Journal: CNS Neuroscience & Therapeutics

Article Title: Thioredoxin‐interacting protein induced α‐synuclein accumulation via inhibition of autophagic flux: Implications for Parkinson's disease

doi: 10.1111/cns.12721

Figure Lengend Snippet: Thioredoxin‐interacting protein (TXNIP) increased in the midbrain of A53T mice and in Parkinson's disease (PD) cellular model. (A) Western blot analysis of TXNIP in 5‐month‐old WT and A53T mice. (B) Western blot analysis of TXNIP in HEK293 cells transfected with α‐synuclein plasmid. (C) The supernatant of HEK293 cells transfected with vector or α‐synuclein plasmid was collected to stimulate SH‐SY5Y cells for 48 hours. Then, TXNIP was detected by Western blot. HEK293 cells were cultured in 6‐well or 96‐well plates and transfected with TXNIP or α‐synuclein plasmid for 48 hours. (D) Western blot analysis of TXNIP. (E) Cytotoxicity was measured by MTT conversion. (F) Cell apoptosis was detected by TUNEL staining. *P<.05, **P<.01 vs WT mice or vector‐transfected cells. n=4 in (A); n=3 in (B‐F). Scale bar, 50 μm

Article Snippet: Plasmids and transfection The plasmids expressing TXNIP, RFP‐LC3, and ATP13A2 were purchased from GenePharma (Suzhou, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, Cell Culture, TUNEL Assay, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Thioredoxin‐interacting protein induced α‐synuclein accumulation via inhibition of autophagic flux: Implications for Parkinson's disease

doi: 10.1111/cns.12721

Figure Lengend Snippet: Thioredoxin‐interacting protein (TXNIP) blocked autophagic flux. (A) HEK293 cells were cotransfected with RFP‐LC3/TXNIP plasmids. LC3 spots were detected by fluorescence microscopy. To inhibit lysosome, HEK293 cells were treated with 20 μmol L−1 NH 4Cl from 7 to 48 hours after TXNIP transfection. (B‐D) LC3, p62, p‐AMPK, AMPK, and Beclin 1 were determined by Western blot analysis. *P<0.05, **P<0.01 vs vector‐transfected cells. n=3. Scale bar, 10 μm

Article Snippet: Plasmids and transfection The plasmids expressing TXNIP, RFP‐LC3, and ATP13A2 were purchased from GenePharma (Suzhou, China).

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Plasmid Preparation

Journal: CNS Neuroscience & Therapeutics

Article Title: Thioredoxin‐interacting protein induced α‐synuclein accumulation via inhibition of autophagic flux: Implications for Parkinson's disease

doi: 10.1111/cns.12721

Figure Lengend Snippet: ATP13A2 attenuated α‐synuclein accumulation induced by Thioredoxin‐interacting protein (TXNIP). (A) HEK293 cells were transfected with α‐synuclein plasmid. α‐Synuclein was detected by Western blot analysis. (B) TXNIP and ATP13A2 were cotransfected into α‐syn‐transfected HEK293 cells to explore their effects on α‐synuclein accumulation. α‐Synuclein was detected by Western blot analysis. *P<0.05 vs vector‐transfected cells; # P<0.05 vs TXNIP‐transfected cells. n=3

Article Snippet: Plasmids and transfection The plasmids expressing TXNIP, RFP‐LC3, and ATP13A2 were purchased from GenePharma (Suzhou, China).

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Thioredoxin‐interacting protein induced α‐synuclein accumulation via inhibition of autophagic flux: Implications for Parkinson's disease

doi: 10.1111/cns.12721

Figure Lengend Snippet: ATP13A2 improved dysfunction of autophagy induced by Thioredoxin‐interacting protein (TXNIP). (A‐C) ATP13A2, LC3, and p62 were detected by Western blot analysis. *P<0.05, **P<0.01 vs vector‐transfected cells; # P<0.05 vs TXNIP‐transfected cells. n=3

Article Snippet: Plasmids and transfection The plasmids expressing TXNIP, RFP‐LC3, and ATP13A2 were purchased from GenePharma (Suzhou, China).

Techniques: Western Blot, Plasmid Preparation, Transfection

Journal: CNS Neuroscience & Therapeutics

Article Title: Thioredoxin‐interacting protein induced α‐synuclein accumulation via inhibition of autophagic flux: Implications for Parkinson's disease

doi: 10.1111/cns.12721

Figure Lengend Snippet: Thioredoxin‐interacting protein (TXNIP) resulted in DA neuron loss in mouse midbrain and induced autophagic dysfunction. (A) TH immunohistochemistry and stereological counts of TH‐positive cells in the midbrain. (B‐D) TXNIP, LC3, p62, ATP13A2, and α‐synuclein were detected by Western blot analysis. *P<.05 vs control mice. n=4. Scale bar, 200 μm

Article Snippet: Plasmids and transfection The plasmids expressing TXNIP, RFP‐LC3, and ATP13A2 were purchased from GenePharma (Suzhou, China).

Techniques: Immunohistochemistry, Western Blot, Control

Journal: Nanomaterials

Article Title: Synergistic Effect of SiO 2 and Fe 3 O 4 Nanoparticles in Autophagy Modulation

doi: 10.3390/nano14121033

Figure Lengend Snippet: LC3 transfected cells were used for the cell experiments. ( A ) Levels of the LC3 gene. ( B ) Testing the cytotoxicity of different concentrations (0–100 µg/mL) of Fe 3 O 4 , SiO 2 , and their combination. ( C ) ROS levels at 50 µg/mL for Fe 3 O 4 , SiO 2 , and their mixture. The data are reported as mean ± SD ( n = 3). The data were standardized using a negative control that did not involve nanoparticle treatment. * p < 0.05, ** p < 0.005, **** p < 0.0001.

Article Snippet: LC3 transfection was conducted using GFP–LC3 Expression Vector (CELL BIOLABS, San Diego, CA, USA, CBA-401).

Techniques: Transfection, Negative Control

Journal: Nanomaterials

Article Title: Synergistic Effect of SiO 2 and Fe 3 O 4 Nanoparticles in Autophagy Modulation

doi: 10.3390/nano14121033

Figure Lengend Snippet: Alteration of LC3 treatment using nanoparticles. ( A ) Western blot images of nanoparticles treated at 50 µg/mL for 24 h. ( B ) Relative band intensity of the western blot. ( C ) Expression levels of LC3 gene at 50 µg/mL for Fe 3 O 4 , SiO 2 , and their combination. All data are presented as mean ± SD ( n = 3). The data were normalized using a negative control that did not involve nanoparticle treatment. * p < 0.05.

Article Snippet: LC3 transfection was conducted using GFP–LC3 Expression Vector (CELL BIOLABS, San Diego, CA, USA, CBA-401).

Techniques: Western Blot, Expressing, Negative Control

Journal: Nanomaterials

Article Title: Synergistic Effect of SiO 2 and Fe 3 O 4 Nanoparticles in Autophagy Modulation

doi: 10.3390/nano14121033

Figure Lengend Snippet: Fluorescence analysis of LC3 marker (green) and nucleus (blue, DAPI) in Fe 3 O 4 , SiO 2 , and their combination nanoparticles. ( A ) Fluorescence images ( B ) The intensity of the fluorescence signal Scale bar: 20 µm. The intensity was calculated dividing green into blue, and data were normalized using a negative control that did not involve nanoparticle treatment. ** p < 0.005.

Article Snippet: LC3 transfection was conducted using GFP–LC3 Expression Vector (CELL BIOLABS, San Diego, CA, USA, CBA-401).

Techniques: Fluorescence, Marker, Negative Control

Journal: Molecular Medicine Reports

Article Title: Liraglutide protects pancreatic β-cells against free fatty acids in vitro and affects glucolipid metabolism in apolipoprotein E −/− mice by activating autophagy

doi: 10.3892/mmr.2015.3944

Figure Lengend Snippet: LRG protects INS-1 cells by activating autophagy. (A) INS-1 cells were transfected with the GFP-LC3 expression plasmid and then treated with normal saline or LRG. Cells were observed under fluorescence microscopy (magnification, ×200). (B) INS-1 cells were transfected with the GFP-LC3 expression plasmid and then treated with different concentrations of LRG: L1, 10; L2, 20; L3, 40 or or L4, 80 µ mol/l for 36 h. The LC3 protein was detected using immunoblotting. (C) Gray scanning of LC3A and LC3B normalized to that of GAPDH. (D) INS-1 cells were transfected with the GFP-LC3 expression plasmid and were then treated with different concentrations of LRG for 36 h. p62, The ATG7 and Beclin1 proteins were detected using immunoblotting and gray scanning, normalized to that of GAPDH; (E) ECM images of INS-1 cells in the CON, FFA, LRG and FFA + LRG groups. Arrows indicate autophagosomes (magnification, ×8,000). LRG, liraglutide; GFP, green fluorescent protein; LC3, light chain 3; CON, control; FFA, free fatty acids.

Article Snippet: The LC3 expression plasmid containing GFP, provided by Professor Jin Shengkan at the University of Medicine and Dentistry of New Jersey (New Brunswick, NJ, USA) was transfected into the INS-1 cells in the different treatment groups using LipofectamineTM 2000 Transfection reagent (Gibco Life Technologies).

Techniques: Transfection, Expressing, Plasmid Preparation, Saline, Fluorescence, Microscopy, Western Blot, Control

Journal: Journal of Inflammation Research

Article Title: Effect of Vitamin D Deficiency and Supplementation in Lactation and Early Life on Allergic Airway Inflammation and the Expression of Autophagy-Related Genes in an Ovalbumin Mouse Model

doi: 10.2147/JIR.S321642

Figure Lengend Snippet: Expression of LC3 and Beclin-1 protein in the lung tissue of OVA-sensitized and challenged mice by IH and IF. ( A ) Lung tissues were subjected to immunohistochemical analysis with LC3 and Beclin-1 antibody. LC3 and Beclin-1 immunohistochemical images ( A , B ) and H-score ( C , D ) are shown (immunoreactivity was detected primarily in the cytoplasm of alveolar epithelial cells which indicated by a black arrow, macrophages by a red arrow, and the epithelial cells at the apical region of the airway by a blue arrow). ( E ) Lung tissues were subjected to immunofluorescence analysis with LC3 and Beclin-1 antibody. LC3 and Beclin-1 immunofluorescence image ( E ) and quantification of fluorescence intensity ( F – H ) are shown. The merged signals of LC3 and Beclin-1 in the immunofluorescence images are marked with yellow and a typical merged signal is indicated by a white arrow. The percentage of co-expression cells was calculated. H-score and the percentage of LC3 and Beclin-1 immunofluorescence expression were quantified and analyzed by ANOVA and the Tukey post-hoc test. Results are expressed as mean ± SD of seven to nine animals in each group (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: LC3 and Beclin-1 expression was also examined by double immunofluorescence staining of LC3 and Beclin-1 (GB13431 and GB11228, respectively, rabbit, Servicebio, Wuhan, China) on slides of lung tissues.

Techniques: Expressing, Immunohistochemical staining, Immunofluorescence, Fluorescence

Journal: Journal of Inflammation Research

Article Title: Effect of Vitamin D Deficiency and Supplementation in Lactation and Early Life on Allergic Airway Inflammation and the Expression of Autophagy-Related Genes in an Ovalbumin Mouse Model

doi: 10.2147/JIR.S321642

Figure Lengend Snippet: Expression of LC3B, Beclin-1, ATG5, NF-κB p65, and LL37 in the lung tissue of OVA-sensitized/challenged mice. ( A – E ) Total cellular RNA was extracted from the lung tissue and then analyzed by real-time PCR. Data are shown as mean ± SEM for seven to nine animals in each group. ( F , L ) Lung tissue lysates from different vitamin D diet groups of mice (n = 6) were subjected to immunoblot analysis with LC3B, Beclin-1, ATG5, NF-κB p65, and ACTIN antibodies. ( G – I , K , L ) The intensity of the indicated antibody’s bands from three independent experiments were quantified (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: LC3 and Beclin-1 expression was also examined by double immunofluorescence staining of LC3 and Beclin-1 (GB13431 and GB11228, respectively, rabbit, Servicebio, Wuhan, China) on slides of lung tissues.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Inflammation Research

Article Title: Effect of Vitamin D Deficiency and Supplementation in Lactation and Early Life on Allergic Airway Inflammation and the Expression of Autophagy-Related Genes in an Ovalbumin Mouse Model

doi: 10.2147/JIR.S321642

Figure Lengend Snippet: In OVA-sensitized/challenged mice, the level of LC3B mRNA was correlated with inflammatory markers in bronchoalveolar lavage fluid (BALF). The correlation was analyzed by Spearman correlation. ( A , B ) Correlation analysis between LC3B mRNA in lung tissue and BAL total cell number or eosinophils. ( C , D ) Correlation analysis between LC3B mRNA and levels of cytokines in BALF ( p < 0.05).

Article Snippet: LC3 and Beclin-1 expression was also examined by double immunofluorescence staining of LC3 and Beclin-1 (GB13431 and GB11228, respectively, rabbit, Servicebio, Wuhan, China) on slides of lung tissues.

Techniques:

Journal: Journal of Inflammation Research

Article Title: Effect of Vitamin D Deficiency and Supplementation in Lactation and Early Life on Allergic Airway Inflammation and the Expression of Autophagy-Related Genes in an Ovalbumin Mouse Model

doi: 10.2147/JIR.S321642

Figure Lengend Snippet: 1,25(OH) 2 D 3 suppressed LC3B, Beclin-1, ATG5, and NF-κB p65, and increased LL37 mRNA expression in macrophages collected from BAL in OVA-sensitized/challenged mice with sufficient dietary vitamin D. ( A – E ) LC3B, Beclin-1, ATG5, NF-κB p65, and LL37 mRNA expression in macrophages with 1,25(OH) 2 D 3 in various doses (0.2, 2, 20, and 200 nM) or time course (12 or 24 h) were detected by rt-PCR normalized to GAPDH. Data are representative of at least three independent experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001; # p < 0.05, ## p < 0.01, and ### p < 0.001).

Article Snippet: LC3 and Beclin-1 expression was also examined by double immunofluorescence staining of LC3 and Beclin-1 (GB13431 and GB11228, respectively, rabbit, Servicebio, Wuhan, China) on slides of lung tissues.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction