Journal: Antioxidants

Article Title: Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection

doi: 10.3390/antiox11071410

Figure Lengend Snippet: H 2 S donor GYY4137 activates and rescues NRF2 in RSV infection. ( a ) Primary human small airway epithelial cells (SAECs) were treated with 1, 3, or 5 mM GYY4137 or its vehicle. Cells were harvested 17 h after treatment and total cell lysates were analyzed by Western blot with anti-NRF2 antibody. The membrane was reprobed with anti-β-actin antibody for loading control. Western blot image is one representative of three independent experiments. The graph shows the densitometric analysis of NRF2 after normalization to β-actin expressed as mean ± SEM. Data were analyzed by one-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR); ( b ) SAECs uninfected and infected with RSV were treated with 5 mM GYY4137 (or vehicle). GYY4137 was added 1 h after RSV infection. Cells were harvested 18 h post-infection (hpi) and total cell lysates were analyzed by Western blot with anti-NRF2 antibody. The membrane was reprobed with anti-β-actin antibody for loading control. Western blot image is one representative of three independent experiments. The graph shows the densitometric analysis of NRF2 after normalization to β-actin expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR; # p < 0.05 vs. RSV); ( c ) SAECs were infected with RSV followed by treatment with 5 mM GYY4137. Cells were harvested 18 hpi and RSV proteins were detected in total cell lysates by Western blot using anti-RSV antibody. RSV proteins corresponding bands are indicated on the right. The membrane was reprobed with anti-β-actin for loading control. Western blot image is one representative of two independent experiments.

Article Snippet: The NRF2 was immunoprecipitated from the total cell lysates (containing 1 mg of total protein) with 8 μg of anti-NRF2 antibody (PM069, MBL International Corporation, Woburn, MA, USA) and the NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody (AUB01-HRP, Cytoskeleton, Denver, CO, USA).

Techniques: Infection, Western Blot, Membrane, Control

Journal: Antioxidants

Article Title: Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection

doi: 10.3390/antiox11071410

Figure Lengend Snippet: H 2 S donor GYY4137 rescues NRF2-dependent gene expression in RSV infection. SAECs uninfected and infected with RSV were treated with 5 mM GYY4137 and harvested 18 hpi to prepare total RNA. SOD1, catalase, NQO1, GCLC, and GCLM gene expression were quantified by RT-qPCR. Graphs show combined data from three independent experiments expressed as mean ± SEM. The results were analyzed by two-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR; # p < 0.05 vs. RSV).

Article Snippet: The NRF2 was immunoprecipitated from the total cell lysates (containing 1 mg of total protein) with 8 μg of anti-NRF2 antibody (PM069, MBL International Corporation, Woburn, MA, USA) and the NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody (AUB01-HRP, Cytoskeleton, Denver, CO, USA).

Techniques: Gene Expression, Infection, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection

doi: 10.3390/antiox11071410

Figure Lengend Snippet: H 2 S donor GYY4137 restores NRF2 ubiquitination and does not affect histone deacetylase (HDAC) activity in RSV infection. ( a ) SAECs uninfected and infected with RSV were treated with 5 mM GYY4137 and harvested 18 hpi. NRF2 was immunoprecipitated from total cell lysates with anti-NRF2 antibody and NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody. The membrane was stripped and reprobed with anti-NRF2 antibody to determine levels of immunoprecipitated NRF2. A sample of the original pre-immunoprecipitation lysate was also analyzed by Western blot to show levels of NRF2 before immunoprecipitation (input). Western blot images are one representative of three independent experiments. Graph shows densitometric analysis of NRF2 ubiquitination after normalization to immunoprecipitated NRF2 expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR; # p < 0.05 vs. RSV); ( b ) SAECs, uninfected and infected with RSV, were treated with 5 mM GYY4137 and harvested 18 hpi. HDAC activity (class I and class II) in nuclear extracts was measured using HDAC Fluorometric Activity Assay Kit (Cayman Chemical). The HDAC activity was normalized by protein concentration. The graph shows combined data from three independent experiments expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey’s test (* p < 0.05 vs. CTR).

Article Snippet: The NRF2 was immunoprecipitated from the total cell lysates (containing 1 mg of total protein) with 8 μg of anti-NRF2 antibody (PM069, MBL International Corporation, Woburn, MA, USA) and the NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody (AUB01-HRP, Cytoskeleton, Denver, CO, USA).

Techniques: Ubiquitin Proteomics, Histone Deacetylase Assay, Activity Assay, Infection, Immunoprecipitation, Western Blot, Membrane, Protein Concentration