nonspecific scramble control  (Santa Cruz Biotechnology)

Journal: Redox biology

Article Title: Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

doi: 10.1016/j.redox.2023.102786

Figure Lengend Snippet: Fig. 5. AMPK activator A769662 mimics the effect of metformin in MGO-stimulated cells (A) Cells were pretreated with metformin (6 mM) or A769662 (25 μM) 30 min prior to MGO (300 μg/ml) treatment for indicated time points. AMPK phosphorylation was measured by immunoblotting. (B) Cells were 30 min pretreated with A769662 (25 μM) prior to MGO (300 μg/ml) for 6 h. Cell viability was measured by Annexin V-FITC/PI using FACS. (C, D, E, F) Cells were pretreated with A769662 (25 μM) for 30 min followed by MGO (300 μg/ml) treatment for 4 h. DCFDA (C), DHE (D), mitoSOX (E), and mitoPY1 (F) were used to measure cellular ROS. (G) Cells were 30 min pretreated with compound C (10 μM) followed by MGO (300 μg/ml) stimulation for 6 h. In some experiments, ARPE-19 cells were treated with siRNA followed by stimulation with MGO (300 μg/ml) for 6 h. Cell viability was measured by Annexin V-FITC/PI using FACS. AMPK expression after siRNA treatment was determined by immunoblotting. (H) Immediately after treatment with A769662 (25 μM), cells were subjected into SFe24 analyzer for OCR measurement, as described in Fig. 3D. Data were the mean ± S.E.M. from at least 3 independent experiments. *p < 0.05, indicating the significant effect of MGO; #p < 0.05, indicating the significant effects of A769662, compound C and AMPK silencing on MGO- induced responses.

Article Snippet: Human si-AMPKα1/2 (sc-45312) and scramble nonspecific siRNA (sc-44236) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and human si-GLO1 (s5825) was from Thermo Fischer Scientific (Waltham, US).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Redox biology

Article Title: Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

doi: 10.1016/j.redox.2023.102786

Figure Lengend Snippet: Fig. 8. Metformin and A769662 reverse MGO-induced GLO1 downregulation. (A) Cells were pretreated with BBGC (10 μM), metformin (6 mM) and/or A769662 (25 μM) 30 min before MGO (100 μg/ml) stimulation. After 4 h, cell viability was determined by Annexin V-FITC/PI staining using FACS. (B) Cells were treated with siRNA to silence GLO1, then treated with MGO (100 or 300 μg/ml) for 6 h. Cell viability was determined by Annexin V-FITC/PI staining using FACS. (C–E) Cells were pretreated with metformin (6 mM) and/or A769662 (25 μM) 30 min prior to MGO (300 μg/ml) stimulation. (C) After incubation for 1, 3, or 6 h, cell lysates were prepared for immunoblotting. (D) After incubation for 2 or 4 h, GLO1 gene expression was measured using PCR analysis. (E) After incubation for 3 or 6 h, GLO1 and GLO2 activities were determined by commercial kits according to the manufacturer’s instructions. Data were the mean ± S.E.M. from at least 3 independent experiments. *p < 0.05, indicating the significant effect of MGO. #p < 0.05, indicating the blockade effects of metformin and A769662.

Article Snippet: Human si-AMPKα1/2 (sc-45312) and scramble nonspecific siRNA (sc-44236) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and human si-GLO1 (s5825) was from Thermo Fischer Scientific (Waltham, US).

Techniques: Staining, Incubation, Western Blot, Gene Expression