Journal: PLoS ONE

Article Title: Electroacupuncture-Like Stimulation at the Baihui (GV20) and Dazhui (GV14) Acupoints Protects Rats against Subacute-Phase Cerebral Ischemia-Reperfusion Injuries by Reducing S100B-Mediated Neurotoxicity

doi: 10.1371/journal.pone.0091426

Figure Lengend Snippet: Representative western blot images show the expression of (A) mitochondrial cytochrome c and (C) cytosolic cytochrome c, TRADD, FADD, cleaved caspase-8 and cleaved caspase-3 in the ischemic cortical penumbra in the Sham-7 d, Model-7 d, EA-7 d, and Non-acup-7 d groups 7 d after reperfusion. COX IV and actin were used as internal controls for the mitochondrial and cytosolic fractions, respectively. The relative mitochondrial expression of (B) cytochrome c, and the relative cytosolic expression of (D) cytochrome c, (E) TRADD, (F) FADD, (G) cleaved caspase-8, and (H) cleaved caspase-3 were evaluated in the ischemic cortical penumbra in the Sham-7 d, Model-7 d, EA-7 d, and Non-acup-7 d groups (n = 4). Data are presented as mean ± SD. * P <0.05 compared with the Sham-7 d group; # P <0.05 compared with the Model-7 d group.

Article Snippet: They were then incubated with a mouse anti-GFAP (1∶1000 dilution, #3670 Cell Signaling Technology), rabbit anti-phospho-SAPK/JNK (p-JNK (Thr183/Tyr185); 1∶1000 dilution, #9251S Cell Signaling Technology), rabbit anti-phospho-p44/42 mitogen-activated protein kinase (MAPK (p-ERK); 1∶1000 dilution, #9101 Cell Signaling Technology), rabbit anti-phospho-p38 MAP kinase (p-p38 MAP kinase (Thr180/Tyr182); 1∶1000 dilution, #9212 Cell Signaling Technology), rabbit anti-cytochrome c (1∶1000 dilution, #4272 Cell Signaling Technology), rabbit anti-tumor necrosis factor receptor type 1-associated death domain (TRADD) (1∶1000 dilution, #3694 Cell Signaling Technology), rabbit anti-Fas-associated death domain (FADD) (1∶1000 dilution, #341282 Calbiochem), rabbit anti-cleaved caspase-8 (1∶1000 dilution, 3259-100 BioVision), or rabbit anti-cleaved caspase-3 (1∶1000 dilution, #9661S Cell Signaling Technology) antibody overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Journal of Neurotrauma

Article Title: MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death

doi: 10.1089/neu.2017.5572

Figure Lengend Snippet: Ang-1 activates the Akt pathway in neurons through both Tie-2 and β1-integrin signaling. (A) RCNs were treated with Ang-1 for the indicated time. Whole-cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Tie-2, phosphor-Tie2 (Y992), β1-integrin, FAK, phosphor-FAK (Tyr397), phospho-Akt (Ser473), phosphor-GSK3α/β, and β-actin. (B) Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared to control untreated levels. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; *p < 0.05; **p < 0.01; ***p < 0.001 versus cnt; ^^^p < 0.001 versus etoposide treated (N = 3). Akt, protein kinase B; Ang-1, angiopoietin-1; ANOVA, analysis of variance; FAK, focal adhesion kinase; GSK3α/β, glycogen synthase kinase 3α/β; RCNs, rat cortical neurons; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; SNK, Student–Newman–Keuls; Tie-2, Tunica interna endothelial cell kinase 2.

Article Snippet: Antibodies The following antibodies were used in this study: Histone H2A.X (ab11175; Abcam); apoptosis-inducing factor (AIF; sc-13116); cytochrome c (sc-13560) and Bim (sc-11425; Santa Cruz Biotechnology, Santa Cruz, CA); cleaved caspase-3 (#9661), cleaved PARP (poly (ADP-ribose) polymerase-1; #9545); phospho-GSK3α/β (glycogen synthase kinase 3α/β; Ser21/9; #9331); forkhead box O3a (FoxO3a; 75D8; #2497); Akt (pan; 11E7; #4685); FAK (#3285); phospho-FAK (Tyr397; #3283); phospho-p53 (Ser15; #9284); p53 (#2524); mitogen-activated protein kinase kinase 1 and 2 (MEK1/2; #8727); Lamin A/C (#4777); cyclooxegenase IV (COX-IV; #4844); phospho-Akt (Ser473; #4060; Cell Signaling Technology; GAPDH (ADI-CSA-335) and α-fodrin (BML-FG6090); active Bax (ALX-804-224-C100; Enzo Life Sciences, Inc., Farmingdale, NY); PUMA (#3041), Noxa (#2437; ProSci Incorporated, Poway, CA); β-actin (A1978); MAP2 (M3696; Sigma-Aldrich); integrin β1 (MAB1965); GFAP (MAB360; Millipore, Billerica, MA); Ang-1 (GTX28451; GeneTex Inc., Irvine, CA); Tie-2 (#AF762); phospho-Tie-2 (#AF2720; R&D Systems); and ionized calcium-binding adaptor molecule 1 (019-19741; Wako Pure Chemical Industries, Osaka, Japan).

Techniques:

Journal: Journal of Neurotrauma

Article Title: MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death

doi: 10.1089/neu.2017.5572

Figure Lengend Snippet: Blocking of Tie-2 and β1-integrin signaling inhibits the neuroprotective effect of Ang-1. RCNs were treated for 30 min with antibodies against Tie2 (50 μg/mL; A); antibodies against β1-integrin (5 μg/mL; B); combination of Tie2 (50 μg/mL) and β1-integrin (5 μg/mL; C) and FAK inhibitor PF573228 (100 nM; D). Next RCNs were treated with Ang-1 and etoposide (Etop) or etoposide alone. Twenty-four hours later, LDH release was measured. Data are expressed as percentage of control untreated neurons. (E) RCNs were treated with etoposide alone; Ang-1 and etoposide and etoposide; and Ang-1 etoposide and Akt inhibitor (2.875 μM). Cell death and cell viability were measured using the LDH and Calcein AM (calcein-acetoxymethyl ester). Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; *p < 0.05; **p < 0.01; ***p < 0.001 versus cnt; ^^p < 0.01; ^^^p < 0.001 versus etoposide treated; #p < 0.05; ##p < 0.01; ###p < 0.001 versus Ang-1 + etoposide treated; ∼∼p < 0.01; ∼∼∼p < 0.001 versus Tie-2 blocking/Ang-1/etoposide treated; &&p < 0.01 versus β1-integrin blocking/Ang-1/etoposide treated (N = 3). Akt, protein kinase B; Ang-1, angiopoietin-1; ANOVA, analysis of variance; FAK, focal adhesion kinase; LDH, lactate dehydrogenase; RCNs, rat cortical neurons; SEM, standard error of the mean; SNK, Student–Newman–Keuls; Tie-2, Tunica interna endothelial cell kinase 2.

Article Snippet: Antibodies The following antibodies were used in this study: Histone H2A.X (ab11175; Abcam); apoptosis-inducing factor (AIF; sc-13116); cytochrome c (sc-13560) and Bim (sc-11425; Santa Cruz Biotechnology, Santa Cruz, CA); cleaved caspase-3 (#9661), cleaved PARP (poly (ADP-ribose) polymerase-1; #9545); phospho-GSK3α/β (glycogen synthase kinase 3α/β; Ser21/9; #9331); forkhead box O3a (FoxO3a; 75D8; #2497); Akt (pan; 11E7; #4685); FAK (#3285); phospho-FAK (Tyr397; #3283); phospho-p53 (Ser15; #9284); p53 (#2524); mitogen-activated protein kinase kinase 1 and 2 (MEK1/2; #8727); Lamin A/C (#4777); cyclooxegenase IV (COX-IV; #4844); phospho-Akt (Ser473; #4060; Cell Signaling Technology; GAPDH (ADI-CSA-335) and α-fodrin (BML-FG6090); active Bax (ALX-804-224-C100; Enzo Life Sciences, Inc., Farmingdale, NY); PUMA (#3041), Noxa (#2437; ProSci Incorporated, Poway, CA); β-actin (A1978); MAP2 (M3696; Sigma-Aldrich); integrin β1 (MAB1965); GFAP (MAB360; Millipore, Billerica, MA); Ang-1 (GTX28451; GeneTex Inc., Irvine, CA); Tie-2 (#AF762); phospho-Tie-2 (#AF2720; R&D Systems); and ionized calcium-binding adaptor molecule 1 (019-19741; Wako Pure Chemical Industries, Osaka, Japan).

Techniques: Blocking Assay

Journal: Journal of Clinical Investigation

Article Title: Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury

doi: 10.1172/jci125517

Figure Lengend Snippet: Figure 7. Overnight incubation of HK-2 cells with 2 different monoclonal FLCs promotes the expression of ITGB6 on the cell surface. (A and B) Flow cytometric studies demonstrated that both the κ2 and λ3 FLCs increased the number of cells expressing ITGB6 (n = 4 experiments in each group). Pre- treatment of HK-2 cells with ITGB6 shRNA prevented this response. (C) Along with the changes in surface localization of ITGB6, pretreatment of the cells with ITGB6 shRNA also inhibited the production of active TGF-β (n = 4 experiments in each group). Data are expressed as the mean ± SEM. *P < 0.0009 compared with the other 2 corresponding groups (ANOVA).

Article Snippet: ITGB6 was knocked down using shRNA (ITGB6 Human shRNA Plasmid Kit Locus ID 3694, OriGene Technologies), with a nontargeting shRNA (TR30015, OriGene Technologies) serving as a control.

Techniques: Incubation, Expressing, shRNA

Journal: Journal of Clinical Investigation

Article Title: Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury

doi: 10.1172/jci125517

Figure Lengend Snippet: Figure 11. FLCs induce a peritubular inflammatory infiltrate that includes neutrophils and F4/80+HO-1+ macrophages. (A) Neutrophils, detected using anti–NIMP-R14 antibodies (red fluorescence), were present in greater numbers (white arrowheads) only in the Stat1+/+ mice treated with the higher dose of κ2 FLC. Proximal tubule expression of ITGB6 (green fluorescence) was readily detected. The IgG controls (left panel) showed minimal background staining of the samples. (B) M2 macrophage numbers, detected by colocalization of F4/80 (red fluorescence) with HO-1 (green fluorescence), were increased (white arrowheads) only in the Stat1+/+ mice treated with κ2 FLC. The inset shows a magnified image of a F4/80+HO-1+ cell (colocalized stain is shown in yellow). The IgG controls (left panel) showed minimal background staining of the samples. T, tubule. n = 8–10 mice/group. Data are expressed as the mean ± SEM of cells per HPF. *P < 0.0001 compared with the other 3 groups (ANOVA). Scale bars: 50 μm. Original magnification ×1200.

Article Snippet: ITGB6 was knocked down using shRNA (ITGB6 Human shRNA Plasmid Kit Locus ID 3694, OriGene Technologies), with a nontargeting shRNA (TR30015, OriGene Technologies) serving as a control.

Techniques: Fluorescence, Expressing, Staining

Journal: Journal of Clinical Investigation

Article Title: Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury

doi: 10.1172/jci125517

Figure Lengend Snippet: Figure 12. Treatment of Stat1+/+ mice with κ2 FLC promotes an increase in ITGB6 and p-SMAD2/3 in the kidney cortex. (A) Both doses of the κ2 FLCs prompted increases in ITGB6 levels of cortical lysates from Stat1+/+ mice (n = 6 mice/group). Data are expressed as the mean ± SEM. **P ≤ 0.006 compared with each of the other 4 groups (ANOVA). Relative ITGB6 levels were greater (P = 0.0096, ANOVA) in the PBS-treated Stat1+/+ mice compared with levels in the PBS-treated Stat1–/– mice. (B) A dose-dependent effect of κ2 FLC on p-SMAD2/3 was observed in Stat1+/+ mice but produced no changes in p-SMAD2/3 in Stat1–/– mice. Because SMAD2/3 levels were remarkably low in the kidney cortex of Stat1–/– mice, the data were factored by the density of GAPDH in the samples. n = 6 mice/group. Data are expressed as the mean ± SEM. *P < 0.005 compared with each of the other 5 groups in the experiment (ANOVA).

Article Snippet: ITGB6 was knocked down using shRNA (ITGB6 Human shRNA Plasmid Kit Locus ID 3694, OriGene Technologies), with a nontargeting shRNA (TR30015, OriGene Technologies) serving as a control.

Techniques: Produced

Journal: The Journal of Clinical Investigation

Article Title: Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury

doi: 10.1172/JCI125517

Figure Lengend Snippet: (A) All tested FLCs increased active TGF-β by HK-2 cells treated with a nontargeting siRNA, but not by HK-2 cells pretreated with an siRNA that targeted STAT1 (n = 5–6 in each group). Data are expressed as the mean ± SEM. Factorial ANOVA comparing the main effects of the FLC and the interaction effect between the FLC and siRNA on TGF-β showed that all effects were significant at a P value of less than 0.0001. The main effect for the siRNA yielded an F ratio of F(1, 66) = 6203, P < 0.0001. The interaction effect was significant: F(6, 66) = 185.8, P < 0.001. *P < 0.0001 compared with the corresponding group. (B) Western blot and (C) densitometric analyses showed the effect of siRNA treatment on p-STAT1 and p-SMAD2 (n = 4 in each group). Data are expressed as the mean ± SEM. Factorial ANOVA comparing the main effects of the FLC and the interaction effect between the FLC and siRNA on p-STAT1 showed that the main effect for the FLC yielded an F ratio of F(2, 18) = 3.724, P = 0.044, and that the effect of the siRNA yielded an F ratio of F(1, 18) = 0.0001. The interaction effect was not significant. *P = 0.014 compared with the corresponding group. For p-SMAD2, factorial ANOVA comparing the main effects of the FLC and the interaction effect between the FLC and the siRNA showed that all effects were significant at a P value of less than 0.0001. The main effect for the siRNA yielded an F ratio of F(1, 18) = 194.3, P < 0.0001. The interaction effect was significant at F(2, 18) = 32.19, P < 0.0001. *P < 0.0001 compared with the corresponding group.

Article Snippet: ITGB6 was knocked down using shRNA (ITGB6 Human shRNA Plasmid Kit Locus ID 3694, OriGene Technologies), with a nontargeting shRNA (TR30015, OriGene Technologies) serving as a control.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1

doi: 10.1038/srep27597

Figure Lengend Snippet: ( A ) ORF3 suppresses the phosphorylation of IKKβ and IκBα. ( B ) TLR3 pulls down of key downstream molecules detected by a coimmunoprecipitation assay. ( C,D ) TRADD and TAK1 expression levels are down-regulated in ORF3-pretreated cells compared with GFP-pretreated cells after stimulation with Poly(I:C). In contrast, TRIF, TRAF6 and RIP1 expression levels are increased by this stimulation.

Article Snippet: Primary antibodies against TRADD (# 3694, 1:1000 dilution), TRIF (# 4596, 1:1000 dilution), TRAF6 (# 8028, 1:800 dilution) and TAK1 (# 5206, 1:800 dilution) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Co-Immunoprecipitation Assay, Expressing