Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Peroxiredoxin 6 (Prdx6)-D140A mutation abolishes acidic Ca2+-independent phospholipase A2 (aiPLA2) activity without affecting Prdx6 expression or peroxidase activity. A: genotyping results of wild-type (WT) and Prdx6-D140A knockin mice. B: PCR showing excision of the neomycin resistance cassette NeoR. C: WT and Prdx6-D140A knockin lungs stained with hematoxylin and eosin; the scale bar = 100 µm. D: representative Western blot for Prdx6 expression in WT, Prdx6 null (KO), and Prdx6-D140A knockin (KI) lungs. Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and have been previously characterized (6, 58). E–G: enzymatic activities of Prdx6 in WT, Prdx6 null, and Prdx6-D140A knockin lungs: aiPLA2 activity using radiolabeled phospholipid substrate (E) and peroxidase activity with H2O2 (F) and phospholipid hydroperoxide (PLPCOOH) substrates (G). Error bars indicate the means ± SE for n = 3. Statistical significance was tested using ANOVA with Bonferroni post hoc test. *P < 0.05 vs WT.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Activity Assay, Expressing, Knock-In, Staining, Western Blot, Generated

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Genetic inactivation of acidic Ca2+-independent phospholipase A2 (aiPLA2) reduces mortality and prevents sepsis-induced lung inflammation. Wild-type (WT) and peroxiredoxin 6 (Prdx6)-D140A knockin (KI) mice were treated with 10 mg/kg LPS intraperitoneally. A: survival plots constructed using the Kaplan-Meier estimator. B: protein content of bronchoalveolar lavage fluid (BALF). C: total cells in BALF. D: myeloperoxidase (MPO) activity of cells in the BALF. E: lung staining for the presence of PMN as indicated by MPO (green, n = 4) using an antibody from R&D Systems (cat no. MAB3174; Minneapolis, MN). F: staining of lungs with anti-PMN antibody (NIMP-R14, green; cat. no. sc-59338; Santa Cruz Biotechnology) (n = 4). E and F: nuclei are counterstained with propidium iodide (red) and the scale bars = 20 µm. Blue circles and red squares represent individual animals per experiment. Error bars indicate the means ± SD. Group differences (saline vs. LPS) were evaluated by t-test, and the P value for comparison of the 2 groups is indicated; no P value is shown for those comparisons where P > 0.05.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Knock-In, Construct, Activity Assay, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Lung wet-to-dry weight ratio

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques:

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Genetic inactivation of acidic Ca2+-independent phospholipase A2 (aiPLA2) ameliorates sepsis-induced lung VCAM-1 expression, cytokine release, and oxidative stress. Wild-type (WT) and peroxiredoxin 6 (Prdx6)-D140A knockin (KI) mice were treated with 10 mg/kg LPS intraperitoneally. A: VCAM-1 expression measured by immunofluorescence of control (saline) and LPS-treated WT and Prdx6-D140A mice (n = 4). The monoclonal VCAM-1 antibody (catalog no. 32653) was purchased from Cell Signaling Technology (Danvers, MA). B: VCAM-1 expression measured by Western blot in whole lung homogenates of control (saline) and LPS-treated WT and Prdx6-D140A mice; the gels for WT (control and LPS) and mutant mice (control and LPS) were done at different times so that gel densities for the 2 types of mice should not be compared. The rabbit polyclonal VCAM-1 antibody (catalog no. sc-1504-R) was purchased from Santa Cruz Biotechnology. C–E: cytokine/chemokine expression in bronchoalveolar lavage fluid (BALF): TNF-α (C); IL-1 (D); and monocyte chemoattractant protein-1 (MCP-1; E); this cytokine was detected only in WT animals stimulated with LPS. F: staining of lung for 3-nitrotyrosine (green, n = 3) using an antibody generated by Dr. Harry Ischiropoulos (Children’s Hospital of Philadelphia) and previously described (17). In A and F, nuclei are counterstained in red and the scale bars = 20 µm. Blue circles and red squares represent individual animals per experiment. Error bars indicate the means ± SD. Group differences (saline vs. LPS) were evaluated by t-test, and the P value for comparison of the 2 groups is indicated; no P value is shown for those comparisons where P > 0.05.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Expressing, Knock-In, Immunofluorescence, Western Blot, Mutagenesis, Staining, Generated

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: LPS induces peroxiredoxin 6 (Prdx6) phosphorylation and promotes its translocation to the plasma membrane in lung pulmonary microvascular endothelial cells (PMVECs). Wild-type (WT) PMVECs were treated with 1 μg/ml LPS for 8 h. A: representative Western blots using Ab to phosphorylated Prdx6 (P-Prdx6; top), Prdx6 (middle), and GAPDH (bottom); Prdx6 is phosphorylated after treatment of cells with LPS. Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously (6); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology. B: acidic Ca2+-independent phospholipase A2 (aiPLA2) activity in control cells (PBS), in cells after treatment with LPS, and in cells that were pretreated with 10 μM MJ33 for 30 min before the addition of LPS. C: immunofluorescence using an anti-phosphorylated Prdx6 antibody (green) (58) and the membrane marker WGA-Alexa Fluor 594 (red). The scale bar = 20 µm. Note colocalization of the red and green markers at the cell membrane following LPS. The basis for the green nuclear staining is unknown. Error bars indicate the means ± SD for n = 3. Statistical significance was tested using ANOVA with Bonferroni post hoc test. Group differences (LPS vs. control or MJ33) were evaluated by ANOVA with Bonferroni post hoc test, and the P value for comparison of the groups is indicated.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Translocation Assay, Western Blot, Generated, Activity Assay, Immunofluorescence, Marker, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Genetic inactivation of acidic Ca2+-independent phospholipase A2 (aiPLA2) prevents NADPH oxidase, type 2 (Nox2)-mediated oxidant generation and oxidative stress in intact lungs and endothelial cells. A: intravascular oxidant generation was measured using Amplex red in the recirculating perfusate of isolated lungs from wild-type (WT) and peroxiredoxin 6 (Prdx6)-D140A mice at 24 h after intraperitoneal administration of LPS or saline (control). The time axis indicates the time of in vitro lung perfusion. Numbers in parenthesis represent the slope of the change in Amplex red fluorescence vs time calculated by least mean squares; n = 4 for Prdx6-D140A LPS; for all other groups, n = 3. B: extracellular H2O2 generation measured by the change in Amplex red fluorescence in WT, Nox2 null and Prdx6-D140A PMVECs at 8 h after administration of LPS. Data are the %change vs. control (cells treated with PBS for 8 h); n = 4 for WT; n = 3 for Nox2 and Prdx6-D140A; statistical significance was tested using ANOVA with Bonferroni post hoc test; the individual P values are for comparison to WT. C: intracellular H2O2 generation in PMVECs stably expressing pHyPer-Cyto. Cells were stimulated with LPS with or without MJ33 pretreatment for 8 h. An increase in cellular H2O2 is indicated by increased fluorescence. n = 3–4. The y-axis in A and C is in arbitrary fluorescence units (a.u.). D: lipid peroxidation (green fluorescence) was visualized using Liperfluo in WT and Prdx6-D140A PMVECs stimulated with LPS for 16 h; n = 3. Data are reported as change from corresponding PBS control for each cell type (WT and Prdx6-D140A). E: nitrotyrosine staining (green) in WT and Prdx6-D140A PMVECs stimulated with LPS for 16 h. Nuclei (red) are counterstained with propidium iodide. The antibody to 3-nitrotyrosine was a kind gift of Dr. Harry Ischiropoulos (Children’s Hospital of Philadelphia) and has been previously described (17). D and E: the scale bar for the fluorescence images indicates 20 µm and the y-axis on the bar graphs indicates fold-change from corresponding PBS control in cell fluorescence. WT, n = 4; Prdx6-D140A, n = 3. Error bars indicate the means ± SD. Group differences (PBS vs LPS) were evaluated by t-test; no P value is shown for those comparisons where P > 0.05.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Isolation, In Vitro, Fluorescence, Stable Transfection, Expressing, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Genetic inactivation of acidic Ca2+-independent phospholipase A2 (aiPLA2) prevents LPS-induced inflammation in lung endothelial cells. A: immunofluorescence staining for NF-κB (red) in wild-type (WT) and peroxiredoxin 6 (Prdx6)-D140A pulmonary microvascular endothelial cells (PMVECs) stimulated with LPS for different times. Nuclei are counterstained with SYTOX green; images are representative of three biological replicates. B: ICAM-1 staining (green) in WT and Prdx6-D140A PMVECs stimulated with LPS for 8 h. Nuclei are counterstained with propidium iodide (red); n = 3. ICAM-1 antibodies (cat no. ab119871) were obtained from Abcam (Cambridge, MA). C: representative Western blot for nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) expression in WT and Prdx6-D140A PMVECs stimulated with LPS. WT, n = 6; Prdx6-D140A, n = 3. NLRP3 expression was not detected in Prdx6-D140A PMVECs even after stimulation with LPS. NLRP3 antibodies were purchased from Cell Signaling Technologies (cat no. 15101). D: in situ proximity ligation assays (Duolink) to evaluate the colocalization of the NLRP3 and ASC (BioLegend antibody no. 653902) inflammasome subunits. KI, knockin. Red fluorescence indicates proximity (<40 nm) of the 2 proteins. Gray indicates nuclear staining. Images are representative of at least 3 biological replicates. Quantitative analysis was performed using Fiji software. For each biological sample, ten pictures were evaluated and counted. NLRP3:ASC interactions were normalized to nuclei. The scale bars in A, B, and D indicate 25 µm. Error bars indicate the means ± SD. Group differences (PBS vs. LPS) were evaluated by t-test; no p\P value is shown for those comparisons where P > 0.05.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Immunofluorescence, Staining, Western Blot, Binding Assay, Expressing, In Situ, Ligation, Knock-In, Fluorescence, Software

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Schematic for the effect of the D140 mutation of peroxiredoxin 6 (Prdx6-D140A) on the lung response to intraperitoneal LPS. LPS through interaction with Toll-like receptor 4 (TLR4) leads to MAP kinase activation with subsequent phosphorylation of Prdx6 and its translocation to the plasma membrane. An increased Prdx6-PLA2 activity leads to NADPH oxidase, type 2 (Nox2) activation and generation of oxidants, resulting in tissue oxidative stress and lung injury. Oxidative stress also results in NF-κB translocation to the cell nucleus, which promotes the expression of various inflammatory agents, recruitment of PMNs to the lung, and amplification of oxidant-induced lung injury. Mutation of D140 to A140 in Prdx6 abolishes its PLA2 activity and prevents the tissue-damaging cascade associated with Nox2 activation. aiPLA2, acidic Ca2+-independent phospholipase A2.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Activation Assay, Translocation Assay, Activity Assay, Expressing, Amplification

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Peroxiredoxin 6 (Prdx6)-D140A mutation abolishes acidic Ca2+-independent phospholipase A2 (aiPLA2) activity without affecting Prdx6 expression or peroxidase activity. A: genotyping results of wild-type (WT) and Prdx6-D140A knockin mice. B: PCR showing excision of the neomycin resistance cassette NeoR. C: WT and Prdx6-D140A knockin lungs stained with hematoxylin and eosin; the scale bar = 100 µm. D: representative Western blot for Prdx6 expression in WT, Prdx6 null (KO), and Prdx6-D140A knockin (KI) lungs. Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and have been previously characterized (6, 58). E–G: enzymatic activities of Prdx6 in WT, Prdx6 null, and Prdx6-D140A knockin lungs: aiPLA2 activity using radiolabeled phospholipid substrate (E) and peroxidase activity with H2O2 (F) and phospholipid hydroperoxide (PLPCOOH) substrates (G). Error bars indicate the means ± SE for n = 3. Statistical significance was tested using ANOVA with Bonferroni post hoc test. *P < 0.05 vs WT.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Activity Assay, Expressing, Knock-In, Staining, Western Blot, Generated

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Genetic inactivation of acidic Ca2+-independent phospholipase A2 (aiPLA2) reduces mortality and prevents sepsis-induced lung inflammation. Wild-type (WT) and peroxiredoxin 6 (Prdx6)-D140A knockin (KI) mice were treated with 10 mg/kg LPS intraperitoneally. A: survival plots constructed using the Kaplan-Meier estimator. B: protein content of bronchoalveolar lavage fluid (BALF). C: total cells in BALF. D: myeloperoxidase (MPO) activity of cells in the BALF. E: lung staining for the presence of PMN as indicated by MPO (green, n = 4) using an antibody from R&D Systems (cat no. MAB3174; Minneapolis, MN). F: staining of lungs with anti-PMN antibody (NIMP-R14, green; cat. no. sc-59338; Santa Cruz Biotechnology) (n = 4). E and F: nuclei are counterstained with propidium iodide (red) and the scale bars = 20 µm. Blue circles and red squares represent individual animals per experiment. Error bars indicate the means ± SD. Group differences (saline vs. LPS) were evaluated by t-test, and the P value for comparison of the 2 groups is indicated; no P value is shown for those comparisons where P > 0.05.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Knock-In, Construct, Activity Assay, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Lung wet-to-dry weight ratio

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques:

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Genetic inactivation of acidic Ca2+-independent phospholipase A2 (aiPLA2) ameliorates sepsis-induced lung VCAM-1 expression, cytokine release, and oxidative stress. Wild-type (WT) and peroxiredoxin 6 (Prdx6)-D140A knockin (KI) mice were treated with 10 mg/kg LPS intraperitoneally. A: VCAM-1 expression measured by immunofluorescence of control (saline) and LPS-treated WT and Prdx6-D140A mice (n = 4). The monoclonal VCAM-1 antibody (catalog no. 32653) was purchased from Cell Signaling Technology (Danvers, MA). B: VCAM-1 expression measured by Western blot in whole lung homogenates of control (saline) and LPS-treated WT and Prdx6-D140A mice; the gels for WT (control and LPS) and mutant mice (control and LPS) were done at different times so that gel densities for the 2 types of mice should not be compared. The rabbit polyclonal VCAM-1 antibody (catalog no. sc-1504-R) was purchased from Santa Cruz Biotechnology. C–E: cytokine/chemokine expression in bronchoalveolar lavage fluid (BALF): TNF-α (C); IL-1 (D); and monocyte chemoattractant protein-1 (MCP-1; E); this cytokine was detected only in WT animals stimulated with LPS. F: staining of lung for 3-nitrotyrosine (green, n = 3) using an antibody generated by Dr. Harry Ischiropoulos (Children’s Hospital of Philadelphia) and previously described (17). In A and F, nuclei are counterstained in red and the scale bars = 20 µm. Blue circles and red squares represent individual animals per experiment. Error bars indicate the means ± SD. Group differences (saline vs. LPS) were evaluated by t-test, and the P value for comparison of the 2 groups is indicated; no P value is shown for those comparisons where P > 0.05.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Expressing, Knock-In, Immunofluorescence, Western Blot, Mutagenesis, Staining, Generated

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: LPS induces peroxiredoxin 6 (Prdx6) phosphorylation and promotes its translocation to the plasma membrane in lung pulmonary microvascular endothelial cells (PMVECs). Wild-type (WT) PMVECs were treated with 1 μg/ml LPS for 8 h. A: representative Western blots using Ab to phosphorylated Prdx6 (P-Prdx6; top), Prdx6 (middle), and GAPDH (bottom); Prdx6 is phosphorylated after treatment of cells with LPS. Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously (6); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology. B: acidic Ca2+-independent phospholipase A2 (aiPLA2) activity in control cells (PBS), in cells after treatment with LPS, and in cells that were pretreated with 10 μM MJ33 for 30 min before the addition of LPS. C: immunofluorescence using an anti-phosphorylated Prdx6 antibody (green) (58) and the membrane marker WGA-Alexa Fluor 594 (red). The scale bar = 20 µm. Note colocalization of the red and green markers at the cell membrane following LPS. The basis for the green nuclear staining is unknown. Error bars indicate the means ± SD for n = 3. Statistical significance was tested using ANOVA with Bonferroni post hoc test. Group differences (LPS vs. control or MJ33) were evaluated by ANOVA with Bonferroni post hoc test, and the P value for comparison of the groups is indicated.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Translocation Assay, Western Blot, Generated, Activity Assay, Immunofluorescence, Marker, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Genetic inactivation of acidic Ca2+-independent phospholipase A2 (aiPLA2) prevents NADPH oxidase, type 2 (Nox2)-mediated oxidant generation and oxidative stress in intact lungs and endothelial cells. A: intravascular oxidant generation was measured using Amplex red in the recirculating perfusate of isolated lungs from wild-type (WT) and peroxiredoxin 6 (Prdx6)-D140A mice at 24 h after intraperitoneal administration of LPS or saline (control). The time axis indicates the time of in vitro lung perfusion. Numbers in parenthesis represent the slope of the change in Amplex red fluorescence vs time calculated by least mean squares; n = 4 for Prdx6-D140A LPS; for all other groups, n = 3. B: extracellular H2O2 generation measured by the change in Amplex red fluorescence in WT, Nox2 null and Prdx6-D140A PMVECs at 8 h after administration of LPS. Data are the %change vs. control (cells treated with PBS for 8 h); n = 4 for WT; n = 3 for Nox2 and Prdx6-D140A; statistical significance was tested using ANOVA with Bonferroni post hoc test; the individual P values are for comparison to WT. C: intracellular H2O2 generation in PMVECs stably expressing pHyPer-Cyto. Cells were stimulated with LPS with or without MJ33 pretreatment for 8 h. An increase in cellular H2O2 is indicated by increased fluorescence. n = 3–4. The y-axis in A and C is in arbitrary fluorescence units (a.u.). D: lipid peroxidation (green fluorescence) was visualized using Liperfluo in WT and Prdx6-D140A PMVECs stimulated with LPS for 16 h; n = 3. Data are reported as change from corresponding PBS control for each cell type (WT and Prdx6-D140A). E: nitrotyrosine staining (green) in WT and Prdx6-D140A PMVECs stimulated with LPS for 16 h. Nuclei (red) are counterstained with propidium iodide. The antibody to 3-nitrotyrosine was a kind gift of Dr. Harry Ischiropoulos (Children’s Hospital of Philadelphia) and has been previously described (17). D and E: the scale bar for the fluorescence images indicates 20 µm and the y-axis on the bar graphs indicates fold-change from corresponding PBS control in cell fluorescence. WT, n = 4; Prdx6-D140A, n = 3. Error bars indicate the means ± SD. Group differences (PBS vs LPS) were evaluated by t-test; no P value is shown for those comparisons where P > 0.05.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Isolation, In Vitro, Fluorescence, Stable Transfection, Expressing, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Genetic inactivation of acidic Ca2+-independent phospholipase A2 (aiPLA2) prevents LPS-induced inflammation in lung endothelial cells. A: immunofluorescence staining for NF-κB (red) in wild-type (WT) and peroxiredoxin 6 (Prdx6)-D140A pulmonary microvascular endothelial cells (PMVECs) stimulated with LPS for different times. Nuclei are counterstained with SYTOX green; images are representative of three biological replicates. B: ICAM-1 staining (green) in WT and Prdx6-D140A PMVECs stimulated with LPS for 8 h. Nuclei are counterstained with propidium iodide (red); n = 3. ICAM-1 antibodies (cat no. ab119871) were obtained from Abcam (Cambridge, MA). C: representative Western blot for nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) expression in WT and Prdx6-D140A PMVECs stimulated with LPS. WT, n = 6; Prdx6-D140A, n = 3. NLRP3 expression was not detected in Prdx6-D140A PMVECs even after stimulation with LPS. NLRP3 antibodies were purchased from Cell Signaling Technologies (cat no. 15101). D: in situ proximity ligation assays (Duolink) to evaluate the colocalization of the NLRP3 and ASC (BioLegend antibody no. 653902) inflammasome subunits. KI, knockin. Red fluorescence indicates proximity (<40 nm) of the 2 proteins. Gray indicates nuclear staining. Images are representative of at least 3 biological replicates. Quantitative analysis was performed using Fiji software. For each biological sample, ten pictures were evaluated and counted. NLRP3:ASC interactions were normalized to nuclei. The scale bars in A, B, and D indicate 25 µm. Error bars indicate the means ± SD. Group differences (PBS vs. LPS) were evaluated by t-test; no p\P value is shown for those comparisons where P > 0.05.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Immunofluorescence, Staining, Western Blot, Binding Assay, Expressing, In Situ, Ligation, Knock-In, Fluorescence, Software

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Genetic inactivation of the phospholipase A 2 activity of peroxiredoxin 6 in mice protects against LPS-induced acute lung injury

doi: 10.1152/ajplung.00344.2018

Figure Lengend Snippet: Schematic for the effect of the D140 mutation of peroxiredoxin 6 (Prdx6-D140A) on the lung response to intraperitoneal LPS. LPS through interaction with Toll-like receptor 4 (TLR4) leads to MAP kinase activation with subsequent phosphorylation of Prdx6 and its translocation to the plasma membrane. An increased Prdx6-PLA2 activity leads to NADPH oxidase, type 2 (Nox2) activation and generation of oxidants, resulting in tissue oxidative stress and lung injury. Oxidative stress also results in NF-κB translocation to the cell nucleus, which promotes the expression of various inflammatory agents, recruitment of PMNs to the lung, and amplification of oxidant-induced lung injury. Mutation of D140 to A140 in Prdx6 abolishes its PLA2 activity and prevents the tissue-damaging cascade associated with Nox2 activation. aiPLA2, acidic Ca2+-independent phospholipase A2.

Article Snippet: Prdx6 and phosphorylated Prdx6 antibodies were generated by Covance Research Products (Denver, CO) and Proteintech Group (Chicago, IL), respectively, and have been described previously ( 6 ); GAPDH antibodies (cat no. 2118) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Activation Assay, Translocation Assay, Activity Assay, Expressing, Amplification

Journal: PLOS Pathogens

Article Title: Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing

doi: 10.1371/journal.ppat.1012232

Figure Lengend Snippet: (a) White arrows indicate the localization of IBV N protein in AQP2-expressing collecting duct cells. Green fluorescence shows positive staining for IBV N and red fluorescence shows staining for AQP2. (b) White arrows indicate the localization of IBV N protein in CALB1-expressing distal tubule cells. Red fluorescence shows positive staining for IBV N and green fluorescence shows staining for CALB1. (c) Changes in cell communication numbers: The top network diagram shows cell clusters as nodes, with line thickness indicating changes in communication numbers. The lower heatmap details these changes, with rows representing signal-sending cells and columns indicating signal-receiving cells. The color scale reflects the inter-group differences in signal communication frequency between different cell types (number of communications in the infected group—number in control group). The bar plots at the top and right side represent the overall differences in the number of signals sent/received by specific cell clusters. (d) Changes in cell communication strength: Similar to (c), with the top network diagram displaying changes in communication strength (communication strength in the infected group—strength in the control group). In the lower heatmap, the color scale reflects the inter-group differences in signal communication strength between different cell types. The bar plots at the top and right side represent the overall differences in the strength of signals sent/received by specific cell clusters (infected group—control group). (e) Inter-group differences in the communication strength of specific signaling pathways (receptor-ligand pairs) across cell clusters. Rows represent signal pathways and columns correspond to cell clusters, with heatmap colors depicting the strength variation of signals (infect group vs. control group). Upper left triangles for signal sent and lower right triangles for signal received. The bar plot on the right side shows the overall difference in communication strength of these signaling pathways between the IBV-infected group and the control group.

Article Snippet: mouse mab calb1 , 1:200 , Boster Bio (BM0203).

Techniques: Expressing, Fluorescence, Staining, Infection, Control, Protein-Protein interactions

Journal: PLOS Pathogens

Article Title: Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing

doi: 10.1371/journal.ppat.1012232

Figure Lengend Snippet: Antbodies and reagents used in present study.

Article Snippet: mouse mab calb1 , 1:200 , Boster Bio (BM0203).

Techniques: Blocking Assay, Immunofluorescence