Journal: Current eye research

Article Title: Downregulation of AMPK dependent FOXO3 and TFEB involves in the inhibition of autophagy in diabetic cataract.

doi: 10.1080/02713683.2021.2009516

Figure Lengend Snippet: Figure 2. AMPK activity is decreased in anterior capsule LECs from DC patients and suppression of autophagy and AMPK activity is found in SRA 01/04 under HG condition. (A) Proteins were extracted from the anterior capsules of DC and ARC patients (control) and then probed for total AMPK and p-AMPK. GAPDH was used as a loading control. (B) Quantification of p-AMPK/AMPK expression levels in A. Fold change relative to the level of control groups is displayed. ****P < .0001, n = 3. (C) SRA 01/04 were supplemented with 30 mM and 5.5 mM glucose and incubated for 48 hours. For experimental group, cells were treated with 1 mM AICAR and 10 mM Compound C for 48 hours respectively at the same time. Proteins were extracted and then probed for AMPK, p-AMPK, Beclin1 and LC3B. Without AICAR or Compound C treated LG (5.5 mM) cultured LECs were used as control groups. β-actin was used as a loading control. (D) Quantification of p-AMPK/AMPK, Beclin1 and LC3B-II expression levels in C. Fold change relative to the level of control groups is displayed. *P < .05, **P < .01, ***P < .001, ****P < .0001, n = 3. (E) The mRNA levels of Beclin1 and ATG8 were determined using real-time PCR and normalized to GAPDH. Fold change relative to the level of the control groups is displayed. *P < .05, **P < .01, ***P < .001, NS: not significant, n = 3.

Article Snippet: This low and high glucose concentrations were applied in previous studies.25,26 For AMPK activator AICAR and inhibitor Compound C treatment, cells were treated with 1 mM AICAR (Selleckchem, Houston, TX, USA) and 10 μM Compound C (Selleckchem, Houston, TX, USA).

Techniques: Activity Assay, Capsules, Control, Expressing, Incubation, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Current eye research

Article Title: Downregulation of AMPK dependent FOXO3 and TFEB involves in the inhibition of autophagy in diabetic cataract.

doi: 10.1080/02713683.2021.2009516

Figure Lengend Snippet: Figure 3. mTOR and AKT were up-regulated in both anterior capsule LECs from DC patients and SRA 01/04 under HG condition. (A) Proteins were extracted from the anterior capsules of DC and ARC patients (control) and then probed for total AKT and p-AKT, total mTOR and p-mTOR. GAPDH was used as a loading control. (B) Quantification of p-AKT/AKT and p-mTOR/mTOR expression levels in A. Fold change relative to the level of control groups is displayed. **P < .01, ***P < .001, n = 3. (C) SRA 01/04 were supplemented with 30 mM and 5.5 mM glucose and incubated for 48 hours. For experimental group, cells were treated with 1 mM AICAR and 10 mM Compound C for 48 hours respectively at the same time. Proteins were extracted and then probed for p-AKT/AKT and p-mTOR/mTOR. Without AICAR or Compound C treated LG (5.5 mM) cultured SRA 01/04 were used as control groups. β-actin was used as a loading control. (D) Quantification of p-AKT/AKT and p-mTOR/mTOR expression levels in C. Fold change relative to the level of control groups is displayed. *P < .05, **P < .01, ***P < .001, ****P < .0001, n = 3.

Article Snippet: This low and high glucose concentrations were applied in previous studies.25,26 For AMPK activator AICAR and inhibitor Compound C treatment, cells were treated with 1 mM AICAR (Selleckchem, Houston, TX, USA) and 10 μM Compound C (Selleckchem, Houston, TX, USA).

Techniques: Capsules, Control, Expressing, Incubation, Cell Culture

Journal: Current eye research

Article Title: Downregulation of AMPK dependent FOXO3 and TFEB involves in the inhibition of autophagy in diabetic cataract.

doi: 10.1080/02713683.2021.2009516

Figure Lengend Snippet: Figure 4. Down-regulated AMPK inhibits autophagy activity through inhibiting expression of FOXO3 and TFEB in LECs. (A) Proteins were extracted from the anterior capsules of DC and ARC patients (control) and then probed for FOXO3 and TFEB. GAPDH was used as a loading control. (B) Quantification of FOXO3 and TFEB expression levels in A. Fold change relative to the level of control groups is displayed. **P < .01, ***P < .001, n = 3. (C) Total RNA was extracted from the anterior capsules of DC and ARC patients (control). The mRNA levels of FOXO3 and TFEB were determined using real-time PCR and normalized to GAPDH. Fold change relative to the level of the control groups is displayed. ***P < .001, NS: not significant, n = 3. (D) SRA 01/04 were supplemented with 30 mM and 5.5 mM glucose and incubated for 48 hours. For experimental group, cells were treated with 1 mM AICAR and 10 mM Compound C for 48 hours respectively at the same time. Proteins were extracted and then probed for FOXO3 and TFEB. Without AICAR or Compound C treated LG (5.5 mM) cultured SRA 01/04 were used as control groups. β-actin was used as a loading control. (E) Quantification of FOXO3 and TFEB expression levels in D. Fold change relative to the level of control groups is displayed. *P < .05, **P < .01, ***P < .001, ****P < .0001, n = 3. (F) The mRNA levels of FOXO3 and TFEB were determined using real-time PCR and normalized to GAPDH. Fold change relative to the level of the control groups is displayed. *P < .05, **P < .01, ***P < .001, ****P < .0001, NS: not significant, n = 3.

Article Snippet: This low and high glucose concentrations were applied in previous studies.25,26 For AMPK activator AICAR and inhibitor Compound C treatment, cells were treated with 1 mM AICAR (Selleckchem, Houston, TX, USA) and 10 μM Compound C (Selleckchem, Houston, TX, USA).

Techniques: Activity Assay, Expressing, Capsules, Control, Real-time Polymerase Chain Reaction, Incubation, Cell Culture