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

Journal: Cancer Science

Article Title: The human endonuclease III enzyme is a relevant target to potentiate cisplatin cytotoxicity in Y‐box‐binding protein‐1 overexpressing tumor cells

doi: 10.1111/j.1349-7006.2008.00739.x

Figure Lengend Snippet: Interaction of different domains of Y‐box‐binding protein‐1 (YB‐1) with human endonuclease III (hNTH1) in total cell extract. (a) Immunoblot against hNTH1 proteins bound to different GST‐YB‐1 affinity Sepahrose beads. Human MCF7 whole cell extracts (WCE) were incubated with either 50 µg of GST‐YB‐1 or GST‐linked glutathione‐sepharose beads overnight. Proteins bound to the affinity beads were analyzed by SDS‐PAGE with antibodies against hNTH1. (b) Schematic representation of different YB‐1 polypeptides that were used in the YB‐1 affinity chromatography experiments. The black box is the cold shock domain and the gray boxes are the basic/acidic cluster domains. The amino acid residues of the YB‐1 fragments used in this study are indicated on the left. hNTH1 binding is indicated on the right by the ‘+’ sign. The ‘–’ sign indicates no binding detected. (c) Coomassie staining of a gel containing purified hNTH1 after the nickel column step (ProBond resin) and after fractionation on Superdex‐200 (fraction number 29). The molecular weight, in kDa, is indicated on the left. (d) Western blot of purified hNTH1 after the final Superdex‐200 step (fraction 29) with an antibody against hNTH1. (e) Interaction of purified hNTH1 (after the final Superdex‐200 step) with 50 µg of GST‐YB‐1, GST‐CSD (cold shock domain of YB‐1; amino acids 51–129), or GST‐linked glutathione‐sepharose beads.

Article Snippet: The pCMV‐YB‐1 expression vector or the pSuperYB‐1 plasmid were transfected into MCF7 cells with the Amaxa nucleofector kit (Amaxa Biosystems, Gaithersburg, MD, USA) when indicated in the figure legends.

Techniques: Binding Assay, Western Blot, Incubation, SDS Page, Affinity Chromatography, Staining, Purification, Nickel Column, Fractionation, Molecular Weight

Journal: Cancer Science

Article Title: The human endonuclease III enzyme is a relevant target to potentiate cisplatin cytotoxicity in Y‐box‐binding protein‐1 overexpressing tumor cells

doi: 10.1111/j.1349-7006.2008.00739.x

Figure Lengend Snippet: Interaction of the auto‐inhibitory domain of human endonuclease III (hNTH1) with the Y‐box‐binding protein‐1 (YB‐1) protein. (a) Immunoblot against YB‐1 proteins bound to different GST‐hNTH1 affinity Sepahrose beads. Human MCF7 cell extracts (WCE) were incubated with either 50 µg of GST‐hNTH1 constructs or GST‐linked glutathione‐sepharose beads overnight. Proteins bound to the affinity beads were analyzed by SDS‐PAGE with antibodies against YB‐1. (b) IVTT/GST interaction assay. In vitro‐transcribed/translated 35S‐labeled hNTH1 protein fragments bound to YB‐1‐GST affinity beads were separated by SDS‐PAGE and visualized using a PhosphoImager. (c) Schematic representation of the different hNTH1 polypeptides that were used in the hNTH1 affinity chromatography experiments. The black box is the auto‐inhibitory domain and the gray box is the catalytic domain. The amino acid residues of the hNTH1 fragments used in this study are indicated on the left. YB‐1 binding is indicated on the right by the ‘+’ sign. The ‘–’ sign indicates no binding detected.

Article Snippet: The pCMV‐YB‐1 expression vector or the pSuperYB‐1 plasmid were transfected into MCF7 cells with the Amaxa nucleofector kit (Amaxa Biosystems, Gaithersburg, MD, USA) when indicated in the figure legends.

Techniques: Binding Assay, Western Blot, Incubation, Construct, SDS Page, In Vitro, Labeling, Affinity Chromatography

Journal: Cancer Science

Article Title: The human endonuclease III enzyme is a relevant target to potentiate cisplatin cytotoxicity in Y‐box‐binding protein‐1 overexpressing tumor cells

doi: 10.1111/j.1349-7006.2008.00739.x

Figure Lengend Snippet: Co‐precipitation of human endonuclease III (hNTH1) protein with TAP‐YB‐1 protein under different DNA‐damaging conditions. (a) Detection of endogenous hNTH1 and Y‐box‐binding protein‐1 (YB‐1) proteins in whole cell lystes after transfection of TAP‐YB‐1 construct into MCF7 cells. (b) Detection of TAP‐YB‐1 and hNTH1 proteins after streptavidin affinity precipitation of the TAP constructs. (c–f) Detection of TAP‐YB‐1 and hNTH1 proteins after streptavidin affinity precipitation of the TAP constructs in untreated cells and in MCF7 cells treated (4 h) with either increasing doses of cisplatin (c), UV light (d), mitomycin C (e), or camptothecin (f). Under each Western blot, scanning analyses of the Western blots (from two independent experiments) are presented. Data are expressed as the mean (±SD) ratio of hNTH1 signals over the affinity‐precipitated TAP‐YB‐1 signals.

Article Snippet: The pCMV‐YB‐1 expression vector or the pSuperYB‐1 plasmid were transfected into MCF7 cells with the Amaxa nucleofector kit (Amaxa Biosystems, Gaithersburg, MD, USA) when indicated in the figure legends.

Techniques: Binding Assay, Transfection, Construct, Affinity Precipitation, Western Blot

Journal: Cancer Science

Article Title: The human endonuclease III enzyme is a relevant target to potentiate cisplatin cytotoxicity in Y‐box‐binding protein‐1 overexpressing tumor cells

doi: 10.1111/j.1349-7006.2008.00739.x

Figure Lengend Snippet: Impact of depleting either endogenous Y‐box‐binding protein‐1 (YB‐1) or human endonuclease III (hNTH1) proteins in MCF7 on survival after different DNA‐damaging conditions. (a) Western blots showing intracellular levels of YB‐1 48 h after transfection with a shRNA specific to YB‐1 or with a control construct. The lower molecular weight band is an unspecific protein recognized by the anti‐YB‐1 antibody. (b–e) MTT cell survival assays of MCF7 cells transfected with a shRNA specific to YB‐1 or with a control construct after of treatment with either cisplatin (b), UV light (c), camptothecin (d), or mitomycin C (e). Cells were first transfected with the appropriate constructs. Forty‐eight hours later, cells were plated in 96‐well plates. Cells were then treated with the drugs for 16 h in culture before the MTT assays. Results represent the mean ± SD of triplicate experiments. (f) Western blots showing intracellular levels of hNTH1 after 48 h after transfection with a siRNA specific to hNTH1 or with a scrambled control siRNA. The anti β‐actin was used as loading control for the blot. (g–j) MTT cell survival assays of MCF7 cells transfected with a siRNA specific to hNTH1 or with a scrambled control siRNA after treatment with either (g) cisplatin, (h) UV light, (i) camptothecin, or (j) mitomycin C. Cells were first transfected with the siRNAs. Forty‐eight hours later, cells were plated in 96‐well plates. Cells were then treated with the drugs for 16 h in culture before the MTT assays. Results represent the mean ± SD of triplicate experiments.

Article Snippet: The pCMV‐YB‐1 expression vector or the pSuperYB‐1 plasmid were transfected into MCF7 cells with the Amaxa nucleofector kit (Amaxa Biosystems, Gaithersburg, MD, USA) when indicated in the figure legends.

Techniques: Binding Assay, Western Blot, Transfection, shRNA, Construct, Molecular Weight

Journal: Cancer Science

Article Title: The human endonuclease III enzyme is a relevant target to potentiate cisplatin cytotoxicity in Y‐box‐binding protein‐1 overexpressing tumor cells

doi: 10.1111/j.1349-7006.2008.00739.x

Figure Lengend Snippet: Impact of depleting endogenous human endonuclease III (hNTH1) proteins in Y‐box‐binding protein‐1 (YB‐1) overexpressing MCF7 cisplatin‐resistant cells. MCF7 cells were transfected with a siRNA specific to hNTH1 or scrambled control siRNA. Forty‐eight hours later, cells were transfected with YB‐1 expression vector or empty expression control vector. (a) Transfected cells were treated with 4 µM cisplatin for 16 h before counting with a hemacytometer (*P < 0.02; P < 0.05). The ‘–’ sign indicates scrambled siRNA or empty expression vector. The ‘+’ sign indicates sihNTH1 or YB‐1 expression vector. Experiments were done in triplicates. (b) Transfected cells were plated in 96‐well plates. Cells were then treated with the drugs for 16 h in culture before the MTT assays. Results represent the mean ± SD of the triplicate experiments.

Article Snippet: The pCMV‐YB‐1 expression vector or the pSuperYB‐1 plasmid were transfected into MCF7 cells with the Amaxa nucleofector kit (Amaxa Biosystems, Gaithersburg, MD, USA) when indicated in the figure legends.

Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation

Journal: Cancer Science

Article Title: The human endonuclease III enzyme is a relevant target to potentiate cisplatin cytotoxicity in Y‐box‐binding protein‐1 overexpressing tumor cells

doi: 10.1111/j.1349-7006.2008.00739.x

Figure Lengend Snippet: Cell growth of MCF7 cells overexpressing Y‐box‐binding protein‐1 (YB‐1). (a) Example of Western blots showing intracellular levels of YB‐1 48 h after transfection with either an empty expression vector or a YB‐1 expression vector. The anti β‐actin is used as loading control for the blot (bottom panel). On average, transfection of YB‐1 expression vector increased the levels of YB‐1 proteins by two‐fold. (b) Cell growth of transfected MCF7 cells. Cells were transfected with either an empty expression vector or a YB‐1 expression vector by electroporation with a nucleofector kit (see ‘Materials and Methods’ for details). Approximately, 3 × 105 transfected cells were plated on 90‐mm Petri dishes. Cells were counted with an hemocytometer 24 and 48 h after the transfections. Results represent the mean ± SD of four independent transfection experiments. All cultures reached confluence at 72 h (data not shown).

Article Snippet: The pCMV‐YB‐1 expression vector or the pSuperYB‐1 plasmid were transfected into MCF7 cells with the Amaxa nucleofector kit (Amaxa Biosystems, Gaithersburg, MD, USA) when indicated in the figure legends.

Techniques: Binding Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Electroporation