Journal: Free radical biology & medicine

Article Title: Differential Peroxiredoxin Hyperoxidation Regulates MAP Kinase Signaling in Human Articular Chondrocytes

doi: 10.1016/j.freeradbiomed.2019.01.005

Figure Lengend Snippet: Confluent human articular chondrocytes were cultured in serum-free media overnight and treated for the indicated times with 25 μM menadione or 25 μM DMNQ. After experimental exposures, cell monolayers were incubated for 10 min in an alkylating buffer containing 100 mM NEM in order to alkylate reduced thiols. Cell lysates were then prepared in lysis buffer containing 100 mM NEM. (A) Hyperoxidized Prxs were identified by reducing immunoblots using an antibody to PrxSO2/3. (B) Results of densitometric analysis from PrxSO2/3 immunoblots. Data are mean±SEM and expressed as relative intensity compared to untreated controls from n=4 independent experiments. Asterisks represent significant differences compared to control (*p<0.05, ***p<0.001, ****p<0.0001) (ANOVA). (C-E) Under non–reducing conditions, immunoblots for total Prx1, Prx2 and Prx3 allowed for identification of Prx reduced monomers (labelled RM on blots) and hyperoxidized monomers (labelled HM on blots). Prx1-Prx3 presented with an oxidized dimer (labelled D on blots) (C, D, E). (F) Densitometric comparative analysis of Prx1, Prx2 and Prx3 hyperoxidized monomers observed at 60 min treatment with menadione or DMNQ. Asterisks represent significant differences between menadione and DMNQ treatments (***p<0.001, ****p<0.0001) (ANOVA) from a minimum of n=3 independent experiments. (G) Menadione and DMNQ-induced phosphorylation of Prx1 was identified by non-reducing immunoblots using an antibody to phospho-Prx1 (Tyr194). Total Prx1 was used as a loading control. (H) Results of densitometric analysis from phospho-Prx1 immunoblots. Data are mean±SEM normalized to total-Prx1 and expressed as relative intensity compared to untreated controls. Asterisks represent significant differences compared to control (****p<0.0001). # represent significant differences between menadione and DMNQ at that time point (# # # #p<0.0001) (ANOVA).

Article Snippet: Adenoviral transduction Human chondrocytes were transduced with adenoviral constructs encoding Prx1 (ad-Prx1), Prx2 (adPrx2) or Prx3 (ad-Prx3) (Vector Biolabs).

Techniques: Cell Culture, Incubation, Lysis, Western Blot

Journal: Free radical biology & medicine

Article Title: Differential Peroxiredoxin Hyperoxidation Regulates MAP Kinase Signaling in Human Articular Chondrocytes

doi: 10.1016/j.freeradbiomed.2019.01.005

Figure Lengend Snippet: Human articular chondrocytes were transduced with adenoviral vectors encoding Prx1, Prx2, Prx3 or a null empty vector control and treated with menadione (25 μM) or DMNQ (25 μM) for the indicated times. (A) Lysates from unstimulated chondrocytes transduced with Prx1–3 adenovirus or null empty control were immunoblotted with antibodies to Prx1-Prx3 or β-tubulin as a protein loading control. (B) Effect of Prx1 overexpression on menadione and DMNQ-induced PrxSO2/3 formation. Representative immunoblots from 7 independent experiments (n=7) with menadione treatment and 4 independent experiments (n=4) with DMNQ treatment. (C) Results of densitometric analysis from PrxSO2/3 immunoblots showing the effect of adenoviral overexpression of Prx1 on menadione and DMNQ-induced PrxSO2/3 formation. (D) Effect of adenoviral overexpression of Prx2 and Prx3 on menadione-induced PrxSO2/3 formation. Representative immunoblots from 6 independent experiments (n=6). (E) Results of densitometric analysis from PrxSO2/3 immunoblots showing the effect of adenoviral overexpression of Prx2 and Prx3 on menadione induced PrxSO2/3 formation. (F) Effect of adenoviral overexpression of Prx2 and Prx3 on DMNQ-induced PrxSO2/3 formation. Representative immunoblots from 4 independent experiments (n=4). (G) Results of densitometric analysis from PrxSO2/3 immunoblots showing the effect of adenoviral overexpression of Prx2 and Prx3 on DMNQ induced PrxSO2/3 formation. All data are mean±SEM normalized to untreated adenoviral control values. Asterisks represent significant differences compared to controls values *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (ANOVA). # represent significant differences between Prx overexpressing vector and null empty vector at that time point (# p<0.05) (ANOVA).

Article Snippet: Adenoviral transduction Human chondrocytes were transduced with adenoviral constructs encoding Prx1 (ad-Prx1), Prx2 (adPrx2) or Prx3 (ad-Prx3) (Vector Biolabs).

Techniques: Transduction, Plasmid Preparation, Over Expression, Western Blot

Journal: Free radical biology & medicine

Article Title: Differential Peroxiredoxin Hyperoxidation Regulates MAP Kinase Signaling in Human Articular Chondrocytes

doi: 10.1016/j.freeradbiomed.2019.01.005

Figure Lengend Snippet: Chondrocytes were transduced with an adenoviral vector encoding Prx2 or a null empty vector control and were treated with IGF-1 alone (50 ng/ml), menadione (25 μM), DMNQ (25 μM) or pre-treated with menadione (25 μM) or DMNQ (25 μM) for 30 min prior to IGF-1 treatment for 60 min. Cell lysates were immunoblotted with antibodies to proteins pertinent to IGF-1 and MAP kinase signaling cascades. (A) Representative immunoblots from 3 independent experiments (n=3) showing the effect of Prx2 overexpression on menadione-induced phosphorylation of Akt, PRAS40, p38, and ERK. (B-E) Results of densitometric analysis showing the effect of Prx2 overexpression on phosphorylation of Akt, PRAS40, p38, and ERK in response to menadione treatment. (F) Representative immunoblots from 5 independent experiments (n=5) showing the effect of Prx2 overexpression on DMNQ-induced phosphorylation of JNK, c-Jun, p38, and ERK. Arrow on c-Jun blots indicates electrophoretic shift of the c-Jun monomer which is indicative of serine and threonine phosphorylation (maximal phosphorylation). (G-J) Results of densitometric analysis showing the effect of Prx2 overexpression on phosphorylation of JNK, c-Jun, p38, and ERK in response to DMNQ treatment. Phospho-proteins are normalized to respective total protein. For c-Jun, β-actin was used as a loading control. Data are mean±SEM expressed as relative intensity compared to ad-Prx2 control. Asterisks represent significant differences compared to control (*p<0.05, **p<0.01, ***p<0.001). # represent significant differences between highlighted conditions (# # # p<0.001, # # # # p<0.0001) (ANOVA).

Article Snippet: Adenoviral transduction Human chondrocytes were transduced with adenoviral constructs encoding Prx1 (ad-Prx1), Prx2 (adPrx2) or Prx3 (ad-Prx3) (Vector Biolabs).

Techniques: Transduction, Plasmid Preparation, Western Blot, Over Expression

Journal: Free radical biology & medicine

Article Title: Differential Peroxiredoxin Hyperoxidation Regulates MAP Kinase Signaling in Human Articular Chondrocytes

doi: 10.1016/j.freeradbiomed.2019.01.005

Figure Lengend Snippet: Chondrocytes were transduced with an adenoviral vector encoding Prx3 or a null empty vector control and were treated with IGF-1 alone (50 ng/ml), menadione (25 μM), DMNQ (25 μM) or pre-treated with menadione (25 μM) or DMNQ (25 μM) for 30 min prior to IGF-1 treatment for 60 min. Cell lysates were immunoblotted with antibodies to proteins pertinent to IGF-1 and MAP kinase signaling cascades. (A) Representative immunoblots from 4 independent experiments (n=4) showing the effect of Prx3 overexpression on menadione-induced phosphorylation of Akt, PRAS40, p38, and ERK. (B-E) Results of densitometric analysis showing the effect of Prx3 overexpression on phosphorylation of Akt, PRAS40, p38, and ERK in response to menadione treatment. (F) Representative immunoblots from 3 independent experiments (n=3) showing the effect of Prx3 overexpression on DMNQ-induced phosphorylation of JNK, c-Jun, p38, and ERK. (G-J) Results of densitometric analysis showing the effect of Prx2 overexpression on phosphorylation of JNK, c-Jun, p38, and ERK in response to DMNQ treatment. Phospho-proteins are normalized to respective total protein. For c-Jun, β-actin was used as a loading control. Arrow on c-Jun blots indicates electrophoretic shift of the c-Jun monomer which is indicative of serine and threonine phosphorylation (maximal phosphorylation). Data are mean±SEM expressed as relative intensity compared to ad-Prx3 control. Asterisks represent significant differences compared to control (*p<0.05, **p<0.01, ***p<0.001). # represent significant differences between highlighted conditions (# p<0.05, # # p<0.01, # # # p<0.001) (ANOVA).

Article Snippet: Adenoviral transduction Human chondrocytes were transduced with adenoviral constructs encoding Prx1 (ad-Prx1), Prx2 (adPrx2) or Prx3 (ad-Prx3) (Vector Biolabs).

Techniques: Transduction, Plasmid Preparation, Western Blot, Over Expression

Journal: The American journal of pathology

Article Title: Low-dose-rate, low-dose irradiation delays neurodegeneration in a model of retinitis pigmentosa.

doi: 10.1016/j.ajpath.2011.09.025

Figure Lengend Snippet: Figure 4. Prdx2 plays a major role in the neuroprotective effects induced by low-dose-rate, low-dose (LDR-LD) radiation. A: Time course of Prdx2 mRNA levels in LDR-LD–irradiated retinas (n 6). B: Western blot analysis of Prdx2 in LDR-LD–irradiated retinas (n 9). C–F: In situ hybridization study of Prdx2 in the retina. G–J: Immunohistochemistry of Prdx2 in the retina. K: Relative expression level of Prdx2 mRNA 26 hours after intravitreal siRNA injection. Radiation was administered at 24 hours after injection. NC, negative control injected; Rad, LDR-LD radiation (n 12). A normal C57BL/6 strain was used in experiments A to K. L: PRL thickness of an rd10 (P25) mouse retina. Intravitreal injection of siRNA at P18. LDR-LD radiation was performed 24 hours after the siRNA injection (P19) (n 12). Scale bars: 60 m (C–J). *P 0.05, **P 0.01 versus control. P values were from post hoc analysis.

Article Snippet: For the Prdx2 blot, a rabbit polyclonal antibody to Prdx2 (ProteinTech Group, Chicago, IL) was used followed by goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA).

Techniques: Irradiation, Western Blot, In Situ Hybridization, Immunohistochemistry, Expressing, Injection, Negative Control, Control