control erythroblasts at day M0–5 day. Cells differentiated to Pro-E stage at day M0 and Baso-E stage at day M3. Arrows, Poly-E; Arrow heads, Orho-E. *, Reticulocyte. Bar 10 μm. ( C ) Conventional RT-PCR for MCRs during erythroblasts differentiation between M0 and M6 day.

Article Snippet: In the second passage (for differentiation; D0–D3 in ), cell stocks were thawed and cultured at 2 × 10 5 cells/ml in HPGM supplemented with 3 U/ml human EPO (Kyowa Hakko Kirin), 25 ng/ml SCF, 10 ng/ml recombinant human IL-3 (PeproTech), and 10 ng/ml recombinant human IL-6 (R&D Systems) for 3 days ( ).

Techniques: In Vitro, Staining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Melanocortins Contribute to Sequential Differentiation and Enucleation of Human Erythroblasts via Melanocortin Receptors 1, 2 and 5

doi: 10.1371/journal.pone.0123232

Figure Lengend Snippet: (A) Phosphorylation of ERK, STAT and AKT in erythroblasts. Erythroblasts at M0, M3 and M6 were starved for 3 h in HPGM without cofactors and subsequently incubated for 15 min with or without EPO. ( B ) – ( E ), Synergistic effects on EPO downstream signaling of ACTH, and the signal inhibition by nAbs. After starvation with HPGM without cofactors for 3 h, the cells were incubated for 15 min with nAbs and reacted with EPO for 15 min. EPO-induced phosphorylation of ERK is not altered by the addition of 0.1 nM ACTH39 to erythroblasts at M0. n = 3; error bars, s.e.m (B) . The phosphorylation of ERK is decreased by nAbs of MC2R and MC5R (C) . ACTH39 enhances EPO-induced phosphorylation of STAT5 in erythroblasts at M3 (D) . Phosphorylation of STAT5 is inhibited by nAbs of MC1R (E) . (F) and (G), AKT phosphorylation by ACTH and, the signal inhibition by nAbs. Treatment with ACTH39 for 15 min without starvation causes phosphorylation of AKT in erythroblasts at M6 (F) . The phosphorylation of AKT is inhibited by MC5R-nAb (G) . EPO, 3 U/ml EPO; ACTH, 0.1 nM ACTH39. n = 3; error bars, s.e.m. IgG, 10 μg/ml normal IgG; MC1R, 10 μg/ml anti-MC1R nAb; MC2R, 5 μg/ml anti-MC2R nAb; MC5R, 10 μg/ml anti-MC5R nAb. n = 3; error bars, s.e.sm.

Article Snippet: In the second passage (for differentiation; D0–D3 in ), cell stocks were thawed and cultured at 2 × 10 5 cells/ml in HPGM supplemented with 3 U/ml human EPO (Kyowa Hakko Kirin), 25 ng/ml SCF, 10 ng/ml recombinant human IL-3 (PeproTech), and 10 ng/ml recombinant human IL-6 (R&D Systems) for 3 days ( ).

Techniques: Phospho-proteomics, Incubation, Inhibition