gfp–lc3 expression vector  (Cell Biolabs Inc)

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: American Journal of Cancer Research

Article Title: A Notch inhibitor plus Resveratrol induced blockade of autophagy drives glioblastoma cell death by promoting a switch to apoptosis

doi:

Figure Lengend Snippet: Resveratrol and GSI cotreatment induces the expression of autophagy markers in GBM cells. A. Time course study. U87MG and T98G cells were treated with vehicle (-) or 10 μM RSV plus 2 μM GSI for different times as indicated and Western blot carried out for LCI and LC3II. B. Upper panel. U87MG cells were transiently transfected with LC3-GFP and treated with vehicle (-) or 10 μM RSV plus 2 μM GSI for 18 hours. Images show the results of one representative experiment out of three. Scale bars = 5 μM. Lower panel, Number of LC3-GFP positive puncta manually counted in each of 50 cells/treatment. Data are means of three independent experiments. Columns are mean of three independent experiments. **P≤0.01 vs vehicle. C. Time course study. U87MG and T98G cells were treated with vehicle (-) or 10 μM RSV plus 2 μM GSI for different times as indicated and Western blot carried out for p62/SQSTM1. -S indicates cells maintained in absence of serum for 72 hours. D. Red:green (R/G) fluorescence ratio. U87MG and T98 cells were treated with vehicle (-) or 10 μM RSV plus 2 μM GSI for different times as indicated. Cells were stained and processed for flow cytometric analysis. The numbers represent the fold increase of the red: green fluorescence ratio in treated cells over vehicle controls, and are the mean of triplicate samples from one experiment that was reproduced twice with similar results. *P≤0.05 vs 15’, vs 1 h, vs, 2 h, vs 6 h. E. Staining of T98G cells with acridine orange followed by fluorescence microscopy for appearance of AVO. Images show the results of one representative experiment out of three. DAPI staining was used to visualize the cell nucleus. Scale bars = 25 μM.

Article Snippet: Plasmids CDK4R24C expression vector (Plasmid #11129) was from Addgene, GFP-LC3 Expression Vector (#CBA-401) was from Cell Biolabs, Inc. (San Diego, CA).

Techniques: Expressing, Western Blot, Transfection, Fluorescence, Staining, Microscopy

Journal: RNA Biology

Article Title: Long non-coding RNA TUG1 and its molecular mechanisms in polycystic ovary syndrome

doi: 10.1080/15476286.2020.1783850

Figure Lengend Snippet: Effect of TUG1 knockdown on P21-dependent autophagy. (A and B) The expression levels of P62, LC3-I, LC3-II, and P21 were detected by western blotting in KGN cells at 48 h post-transfection. ImageJ software was used to analyse and normalize band intensity, and protein levels were normalized to the expression of β-tubulin. (C) qRT–PCR quantification of P21 mRNA levels in KGN cells at 48 h post-transfection. GapmeR Ctrl was set to 1. (D) qRT–PCR quantification of P21 mRNA expression levels in total hLGCs obtained from patients with and without PCOS (n = 100 versus 100). (E) Correlations between TUG1 expression and that of P21 were analysed in 200 hLGC samples obtained from patients with PCOS and healthy controls using Pearson’s rank correlation. (F and G) The expression levels of P62, LC3-I, LC3-II, and P21 were detected by western blotting in KGN cells at 48 h post-transfection with GapmeRs and siRNAs. ImageJ software was used to analyse and normalize band intensity, and protein levels were normalized to the expression of GAPDH. (H and I) Confocal laser microscope images of green fluorescent protein (GFP)-LC3 expression (green fluorescence) and DAPI (blue fluorescence) in KGN cells (Scale bars = 10 μm). The numbers of GFP-LC3 puncta were calculated using five randomly selected fields, and were quantified using at least 20 cells per specimen. (J and K) KGN cells transfected with the corresponding GapmeRs were treated for 4 h with or without the lysosomal inhibitor chloroquine (CQ). The expression levels of P62, LC3-I, LC3-II, and P21 were detected by western blotting. ImageJ software was used to analyse and normalize band intensity, and protein levels were normalized to the expression of GAPDH. (L and M) The formation of GFP-LC3 puncta in KGN cells was measured using a confocal laser microscope (Scale bars = 10 μm). The numbers of GFP-LC3 puncta were calculated using five randomly selected fields, and were quantified using at least 20 cells per specimen. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: A green fluorescent protein (GFP)-LC3 expression vector (GeneCopoeia, Rockville, MD, USA) was used to identify the presence of autophagy.

Techniques: Expressing, Western Blot, Transfection, Software, Quantitative RT-PCR, Microscopy, Fluorescence

Journal: Iranian Journal of Medical Sciences

Article Title: Cell Survival Effects of Autophagy Regulation on Umbilical Cord-Derived Mesenchymal Stem Cells Following Exposure to Oxidative Stress

doi: 10.30476/ijms.2019.44947

Figure Lengend Snippet: The transfection of pCMV-GFP-LC3 expression vector into umbilical cord MSCs. (A) pCMV-GFP-LC3 expression vector map containing GFP-fused LC3 gene, Kanamycin resistance gene as well as Geneticin resistance gene. (B) pCMV-GFP-LC3 was transfected into MSCs, known as MSC-GFP-LC3. Green fluorescent of MSC-GFP-LC3 under fluorescence microscope indicated their successful transfection

Article Snippet: pCMV-GFP-LC3 expression vector (Cell Biolabs Inc., USA) was transformed into E. coli strain TOP10 using the cold CaCl2 method.

Techniques: Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy

Journal: Iranian Journal of Medical Sciences

Article Title: Cell Survival Effects of Autophagy Regulation on Umbilical Cord-Derived Mesenchymal Stem Cells Following Exposure to Oxidative Stress

doi: 10.30476/ijms.2019.44947

Figure Lengend Snippet: The determination of optimized Rapa and 3MA dose. MSC-GFP-LC3 was cultivated in the presence of Rapa (autophagy inducer) and 3MA (autophagy inhibitor) separately and was named MSC-LC3-Rapa and MSC-LC3-3MA, respectively. The WST-1 was performed to assay cell survival. (A) Rapa 800 nM dose has cytotoxic effects on MSC0-LC3-Rapa cells, but Rapa 500 nM dose did not lead to severe cell death. (B) 2 mM and 3 mM of 3MA led to low MSC-LC3-3MA viability. 1 mM of 3MA was selected as the optimized non-toxic autophagy inducing dose (P=0.002, P=0.0044, P=0.0007, mean±SEM). The experimental groups were compared with control.

Article Snippet: pCMV-GFP-LC3 expression vector (Cell Biolabs Inc., USA) was transformed into E. coli strain TOP10 using the cold CaCl2 method.

Techniques:

Journal: Iranian Journal of Medical Sciences

Article Title: Cell Survival Effects of Autophagy Regulation on Umbilical Cord-Derived Mesenchymal Stem Cells Following Exposure to Oxidative Stress

doi: 10.30476/ijms.2019.44947

Figure Lengend Snippet: The evaluation of cell survival under normal condition and harsh lethal oxidative stress. (A) The viability of MSC-LC3-Rapa and MSC-LC3-3MA, non-transfected MSCs and MSC-GFP-LC3 (as controls) under normal conditions without any stress was estimated by WST-1 assay. There was no significant difference between the above-mentioned groups in term of cell viability. (B) MSC-LC3-Rapa and MSC-LC3-3MA were exposed to 300 µm/mL of H 2 O 2 as well as control groups. The results of WST-1 assay showed that the MSC-LC3-3MA had a higher survival rate than other groups and inhibition of autophagy strengthened them against severe oxidative stress (P=0.0003, mean±SEM). The MSC-LC3-3MA was compared with MSC-LC3-Rapa.

Article Snippet: pCMV-GFP-LC3 expression vector (Cell Biolabs Inc., USA) was transformed into E. coli strain TOP10 using the cold CaCl2 method.

Techniques: Transfection, WST-1 Assay, Inhibition