Journal: Experimental and Therapeutic Medicine

Article Title: Annexin A2 upregulation protects human retinal endothelial cells from oxygen-glucose deprivation injury by activating autophagy

doi: 10.3892/etm.2019.7909

Figure Lengend Snippet: Role of A2 in OGD-induced activation of autophagy in human retinal endothelial cells. Cells were infected with lentiviral vectors encoding shRNAs against human A2, lentiviral scramble control vectors (vector1), lentiviral A2 expression vectors and lentiviral control vectors (vector2) for 48 h before OGD treatment. Protein levels were detected by western blotting. (A) Expression of Beclin-1, LC3I and LC3II when cells underwent OGD for different periods of time. (B) Transfection efficiency of sh-A2. (C) Expression of Beclin-1, LC3I and LC3II following knockdown of A2. (D) Transfection efficiency of OE-A2. (E) Expression of Beclin-1, LC3I and LC3II after A2 overexpression. *P<0.05 and **P<0.01 vs. Con group; # P<0.05 and ## P<0.01 vs. OGD group. A2, Annexin A2; sh-A2, lentiviral vectors encoding short hairpin RNAs against human A2; OE-A2, lentiviral A2 overexpression vectors; OGD, oxygen-glucose deprivation; Con, control.

Article Snippet: Lentiviral vectors encoding shRNAs against human A2 (sh-A2, sequence 5′-TGAGGGTGACGTTAGCATTAC-3′ for A2 shRNA), lentiviral control vectors (vector1, sequence 5′-GGATCATCATGCTATGCAGTT-3′ for control scramble shRNA), lentiviral A2 overexpression vectors (OE-A2, primers used as follows, sense: 5′-CGGGGTACCATGTCTACTGTTCACGAAATCCTGT-3′, anti-sense: 5′-ATTGGGCCCGTCATCTCCACCACACAGGTAC-3′ and lentiviral control vectors (vector2, pcDNA3.1 for the control plasmid) were packed and obtained from OBiO Technology (Shanghai) Corp., Ltd. HRECs were plated in 24-well plates at a density of 2×10 5 cells/well and allowed to grow at ~40% confluence.

Techniques: Activation Assay, Infection, Control, Expressing, Western Blot, Transfection, Knockdown, Over Expression

Journal: Experimental and Therapeutic Medicine

Article Title: Annexin A2 upregulation protects human retinal endothelial cells from oxygen-glucose deprivation injury by activating autophagy

doi: 10.3892/etm.2019.7909

Figure Lengend Snippet: Effects of A2 knockdown or overexpression combined with the autophagy inhibitor 3-MA treatment on OGD-induced injury in human retinal endothelial cells. (A and B) Cells were infected with a lentiviral vector encoding shRNAs against human A2 or a lentiviral scramble control vector (vector 1) for 48 h, then treated with OGD for another 24 h. (A) Cell viability and (B) expression of cleaved-caspase 3 were measured. (C and D) Cells were infected with a lentiviral A2 overexpression vector or a lentiviral control vector (vector 2) for 48 h, then treated with OGD for another 24 h in the presence or absence of 3-MA (5 mM). (C) Cell viability and (D) expression of cleaved-caspase 3 were measured. **P<0.01 vs. Con group; # P<0.05 vs. OGD group, & P<0.05 vs. OE-A2+OGD group. 3-MA, 3-methyladenine; A2, Annexin A2; OE-A2, lentiviral A2 overexpression vectors; sh-A2, lentiviral vectors encoding short hairpin RNAs against human A2; OGD, oxygen-glucose deprivation; Con, control.

Article Snippet: Lentiviral vectors encoding shRNAs against human A2 (sh-A2, sequence 5′-TGAGGGTGACGTTAGCATTAC-3′ for A2 shRNA), lentiviral control vectors (vector1, sequence 5′-GGATCATCATGCTATGCAGTT-3′ for control scramble shRNA), lentiviral A2 overexpression vectors (OE-A2, primers used as follows, sense: 5′-CGGGGTACCATGTCTACTGTTCACGAAATCCTGT-3′, anti-sense: 5′-ATTGGGCCCGTCATCTCCACCACACAGGTAC-3′ and lentiviral control vectors (vector2, pcDNA3.1 for the control plasmid) were packed and obtained from OBiO Technology (Shanghai) Corp., Ltd. HRECs were plated in 24-well plates at a density of 2×10 5 cells/well and allowed to grow at ~40% confluence.

Techniques: Knockdown, Over Expression, Infection, Plasmid Preparation, Control, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Annexin A2 upregulation protects human retinal endothelial cells from oxygen-glucose deprivation injury by activating autophagy

doi: 10.3892/etm.2019.7909

Figure Lengend Snippet: Role of A2 in OGD-induced activation of autophagy in human retinal endothelial cells. Cells were infected with lentiviral vectors encoding shRNAs against human A2, lentiviral scramble control vectors (vector1), lentiviral A2 expression vectors and lentiviral control vectors (vector2) for 48 h before OGD treatment. Protein levels were detected by western blotting. (A) Expression of Beclin-1, LC3I and LC3II when cells underwent OGD for different periods of time. (B) Transfection efficiency of sh-A2. (C) Expression of Beclin-1, LC3I and LC3II following knockdown of A2. (D) Transfection efficiency of OE-A2. (E) Expression of Beclin-1, LC3I and LC3II after A2 overexpression. *P<0.05 and **P<0.01 vs. Con group; # P<0.05 and ## P<0.01 vs. OGD group. A2, Annexin A2; sh-A2, lentiviral vectors encoding short hairpin RNAs against human A2; OE-A2, lentiviral A2 overexpression vectors; OGD, oxygen-glucose deprivation; Con, control.

Article Snippet: Lentiviral vectors encoding shRNAs against human A2 (sh-A2, sequence 5′-TGAGGGTGACGTTAGCATTAC-3′ for A2 shRNA), lentiviral control vectors (vector1, sequence 5′-GGATCATCATGCTATGCAGTT-3′ for control scramble shRNA), lentiviral A2 overexpression vectors (OE-A2, primers used as follows, sense: 5′-CGGGGTACCATGTCTACTGTTCACGAAATCCTGT-3′, anti-sense: 5′-ATTGGGCCCGTCATCTCCACCACACAGGTAC-3′ and lentiviral control vectors (vector2, pcDNA3.1 for the control plasmid) were packed and obtained from OBiO Technology (Shanghai) Corp., Ltd. HRECs were plated in 24-well plates at a density of 2×10 5 cells/well and allowed to grow at ~40% confluence.

Techniques: Activation Assay, Infection, Control, Expressing, Western Blot, Transfection, Knockdown, Over Expression

Journal: Experimental and Therapeutic Medicine

Article Title: Annexin A2 upregulation protects human retinal endothelial cells from oxygen-glucose deprivation injury by activating autophagy

doi: 10.3892/etm.2019.7909

Figure Lengend Snippet: Effects of A2 knockdown or overexpression combined with the autophagy inhibitor 3-MA treatment on OGD-induced injury in human retinal endothelial cells. (A and B) Cells were infected with a lentiviral vector encoding shRNAs against human A2 or a lentiviral scramble control vector (vector 1) for 48 h, then treated with OGD for another 24 h. (A) Cell viability and (B) expression of cleaved-caspase 3 were measured. (C and D) Cells were infected with a lentiviral A2 overexpression vector or a lentiviral control vector (vector 2) for 48 h, then treated with OGD for another 24 h in the presence or absence of 3-MA (5 mM). (C) Cell viability and (D) expression of cleaved-caspase 3 were measured. **P<0.01 vs. Con group; # P<0.05 vs. OGD group, & P<0.05 vs. OE-A2+OGD group. 3-MA, 3-methyladenine; A2, Annexin A2; OE-A2, lentiviral A2 overexpression vectors; sh-A2, lentiviral vectors encoding short hairpin RNAs against human A2; OGD, oxygen-glucose deprivation; Con, control.

Article Snippet: Lentiviral vectors encoding shRNAs against human A2 (sh-A2, sequence 5′-TGAGGGTGACGTTAGCATTAC-3′ for A2 shRNA), lentiviral control vectors (vector1, sequence 5′-GGATCATCATGCTATGCAGTT-3′ for control scramble shRNA), lentiviral A2 overexpression vectors (OE-A2, primers used as follows, sense: 5′-CGGGGTACCATGTCTACTGTTCACGAAATCCTGT-3′, anti-sense: 5′-ATTGGGCCCGTCATCTCCACCACACAGGTAC-3′ and lentiviral control vectors (vector2, pcDNA3.1 for the control plasmid) were packed and obtained from OBiO Technology (Shanghai) Corp., Ltd. HRECs were plated in 24-well plates at a density of 2×10 5 cells/well and allowed to grow at ~40% confluence.

Techniques: Knockdown, Over Expression, Infection, Plasmid Preparation, Control, Expressing

Journal: Oncotarget

Article Title: Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

doi:

Figure Lengend Snippet: A. HCT116 p53 +/+ or HCT116 p53 −/− cells, MEF p53 +/+ or MEF p53 −/− cells, and U2OS shLuc or U2OS shp53 cells expressing secreted embryonic alkaline phosphatase (SEAP) were transduced with a pSEAP2 control vector and washed 24 h after transduction. The medium was then changed, and the cells were cultured for another 6 h. Culture media were analyzed for SEAP activity, and luminescence was normalized to cell number. The transfection efficiencies of HCT116 p53 +/+ and HCT116 p53 −/− cells were approximately 80% each (data not shown). B. Overexpression of wild-type p53 inhibited SEAP activity. SEAP activities of cells that constitutively expressed the indicated p53 molecules were analyzed using the same procedure described in (A). C. HCT116 p53 −/− cells that expressed SEAP were transfected with siControl, siATF6, siPERK, or siIRE1α, cultured for 24 h, and following a change of medium, the cells were cultured for another 6 h. Whole cell lysates were analyzed using western blotting with the indicated antibodies, and culture supernatants were analyzed for SEAP activity. Values shown are the mean ± s.d. of three different experiments simultaneously measured. The P value was calculated using two-way ANOVA.

Article Snippet: Cells were transduced with the pSEAP2-Control Vector (Clontech).

Techniques: Expressing, Transduction, Plasmid Preparation, Cell Culture, Activity Assay, Transfection, Over Expression, Western Blot