Proteasomal Degradation of O-GlcNAc Transferase Elevates Hypoxia-induced Vascular
Endothelial Inflammatory Response
Liu: OGT degradation enhances hypoxia-induced inflammation
Hongtao Liu,1 Zhongxiao Wang,2 Shujie Yu,1 Jian Xu1‡
1
Endocrinology and Diabetes, Department of Medicine, Harold Hamm Diabetes Center; 2Department of
Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Supplementary file for online publication
1. Methods
1.1 Cell culture
Cells were grown at 70-80% confluent and used between passages 3 and 8 as previously
reported.1-3 All cells were incubated in a humidified atmosphere of 5% CO2 at 37℃. For hypoxia
experiments, all the conditions were the same except for a culture environment where only 1% O2 was
provided through a hypoxia chamber in a regular incubator.
1.2 Western blot analysis
Cultured cells and tissues from the lung were homogenized on ice in cell-lysis buffer. Protein
quantification, Western blotting, and band densitometry were performed as previously reported.1, 2
1.3 The 26S proteasome functionality assessment and 26S proteasome activity assay
The 26S proteasome functionality was assessed with endothelial cells expressing the vector
UbG76V-GFP as a surrogate protein substrate for 26S proteasomes.4 The system was modified so that
either enhanced or suppressed proteasomal functionality could be detected by evaluating the pattern of the
reporter proteins with Western blot.5 The 26S proteasome activity was determined by the measurement of
the chymotrypsin-like activity in cell lysates with a fluorogenic substrate as previously described.1, 2, 5
1.4 Infection with siRNA duplex in cells
All cells were incubated at 37°C in a humidified atmosphere of 5% CO2. In all experiments, the cells were
between passages 3-8 grown to 70 – 80% confluence as previously reported.5, 6 Transfection of control or β-TrCP1
siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA) as
described previously.5, 6 All cells with siRNA transfection were incubated in a humidified atmosphere of 5% CO2 at
37℃.
1.5 Inflammatory cytokine/chemokine/adhesion molecule gene and protein expression analysis
Analysis of inflammatory cytokine/chemokine/adhesion molecule gene expression was conducted
as previously reported6, 7 with minor modifications. Briefly, the total RNA extraction from cultured cells
and lung tissues was achieved with Total RNA Kit I (Omega Bio-Tek, Norcross, GA) followed by cDNA
synthesis and real-time PCR amplification. iScriptTM cDNA Synthesis Kit (170-8891), iQTM SYBR Green
Supermix (170-8880), and the Real Time Detection System were obtained from Bio-Rad (Hercules, CA).
Sequences of promoter-specific primers for mouse and human MCP-1, IL-6, IL-8, and E-selectin were
respectively listed in Supplementary Table 1. PCR product was analyzed by running 6 μl of each reaction
mixture on a 1.5% agarose gel containing 1% GoldViewTM. Band intensity was quantified with ImageJ
system (NIH, USA) and presented as a percentage of GAPDH expression.
Quantification of IL-8, IL-6, E-selectin and MCP-1 in cell lysates or tissue homogenates were
performed using mouse or human specific ELISA kits according to instructions of the manufacture,
including Human CXCL8/IL-8 Quantikine ELISA Kit, Human IL-6 Quantikine ELISA Kit, Mouse IL-6
Quantikine ELISA Kit, and Human CCL2/MCP-1 Quantikine ELISA Kit from R&D System
(Minneapolis, MN, USA), and Mouse IL-8 ELISA 96T, Human E-Selectin ELISA 96T, and Mouse ESelectin ELISA Kit from Fisher Scientific (Pittsburgh, PA, USA).
1.6 Immunofluorescence staining
Imaging through immunofluorescence staining was performed as previously reported3, 6 with minor
modifications. Briefly, the lung tissues were removed and fixed with 4% paraformaldehyde in PBS.
Following fixation and dissection, the lung tissues were quickly frozen by immersion in Tissue-Tek®
O.C.T. Compound (VWR, PA, USA) and stored at -80°C. Frozen lung tissues were sliced serially at 8 μm
using a cryostat (LEICA CM3000, Nussloch, Germany). Sections were washed with PBS and blocked by
incubation with 1.5% goat serum in PBS containing 0.3% Triton for 30 min, then incubated with specific
rabbit polyclonal anti-OGT antibody or anti-HIF 1α antibody overnight followed by incubation with
fluorescent-conjugated secondary antibody (Alexa Fluor 594; 1:500 dilution, 1 h, Molecular Probes, OR,
USA; Alexa Fluor 488; 1:500 dilution, 1 h, Molecular Probes, OR, USA). Nucleus was stained with DAPI
(ProLong® Gold antifade reagent with DAPI, Invetrogen). The slides were assessed with fluorescence
microscopy (Olympus Ax70, Tokyo, Japan).
2. Supplementary Table 1. Primers for RT-PCR of IL-8, IL-6, E-selectin and MCP-1
The promoter-specific primers were synthesized by Sigma (St. Louis, MO, USA), used for Abbreviations:
m, mouse; h, humans.
Supplementary Figure Legends
Figure S1. Hypoxia-induced OGT reduction in HUVEC is mediated by β-TrCP1. Western blot of
lysates from siRNA-treated HUVEC (control siRNA or β-TrCP1 siRNA) with or without hypoxia
exposue (1% O2 for 24h). ANOVA was used to compare means of different experimental groups, and
Tukey’s Tests were used as post-hoc tests. * indicates significant difference vs control.
Figure S2. Administration of glucosamine mitigated hypoxia-enhanced production of IL-8,Eselection, and IL-6 proteins in vivo. Male and age matched (7 d) wild type (C57BL/6J) mice were used
in an oxygen-induced retinopathy (OIR) model: mice were first subjected to high oxygen treatment (75%
O2 chamber) for 5 days and transferred to room air environment for another 5 days, while the control mice
were kept under room air at all times. Some hypoxia-exposed mice received treatment (glucosamine: 1
mg/g/d; vehicle: saline; i.p., 5d, n=5/group) when returning to room air environment. After treatment,
lung tissues were collected for ELISA analysis of IL-8, E-selectin, and IL-6 proteins. The results
(n=5/group) were analyzed by ANOVA with Tukey’s Tests as post-hoc tests. *P< 0.05 vs the vehicle- or
control siRNA-treated.
Figure S3. The presence of high glucose does not change OGT reduction induced either by cobalt or
hypoxia in HUVEC. Western blot of lysates from high glucose (25 mM)-treated HUVEC with or without
exposure to (A) cobalt (200 µM for 12 h) or (B) hypoxia (1% O2 for 24h). The results (n=3) were
analyzed by ANOVA with Tukey’s Tests as post-hoc tests. * indicates significant difference vs control.
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