Journal: Viruses

Article Title: Recombinant Pseudorabies Virus with TK/gE Gene Deletion and Flt3L Co-Expression Enhances the Innate and Adaptive Immune Response via Activating Dendritic Cells

doi: 10.3390/v13040691

Figure Lengend Snippet: Characterization of the recombinant HNX-TK − /gE − -Flt3L. ( A ) Detection of the transcription of the Flt3L gene in HNX-TK − /gE − -Flt3L virus- and HNX-infected cells through RT-qPCR (**** < 0.001). ( B ) Detection of the Flt3L protein and viral gB protein expression through an immunofluorescence assay (IFA). Green: gB-positive cells; red: Flt3L-positive cells; blue: DAPI-stained PK-15 cell nucleus. DAPI, 4’,6-diamidino-2-phenylindole. ( C ) Western blot analysis of β-actin, gD, and Flt3L of the PK-15 cells infected with HNX-TK − /gE − -Flt3L, HNX, or DMEM as a control. ( D ) One step growth curves of HNX-TK − /gE − -Flt3L and HNX in PK-15 cells.

Article Snippet: The blots were blocked by skim milk (BD, Bioscience, Inc., Saint Louis, MO, USA), and this was followed by incubation with rabbit anti-Flt3L polyclonal antibody (Bioss, Beijing, China), mouse anti-gD monoclonal antibody (Keqian Ltd., Wuhan, China), and mouse anti-β-actin monoclonal antibody (Bioss).

Techniques: Recombinant, Infection, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Neuroendocrinology

Article Title: Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain

doi: 10.1159/000521236

Figure Lengend Snippet: Effects of DHT on insulin and leptin signaling in the hypothalamus of 6-week-old, prepubertal, nonobese rats. a, e Representative immunoblots showing phosphorylated and total IR, AKT, GSK, JAK2, and Stat3 levels, with β-actin as the loading control. b–d, f, g Densitometry analysis and comparison of phosphorylated forms versus total amounts of different proteins. DHT treatment significantly decreased the phosphorylation levels of these signaling proteins. h Expression levels of feeding-related genes in the hypothalamus of 6-week-old DHT and control rats. Note the significant increase in the mRNA levels of NPY and Agrp genes. Experiments were performed in 3 rats for each group. Data are expressed in mean ± SEM ( n = 6). * p < 0.05, ** p < 0.01. SEM, standard error.

Article Snippet: β-Actin , Abcam , ab8226 , 1:10,000.

Techniques: Western Blot, Expressing

Journal: Neuroendocrinology

Article Title: Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain

doi: 10.1159/000521236

Figure Lengend Snippet: Effect of DHT on insulin and leptin signaling in the hypothalamus of 7-week-old pair-fed rats. a, f Representative immunoblots showing phosphorylated and total IR, AKT, GSK, JAK2, and Stat3 levels, with β-actin as the loading control. b–e, g–j Densitometry analysis and comparison of phosphorylated versus total amounts of proteins. DHT treatment significantly decreased the phosphorylation levels of these signaling factors after pair feeding. Experiments were performed in 3 rats for each group. Data are expressed in mean ± SEM. * p < 0.05, ** p < 0.01. SEM, standard error.

Article Snippet: β-Actin , Abcam , ab8226 , 1:10,000.

Techniques: Western Blot

Journal: Neuroendocrinology

Article Title: Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain

doi: 10.1159/000521236

Figure Lengend Snippet: Effect of central leptin on the hypothalamus. a Leptin levels in the CSF were significantly decreased in DHT rats than in control rats. b ICV injection of leptin inhibited the improvement of food intake caused by DHT. c Representative immunoblots showing phosphorylated and total Stat3 levels, with β-actin as the loading control. d Densitometry analysis and comparison of phosphorylated forms versus total amounts of proteins. The phosphorylation of STAT3 was also increased in the DHT rats. Data were expressed as mean ± SEM. * p < 0.05. SEM, standard error.

Article Snippet: β-Actin , Abcam , ab8226 , 1:10,000.

Techniques: Injection, Western Blot

Journal: The Journal of cell biology

Article Title: Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation.

doi: 10.1083/jcb.202305048

Figure Lengend Snippet: Figure 1. Transcriptional reprogramming of neuronal metabolism during glucose deprivation. (A) Schematic for glucose deprivation of rat cortical neurons. A subset of ∼250 genes significantly upregulated during glucose deprivation (P value <0.01) were selected for pathway analysis using annotated gene sets from the Molecular Signatures Database (MSigDB). n = 3 cortical samples from one rat litter. (B) Enrichment of genes in MSigDB Reactome pathways with adjusted P value <0.05. (C) Enrichment of the target genes of the CREB transcription factor in glucose-deprived neurons (adjusted P value <0.05). The target gene Pgc1α (PPARGC1A) is highlighted in red. (D) Immunostaining of cortical neuronal cultures (treated as in A) with an anti-phospho-CREB antibody and Hoechst nuclear stain. Arrowheads denote pCREB-positive nuclei. (E) Fraction of nuclei with positive p-CREB staining in fields of view (FOV), determined as described in Materials and methods. % total ± SEM: +glucose (3 h), 14.87 ± 2.29, −glucose (3 h), 27.41 ± 3.3. n = 17–22 FOVs. (F) Relative mRNA expression of Pgc1α in neuronal cultures treated as in A, with or without the AMPK inhibitor, dorsomorphin. Values are normalized to β-actin mRNA and expressed relative to the +glucose condition. Average normalized mRNA level ± SEM: −glucose (1 h), 2.83 ± 0.42; −glucose (3 h), 3.67 ± 0.46; −glucose + dorsomorphin (3 h), 1.32 ± 0.22. n = 3–10 cortical samples. Bar graphs are plotted as mean ± SEM. Mann–Whitney U test (E), one-way ANOVA (F). See Table 1.

Article Snippet: The following primary antibodies were used: Sirt3 (1:1,000, 5490S; Cell Signaling Technology, rabbit, reactivity for human, mouse, and rat), anti-acetyl-lysine (1:1,000, 9441S; Cell Signaling Technology rabbit, all species expected), ATPase5β (1:1,000, HPA001520; Sigma-Aldrich, rabbit, reactivity for rat, mouse, and humans), β-actin (1:5,000; HCA147P; Bio-Rad, Human Combinatorial Antibody Library/ HuCAL, reactivity for rat, mouse, and humans), α-tubulin (1:2,000, T6074; Sigma-Aldrich, mouse, reactivity for mouse, chicken, Chlamydomonas, African green monkey, human, rat, bovine, sea urchin, and kangaroo rat).

Techniques: Immunostaining, Staining, Expressing, MANN-WHITNEY

Journal: The Journal of cell biology

Article Title: Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation.

doi: 10.1083/jcb.202305048

Figure Lengend Snippet: Figure 2. Glucose deprivation stimulates neuronal Sirt3 expression and deacetylation of mitochondrial proteins. (A) Relative Sirt3 mRNA expression in control and glucose-deprived neu- rons. Values are normalized to β-actin mRNA and expressed relative to control (+glucose). n = 3–10 cortical samples. Average normalized mRNA level ± SEM: −glucose (1 h), 2.58 ± 0.62; −glucose (3 h), 2.20 ± 0.27; −glucose + dorsomorphin (3 h), 1.12 ± 0.08. n = 3–11 cortical samples. (B) Relative Sirt3 mRNA expression in cultures transduced with adenoviral particles encoding GFP (control) and GFP-PGC1α, normalized to 18s rRNA and ex- pressed relative to control. Average normalized mRNA level ± SEM: GFP-PGC1α, 1.93 ± 0.30. n = 16 cortical samples/condition. (C) Immunoblotting of Sirt3 protein expression in cortical neurons with antibodies against Sirt3 and the cytosolic and mitochondrial controls, β-actin, and ATPase5β, respectively. (D) Sirt3 band intensity normalized to the ATPase5β and expressed relative to control. Average normalized Sirt3 band intensity ± SEM: +glucose, 0.99 ± 0.05; −glucose, 1.74 ± 0.16. n = 7 cortical samples/condition. (E) A paradigm for the analysis of Sirt3 expression in hippocampi of mice fed ad libitum (ad lib) or alternate-day fasted for 6 mo (ADF). Schematic created with https:// biorender.com. (F) Immunoblotting of Sirt3 protein in mouse hippocampal lysates. (G) Sirt3 band intensity normalized to the ATPase5β band and expressed relative to the ad lib mice. Average normalized Sirt3 band intensity ± SEM: ad lib, 1 ± 0.24; ADF (6 mo), 1.88 ± 0.19. n = 6 mice/condition. (H) Mitochondrial and cytosolic fractions isolated from cortical neuronal cul- tures maintained for 3 h with (+) or without (−) glucose. High and low exposures were 60 and 25 s, respectively. (I) Intensity of lysine acetylation bands normalized to α-tubulin or ATPase5β plot- ted relative to control. Average normalized Ac-K intensity: cytosolic fraction (−glucose), 1.21 ± 0.15; mitochondrial fraction (−glucose), 0.66 ± 0.08. n = 5 cortical samples. One-way ANOVA (A), two- tailed, unpaired t test (B), Mann–Whitney U test (D and G), one sample t test (I). See Table 1. Source data are available for this figure: SourceData F2.

Article Snippet: The following primary antibodies were used: Sirt3 (1:1,000, 5490S; Cell Signaling Technology, rabbit, reactivity for human, mouse, and rat), anti-acetyl-lysine (1:1,000, 9441S; Cell Signaling Technology rabbit, all species expected), ATPase5β (1:1,000, HPA001520; Sigma-Aldrich, rabbit, reactivity for rat, mouse, and humans), β-actin (1:5,000; HCA147P; Bio-Rad, Human Combinatorial Antibody Library/ HuCAL, reactivity for rat, mouse, and humans), α-tubulin (1:2,000, T6074; Sigma-Aldrich, mouse, reactivity for mouse, chicken, Chlamydomonas, African green monkey, human, rat, bovine, sea urchin, and kangaroo rat).

Techniques: Expressing, Control, Transduction, Western Blot, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: International Journal of Molecular Medicine

Article Title: MicroRNA-17 contributes to the suppression of the inflammatory response in lipopolysaccharide-induced acute lung injury in mice via targeting the toll-like receptor 4/nuclear factor-κB pathway

doi: 10.3892/ijmm.2020.4599

Figure Lengend Snippet: TLR4 is a direct target of miR-17. (A and B) Putative binding site of miR-17 and TLR4. (C) Luciferase activity was detected by a dual luciferase assay in RAW264.7 cells co-transfected with firefly luciferase constructs containing the TLR4 wt or mut 3′-UTRs and miR-17 mimics, mimics NC, miR-17 inhibitor or inhibitor NC, as indicated (n=3). (D) The expression of the TLR4 protein was detected by western blotting following transfection with miR-17 mimics and miR-17 inhibitor. Data are expressed as mean ± standard deviation of three independent experiments, ** P<0.01 vs. mimics NC, ## P<0.01 vs. inhibitor NC. (E) The expression of the TLR4 protein was measured by western blotting in lung tissues from agomir-17- and agomir-NC-injected ALI mice. β-actin was used as the internal control. Data are expressed as mean ± standard deviation of three independent experiments (n=5 mice per group). * P<0.05, ** P<0.01 vs. mimics NC, ## P<0.01 vs. inhibitor NC. ALI, acute lung injury; TLR, toll-like receptor; wt, wild-type; mut, mutant; UTR, untranslated region.

Article Snippet: The membrane was blocked with 5% skimmed milk for 2 h at room temperature, followed by incubation with primary antibodies against TLR4 (cat. no. 14358, 1:2,000 dilution), nuclear p-p65 (Ser-468, cat. no. 3039, 1:1,000 dilution), p-IκB-α (Ser-32, cat. no. 2859, 1:1,000 dilution), IκB-α (cat. no. 4814, 1:1,000 dilution), Histone H3 (cat. no. 9728, 1:1,000 dilution) and β-actin (cat. no. 3700, 1:1,000) (all from Cell Signaling Technology, Inc.) at 4°C overnight, followed by HRP-conjugated goat anti-rabbit IgG (1:10,000; cat. no. 205718; Abcam). β-actin and Histone H3 served as internal controls.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Construct, Expressing, Western Blot, Standard Deviation, Injection, Control, Mutagenesis

Journal: International Journal of Molecular Medicine

Article Title: MicroRNA-17 contributes to the suppression of the inflammatory response in lipopolysaccharide-induced acute lung injury in mice via targeting the toll-like receptor 4/nuclear factor-κB pathway

doi: 10.3892/ijmm.2020.4599

Figure Lengend Snippet: Overexpression of miR-17 inhibits the NF-κB pathway in ALI mice. The mice were injected intravenously with agomiR-17 or agomiR-NC for 24 h, followed by exposure to LPS. Subsequently, the mice were sacrificed and lung tissues were harvested for analysis. (A) The levels of nuclear p-p65, IκB-α and p-IκB-α were measured by western blot analysis. The bands were semi-quantitatively analyzed using ImageJ software. β-actin protein was used as the internal control of the cytoplasmic proteins; Histone H3 protein was used as the internal control of nuclear proteins. The histogram presents the changes in the relative ratio of the phosphorylated protein levels to the total expression level (phospho/total protein ratio). (B) NF-κB activity was assessed using the NF-κB activity assay. Data are expressed as mean ± standard deviation of three independent experiments (n=5 mice per group). * P<0.05, ** P<0.01, vs. control group; ## P<0.01, vs. LPS + agomiR-17 group. ALI, acute lung injury; NF-κB, nuclear factor-κB.

Article Snippet: The membrane was blocked with 5% skimmed milk for 2 h at room temperature, followed by incubation with primary antibodies against TLR4 (cat. no. 14358, 1:2,000 dilution), nuclear p-p65 (Ser-468, cat. no. 3039, 1:1,000 dilution), p-IκB-α (Ser-32, cat. no. 2859, 1:1,000 dilution), IκB-α (cat. no. 4814, 1:1,000 dilution), Histone H3 (cat. no. 9728, 1:1,000 dilution) and β-actin (cat. no. 3700, 1:1,000) (all from Cell Signaling Technology, Inc.) at 4°C overnight, followed by HRP-conjugated goat anti-rabbit IgG (1:10,000; cat. no. 205718; Abcam). β-actin and Histone H3 served as internal controls.

Techniques: Over Expression, Injection, Western Blot, Software, Control, Expressing, Activity Assay, Standard Deviation