Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 2. The expression of Prx3 is regulated by VHL. (A–C) The mRNA and protein levels of the indicated genes were respectively detected by real-time quantitative RT-PCR and Western blot. RCC4/VHL and Caki-1 cells were infected with retroviral vectors harboring shRNAs against VHL (shVHL) or scrambled negative control (NC) (B and C). EV represents empty vector. The column represents mean with bar as S.D. of three independent experiments with triplicate samples (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Retroviral, Negative Control, Plasmid Preparation

Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 3. Prx3 is negatively regulated by HIF-1a. The mRNA and protein levels of the indicated genes were respectively detected by real-time quantitative RT-PCR and Western blot. (A) RCC4/VHL cells were exposed to normal air or hypoxia (1% O2) for 12 h. (B) RCC4 cells were stably transfected with shRNAs against HIF-1a (a1and a2). (C) Caki-1 cells transfected with shRNAs against HIF-1a (a1 and a2) were incubated in normoxia or hypoxia for 12 h. Columns, means of three determinations; bars, S.D. (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Quantitative RT-PCR, Western Blot, Stable Transfection, Transfection, Incubation

Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 4. Two HREs in promoter of PRDX3 are essential for HIF-1a transactivity. (A) 293T cells were transfected with luciferase reporter plasmids driven by four putative HREs in PRDX3 promoter, which are shown on the top and middle panels, and were grown in the normal air or hypoxia for 12 h. In top diagram of putative HREs (black ovals), empty circles represent the transcriptional start point of PRDX3. (B) 293T cells were transfected with luciferase reporter plasmids driven by four putative HREs in PRDX3 promoter together with the indicated doses of HIF-1a and grown in the normal air for 36 h. Protein levels of HIF-1a were detected by western blot with Actin as a loading control (low panel). (C) Luciferase reporter plasmids driven by the HREs or CG/AA mutated HRE sequences as indicated were transfected together with HIF-1a expressing vector or empty vector into 293T cells for 36 h in normal air. All the relative luciferase activities of PRDX3 promoter were normalized by pSV40-Renilla and estimated as the relative folds against cells under normal air (A, lower panel) or empty vector-transfected cells (B and C). (D) RCC4/VHL cells were grown under normoxia and hypoxia for 12 h. Chromatin immunoprecipitation assay was performed as described in Section 2. The column represents mean with bar as S.D. of three independent experiments with triplicate samples (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Transfection, Luciferase, Western Blot, Control, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia

doi: 10.1523/JNEUROSCI.2695-17.2017

Figure Lengend Snippet: Role of peptidergic (IB4−) and nonpeptidergic (IB4+) nociceptors in LMWH-induced hyperalgesia. A, Separate groups of rats received a single intrathecal injection of one of two neurotoxins, isolectin B4-saporin (IB4sap, selective for nonpeptidergic neurons, 3.2 μg, light gray bar), or [Sar9,Met(O2)11] substance P-saporin (SSPsap, selective for SP-containing fibers, 100 ng, dark gray bar), or their combination (black bar). A control group (white bar) received saline. LMWH (1 μg) was injected intradermally on the dorsum of the hindpaw 14 d later. Significant attenuation of LMWH-induced hyperalgesia was observed in all neurotoxin-treated groups (IB4sap: t(22) = 4.162, ***p = 0.0002; SSPsap: t(22) = 5.341, ****p < 0.0001; combination: t(22) = 7.762, ****p < 0.0001, compared with the control group; unpaired Student's t test). B, One week later, the control group (white bar) and the group treated with the combination of toxins (black bar), shown in A, were each further divided into 2 groups, which received an intradermal injection of either 8-bromo cAMP or ψεRACK (both 1 μg), on the dorsum of the hindpaw. At that time point, the mechanical nociceptive thresholds were not significantly different from pre-LMWH baseline (8-bromo cAMP: control group: t(5) = 0.9715, p = 0.1880; combination group: t(5) = 1.321, p = 0.1219; ψεRACK: control group: t(5) = 1.305, p = 0.1244; combination group: t(5) = 0.6697, p = 0.2664, all nonsignificant, when the mechanical nociceptive threshold before LMWH injection and immediately before 8-bromo cAMP or ψεRACK injection are compared, paired Student's t test). Robust mechanical hyperalgesia was observed in all groups, 30 min after injection (8-bromo cAMP, control: t(5) = 21.02, p < 0.0001; combination: t(5) = 4.776, p = 0.0025; ψεRACK, control: t(5) = 31.30, p < 0.0001; combination: t(5) = 6.173, p = 0.0008, when the mechanical nociceptive thresholds were compared with baseline thresholds; paired Student's t test), although in the groups pretreated with the combination of the two toxins, there was attenuation compared with the control groups (8-bromo cAMP-treated groups: t(10) = 3.907, *p = 0.0015; ψεRACK-treated groups: t(10) = 3.501, **p = 0.0029; unpaired Student's t test). A, n = 12 paws per group. B, n = 6 paws per group.

Article Snippet: Both IB4-saporin and SSP-saporin (from Advanced Targeting Systems) were diluted in saline to doses previously shown to deplete nonpeptidergic (3.2 μg/rat for IB4-saporin) and peptidergic (100 ng/rat for SSP-saporin) fibers ( Vulchanova et al., 2001 ; Wiley et al., 2007 ; Choi et al., 2012 ).

Techniques: Injection

Journal: Scientific Reports

Article Title: Differential neutrophil responses to bacterial stimuli: Streptococcal strains are potent inducers of heparin-binding protein and resistin-release

doi: 10.1038/srep21288

Figure Lengend Snippet: Resistin levels in cell culture supernatants of neutrophils from healthy donors, pre-treated for 30 min with inhibitors targeting the signalling molecules Src (PP1), PI3K (LY294002), MAPK p38 (SB203580), ERK (PD98059), and JNK (SP600125) ( A ) or antibodies against β2-integrins (anti-human CD18) and TLR-2 (anti-Human CD282) ( B ), and stimulated with fixed GAS for 2 h. In A, open and filled symbols show 5448 and the T1 isolate, respectively. Mean ± SD of results from 3–9 experiments using different donors presented as per cent relative to untreated cells, the dashed line indicates 100 per cent. The inhibitors did not influence the cell response, as unstimulated cells had similar background levels in the presence or absence of the inhibitors. Wilcoxon matched-pairs signed rank test was used for comparison between groups, *P > 0.05,**P < 0.01.

Article Snippet: The following blocking antibodies and pharmacological inhibitors were used: anti-β2 integrin (anti-human CD18, clone IB4, Merk Millipore, Billerica, MA, USA), anti-TLR2 (anti-human CD282, clone TL2.1, eBioscience, San Diego, CA, USA), LY294002 (PI3K), PD98059 (ERK1/2), SB203580 (MAPK p38), PP1 (Src-family kinases) and SP600125 (JNK) (all from Sigma-Aldrich, St Louis, MO, USA).

Techniques: Cell Culture, Comparison