Journal: bioRxiv

Article Title: Deciphering the Genome Protection Roles of Autophagy in Primary Human Dermal Fibroblasts (HDFs) against Ultraviolet-(B) –Induced Skin Photodamage

doi: 10.1101/2020.09.28.316273

Figure Lengend Snippet: (3A-3E) Western blotting analysis of key autophagy marker proteins showing time and intensity dependent response of autophagy in UV-B 10, 20 and 30mJ/cm 2 exposed HDFs. (3F & 3G) GFP-RFP-LC3B PUNCTA assay for quantification of puncta (+) cells in UV-B 30mJ/cm 2 exposed HDFs and effect of Rapamycin (100nM) and Chloroquine (50µM) on autophagy response in 6h UV-B post-irradiation. (3H & 3I) Immunoflorescence analysis of autophagy proteins P62 and LC3B and DNA damage marker proteins pχH2AX and pP53 in UV-B 30mJ/cm 2 exposed HDFs in 1h UV-B post-irradiation in florescent microscopy. (3J & 3K) Reactive Oxygen Species (ROS) estimation in UV-B 30mJ/cm 2 exposed HDFs treated with Salubrinal (25µM) and Everolimus (200nM) in 1h UV-B post-irradiation. (**p≤ 0.01, #p≤ 0.001, were considered statistically significant).

Article Snippet: Fura 3 AM, DAPI, ER tracker, Acridine Orange, Ethidium Bromide, Salubrinal, Rapamycin, Chloroquine, Bafilomycin A1, Everolimus, GFP RFP LC3B Puncta kit from (Thermo Scientific).

Techniques: Western Blot, Marker, Irradiation, Microscopy

Journal: Molecular and cellular biochemistry

Article Title: Early-life bisphenol A exposure causes neuronal pyroptosis in juvenile and adult male rats through the NF-κB/IL-1β/NLRP3/caspase-1 signaling pathway: exploration of age and dose as effective covariates using an in vivo and in silico modeling approach.

doi: 10.1007/s11010-024-05039-4

Figure Lengend Snippet: Fig. 8 The molecular docking analysis of BPA and neuro- inflammatory, autophagic as well as, pyroptotic molecules displaying 2D and 3D binding interactions of BPA [PubChem CID: 6623] against A NF-kB [PDB ID: 1NFK], B IL-1β [PDB ID: 2MIB], C IL-2 [PDB ID: 4YQX], D IL-12 [PDB ID: 3HMX], E COX-2 [PDB ID: 1PXX], F NLRP3 [PDB ID: 7vtq], G Beclin-1 [PDB ID: 2PON], H LC3A [PDB ID: 6TBE], I LC3B [PDB ID: 5XAC], J Caspase-1 [PDB ID: 6VIE]. The green dotted lines denote hydrogen bonds between ligand and aminoacids, whereas bink/purple dotted lines repre- sent hydrophobic interactions. Electrostatic interactions are shown as orange dotted lines. The red dotted line indicate an unfavorable donor-donor

Article Snippet: Fine Test (cat no. ER1965) provided the NLRP3 kit, LSBio (cat no. LS-F9917; LS-F19802) provided the LC3A and LC3B kits, respectively, and Cusabio (cat no. CSB-EL002658RA provided the beclin-1 kit.

Techniques: Binding Assay

Journal: Autophagy

Article Title: MIR517C inhibits autophagy and the epithelial-to-mesenchymal (-like) transition phenotype in human glioblastoma through KPNA2-dependent disruption of TP53 nuclear translocation.

doi: 10.1080/15548627.2015.1108507

Figure Lengend Snippet: Figure 2. Induction of autophagy by TMZ depended on the phenotype of TP53. (A) Western blot analysis of LC3B-II in U87 and U251 cells after exposure to different concentrations of TMZ. The lower histogram shows densitometric analysis of western blot results (#, P > 0.05; *, P < 0.05; **, P < 0.001). (B) Effects of TMZ, 3-MA, and CQ on the conversion of LC3B-II in U87 and U251 cells. The lower histogram shows densitometric analysis of western blot results (#, P > 0.05; *, P < 0.05; **: P < 0.001). (C) Immunofluorescence analysis of LC3B in U87 cells after treatment with TMZ, 3-MA, and CQ. The LC3B particles were quantized by NIH ImageJ software (*, P < 0.05; **, P < 0.001). (D) Representative transmission electron microscope (TEM) images of U87 and U251 cells after exposure to 0 or 300 mM TMZ. Red arrows indicate formation of autophagic vacuoles. (E) Expression of autophagy-related proteins in U87 cells treated with TMZ and TP53 siRNA. The histogram shows densitometric analysis of western blot results (#, P > 0.05; *, P < 0.05; **, P < 0.001). (F) TEM images of U87 cells after exposure to TMZ and TP53 siRNA. Red arrows indicate formation of autophagic vacuoles.

Article Snippet: After the blocking solution was removed, cells were incubated with primary antibodies targeting LC3B (1:200; Cell Signaling Technology, 4445) or TP53 (1:1500; Cell Signaling Technology, 9282) diluted in KB for 60 min at 37 C. Cells were then washed with KB twice.

Techniques: Western Blot, Software, Transmission Assay, Microscopy, Expressing

Journal: Autophagy

Article Title: MIR517C inhibits autophagy and the epithelial-to-mesenchymal (-like) transition phenotype in human glioblastoma through KPNA2-dependent disruption of TP53 nuclear translocation.

doi: 10.1080/15548627.2015.1108507

Figure Lengend Snippet: Figure 4. ATG7 siRNA inhibited cell migration, infiltration, and the expression of EMT-related proteins in U87 cells but not in U251 cells. (A) Representa- tive phase-contrast microscope images of U87 cells with ATG7 siRNA and NC, which were cultured in TMZ (150 mM) medium. The related parameters (length, width, and area of cells and length of pseudopodia) were measured by NIH ImageJ software. (B) Wounding healing assay of U87 cells with ATG7 siRNA and NC in normal medium (top) and in TMZ (bottom). Quantification of the percentage of cell wound closure in U87 and U251 cells (*, P < 0.05;, **: P < 0.001). (C) The infiltrative ability of U87 cells with ATG7 siRNA and NC was measured. The right histogram represents the cell count of infiltrative cells in 6 high-resolution fields for 3 independent experiments. Error bars represent SDs. (D) Western blotting was used to detect the expression of autophagy- and EMT-related proteins in U87 cells transiently transfected with ATG7 siRNA in normal medium or in the presence of TMZ. Densitometric analysis of the western blots shown in the right histogram (#, P > 0.05; *, P < 0.05; **, P < 0.001). (E) Western blotting was used to detect the expression of autophagy- and EMT-related proteins in U251 cells transiently transfected with ATG7 siRNA in normal medium or in the presence of TMZ (150 or 300 mM). Densitometric analysis of the western blots shown in the right histogram (#, P > 0.05; **, P < 0.001). (F) siRNAs targeting ATG7 and TP53 were cotransfected into U87 cells, and LC3B conversion was detected by western blotting. Densitometric analysis is shown in the right histogram (#, P > 0.05; *, P < 0.05; **, P < 0.001).

Article Snippet: After the blocking solution was removed, cells were incubated with primary antibodies targeting LC3B (1:200; Cell Signaling Technology, 4445) or TP53 (1:1500; Cell Signaling Technology, 9282) diluted in KB for 60 min at 37 C. Cells were then washed with KB twice.

Techniques: Migration, Expressing, Microscopy, Cell Culture, Software, Cell Counting, Western Blot, Transfection

Journal: Autophagy

Article Title: MIR517C inhibits autophagy and the epithelial-to-mesenchymal (-like) transition phenotype in human glioblastoma through KPNA2-dependent disruption of TP53 nuclear translocation.

doi: 10.1080/15548627.2015.1108507

Figure Lengend Snippet: Figure 6. MIR517C inhibited TMZ-induced autophagy and the EMT. (A) Western blot analysis was used to detect the expression of key autophagy-related proteins in U87 and U251 cells following overexpression (oe) or knockdown (kn) of MIR517C. NC cells were used as a control. (B) Levels of EMT-related proteins were detected by western blotting in U87 and U251 cells following overexpression (oe) or knockdown (kn). NC cells were used as a control. (C) Densitometric analysis of the western blots shown in (A). #, P > 0.05; *, P < 0.05; **, P < 0.001. (D) Densitometric analysis of the western blots shown in (B). #, P > 0.05; *, P < 0.05; **, P < 0.001. (E) Immunofluorescence analysis of LC3B expression in U87 and U251 cells following overexpression (oe) or knockdown (kn) of MIR517C. LC3B particle data were subjected to statistical analysis (#, P > 0.05; *, P < 0.05; **, P < 0.001). (F) Representative transmis- sion electron microscope images of U87 and U251 cells following overexpression (oe) or knockdown (kn) of MIR517C. NC cells were used as a control. Red arrows indicate formation of autophagosomes, and green arrows indicate autolysosomes.

Article Snippet: After the blocking solution was removed, cells were incubated with primary antibodies targeting LC3B (1:200; Cell Signaling Technology, 4445) or TP53 (1:1500; Cell Signaling Technology, 9282) diluted in KB for 60 min at 37 C. Cells were then washed with KB twice.

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Control, Microscopy

Journal: Autophagy

Article Title: MIR517C inhibits autophagy and the epithelial-to-mesenchymal (-like) transition phenotype in human glioblastoma through KPNA2-dependent disruption of TP53 nuclear translocation.

doi: 10.1080/15548627.2015.1108507

Figure Lengend Snippet: Figure 7. MIR517C inhibited autophagy through targeting of KPNA2. (A) Two-dimensional electrophoresis analysis of U87 cells transfected with MIR517C mimics or NC. Distinct protein spots could be observed (red dotted area, red arrow). The target spot was confirmed as KPNA2 by mass spectrometry anal- ysis. (B) Western blotting confirmed the expression of KPNA2 protein in U87 and U251 cells following overexpression (oe) or knockdown (kn) of MIR517C. NC cells were used as a control. The right histogram shows densitometric analysis (#, P > 0.05; *, P < 0.05; **, P < 0.001). (C) U87 and U251 cells exhibiting KPNA2 overexpression or knockdown were established, and autophagy-related protein levels were measured by western blotting. (D) Densitometric analysis of the western blots shown in (C). #, P > 0.05; *, P < 0.05; **, P < 0.001. (E) Immunofluorescence analysis of LC3B expression in U87 cells following overexpression or knockdown of KPNA2. LC3B particle data were subjected to statistical analysis (*, P < 0.05; **, P < 0.001). (F) Sequence matching between MIR517C and the 30-UTR of KPNA2 is shown. U87 cells were transfected with MIR517C mimic, a mimic control (NC), AntagomiR, or nontargeting AntagomiR (AntagomiR NC). The level of MIR517C was assayed by Taq Man qRT-PCR. (G) Luciferase assays in U87 cells cotransfected with pGL3-control reporter constructs containing the KPNA2 30-UTR MIR517C-binding site (pGL3-KPNA2-UTR) or a mutated binding site (pGL3-KPNA2-UTRmt) and MIR517C mimic, mimic control, AntagomiR, or nontargeting AntagomiR for 72 h. Samples were run in triplicate, and 3 independent experiments were performed.

Article Snippet: After the blocking solution was removed, cells were incubated with primary antibodies targeting LC3B (1:200; Cell Signaling Technology, 4445) or TP53 (1:1500; Cell Signaling Technology, 9282) diluted in KB for 60 min at 37 C. Cells were then washed with KB twice.

Techniques: Electrophoresis, Transfection, Mass Spectrometry, Western Blot, Expressing, Over Expression, Knockdown, Control, Sequencing, Quantitative RT-PCR, Luciferase, Construct, Binding Assay

Journal: Scientific Reports

Article Title: Cerebrospinal Fluid Levels of Autophagy-related Proteins Represent Potentially Novel Biomarkers of Early-Stage Parkinson’s Disease

doi: 10.1038/s41598-018-35376-6

Figure Lengend Snippet: Comparison of the CSF levels of autophagy-related proteins between the PD and control groups. LC3B, Beclin1, ATG5, and LAMP-2 levels were significantly decreased in patients with PD compared with normal controls.

Article Snippet: Similarly, Beclin1, LAMP-2 and LC3B ELISA Kits (MYBioSource, San Diego, CA, USA) were used with a similar method to detect autophagy biomarkers.

Techniques: Comparison, Control

Journal: Scientific Reports

Article Title: Cerebrospinal Fluid Levels of Autophagy-related Proteins Represent Potentially Novel Biomarkers of Early-Stage Parkinson’s Disease

doi: 10.1038/s41598-018-35376-6

Figure Lengend Snippet: ROC curve showing the diagnostic performance of CSF biomarkers. With 29.8 pg/mL as the cut-off value for the differential diagnosis of PD from normal controls, the CSF level of LC3B exhibited high sensitivity (96.9%) and specificity (89.3%), with an AUC of 0.982 (0.959−1.000). ROC, receiver operating characteristic; PD, Parkinson’s disease.

Article Snippet: Similarly, Beclin1, LAMP-2 and LC3B ELISA Kits (MYBioSource, San Diego, CA, USA) were used with a similar method to detect autophagy biomarkers.

Techniques: Diagnostic Assay, Biomarker Discovery

Journal: Scientific Reports

Article Title: Cerebrospinal Fluid Levels of Autophagy-related Proteins Represent Potentially Novel Biomarkers of Early-Stage Parkinson’s Disease

doi: 10.1038/s41598-018-35376-6

Figure Lengend Snippet: Results of the discriminant analysis using the CSF biomarker levels for a differential diagnosis of PD from normal controls.

Article Snippet: Similarly, Beclin1, LAMP-2 and LC3B ELISA Kits (MYBioSource, San Diego, CA, USA) were used with a similar method to detect autophagy biomarkers.

Techniques: Biomarker Discovery

Journal: Scientific Reports

Article Title: Cerebrospinal Fluid Levels of Autophagy-related Proteins Represent Potentially Novel Biomarkers of Early-Stage Parkinson’s Disease

doi: 10.1038/s41598-018-35376-6

Figure Lengend Snippet: Partial correlation analysis of clinical data and FP-CIT PET uptake values in relation to CSF autophagy-related proteins in patients with PD.

Article Snippet: Similarly, Beclin1, LAMP-2 and LC3B ELISA Kits (MYBioSource, San Diego, CA, USA) were used with a similar method to detect autophagy biomarkers.

Techniques:

Journal: Scientific Reports

Article Title: Cerebrospinal Fluid Levels of Autophagy-related Proteins Represent Potentially Novel Biomarkers of Early-Stage Parkinson’s Disease

doi: 10.1038/s41598-018-35376-6

Figure Lengend Snippet: Correlation of LC3B level with quantitative FP-CIT PET data. Among the autophagy-related proteins, LC3B showed significant correlations with AIs in both the caudate and putamen, regardless of age.

Article Snippet: Similarly, Beclin1, LAMP-2 and LC3B ELISA Kits (MYBioSource, San Diego, CA, USA) were used with a similar method to detect autophagy biomarkers.

Techniques:

Journal: GeroScience

Article Title: Basal autophagic flux measured in blood correlates positively with age in adults at increased risk of type 2 diabetes

doi: 10.1007/s11357-023-00884-5

Figure Lengend Snippet: Participant characteristics

Article Snippet: To determine LC3B-II sample concentration, samples were loaded in triplicate on a FastScan Total LC3B ELISA Kit (Cell Signaling Technology, 35172).

Techniques:

Journal: Human molecular genetics

Article Title: Autophagy is increased in laminin α2 chain-deficient muscle and its inhibition improves muscle morphology in a mouse model of MDC1A.

doi: 10.1093/hmg/ddr427

Figure Lengend Snippet: Figure 1. Autophagy is increased in skeletal muscle from dy3K/dy3K mice. (A) Relative amounts of Bnip, Bnip3l, p62, LC3B, Gabarapl1, Atg4b, Vps34, Beclin, Cathepsin L and Lamp2a mRNAs in quadriceps muscle from 3.5-week-old wild-type, dy3K/dy3K mice and from 4.5-week-old 3-MA-injected wild-type and dy3K/dy3K mice (n ¼ 6 for each group). The GAPDH gene expression served as a reference. Non-significant variations are not labeled. (B, left panel) Co-immunostaining on cross-sections of quadriceps muscle from non-injected wild-type (a, n ¼ 5) and dy3K/dy3K (b, n ¼ 5) mice and 3-MA injected wild-type (c, n ¼ 6) and dy3K/dy3K (d and e, n ¼ 6) mice. LC3B (in red) is present in autophagosomes and laminin g1 chain (in green) serves as delineating fiber bound- aries. Bar ¼ 40 mm. (C) Densitometry analysis of LC3B and Vps34 western blot analysis in quadriceps muscle from wild-type and dy3K/dy3K mice (3.5-week-old; n ¼ 6 per group). Results are expressed in arbitrary units (AU). Labeling of tubulin served as internal loading control. (D) Densitometric analysis of LC3B, Vps34, Cathepsin L and Beclin-1 in human primary myoblasts and myotubes from a control and a laminin a2 chain-deficient patient. Data represent the mean of four different culture wells and are expressed in arbitrary units (AU). (E) Co-immunostainings on cross-sections of muscle biopsies from patients with MDC1A and inclusion-body myositis (IBM) and from a healthy control. LC3B (in red) was detected with two different antibodies in serial sections of MDC1A muscle (a and b). IBM muscle was used as a positive control for LC3B accumulation (d), whereas little LC3B staining was seen in the healthy control (c). (F) Relative amounts of Bnip, Bnip3l, p62, LC3B, Gabarapl1, Atg4b, Vps34, Beclin, Cathepsin L and Lamp2a mRNAs in 5-week-old wild-type and dy3KdE3 mice (n ¼ 3 for each group). The GAPDH gene expression served as a reference. ∗P , 0.05; ∗∗P , 0.001.

Article Snippet: Human muscle sections, obtained from muscle biopsies from a 2.5-year-old healthy control; two MDC1A patients (2- and 3.5-year-old, respectively) with complete deficiency of laminin a2 chain and from a 21-year-old patient with inclusion-body myositis, were stained with rabbit monoclonal antibody against LC3B (1/ 100, #3868, Cell Signaling Technology); rabbit polyclonal antibody against LC3B (1/200, #100-2220, Novus Biologicals) and rat monoclonal antibody against perlecan (1/100, MAB 1948, Chemicon).

Techniques: Injection, Gene Expression, Labeling, Immunostaining, Western Blot, Control, Positive Control, Staining