Journal: PLOS One

Article Title: Metformin attenuates TBHP-induced oxidative injury in human lens epithelial cells and is associated with SIRT1/FOXO1-related autophagy

doi: 10.1371/journal.pone.0346822

Figure Lengend Snippet: (a) MDC puncta: TBHP reduces puncta; MET restores/increases puncta (rep. images and quant.; Scale bar, 50 μm). (b-d) IF for Beclin-1, LC3B, p62: TBHP lowers Beclin-1/LC3B and elevates p62; MET reverses these trends; 3-MA attenuates MET-induced changes (rep. images and quant.; Scale bar, 50 μm). (e) WB with densitometry (to GAPDH): TBHP decreases Beclin-1 and LC3B-II/I and increases p62; MET increases Beclin-1 and LC3B-II/I and decreases p62; 3-MA blunts these effects. (f) Flux assay with lysosomal blockade: vs TBHP, MET increases LC3B-II and reduces p62; with CQ, LC3B-II remains high and p62 accumulates; with BafA1, p62 accumulates while LC3B-II shows no further rise under short exposure—consistent with degradation blockade during active autophagosome formation. Data are mean ± SD (n = 3). Stats: one-way ANOVA (Tukey). Exact P values are shown in panels.

Article Snippet: Cells were fixed with 4% paraformaldehyde (Servicebio, China; Cat# G1101) for 15 min and permeabilized with 0.2% Triton X-100 (Servicebio, China; Cat# GC204003 ) for 10 min. After blocking with 2.5% normal goat serum (Invitrogen, USA; Cat# R37624 ) for 1 h at room temperature, cells were incubated overnight at 4 °C with primary antibodies against Beclin 1 (Affinity Biosciences, China; Cat# AF5128), LC3B (Affinity Biosciences, China; Cat# AF4650), p62/SQSTM1 (Abcam, UK; Cat# ab109012), SIRT1 (Abcam, UK; Cat# ab189494), and FOXO1 (Abcam, UK; Cat# ab179450) (all 1:200).

Techniques: Flux Assay

Journal: PLOS One

Article Title: Metformin attenuates TBHP-induced oxidative injury in human lens epithelial cells and is associated with SIRT1/FOXO1-related autophagy

doi: 10.1371/journal.pone.0346822

Figure Lengend Snippet: (a) ROS: EX-527 partially abrogates the MET-induced reduction (rep. images and quant.; Scale bar, 50 μm). (b) Biochemistry: under TBHP, MET lowers MDA/MPO; EX-527 increases both vs MET (SOD/GSH show no significant difference between MET and MET + EX-527). (c) TUNEL (DAPI): EX-527 diminishes the anti-apoptotic effect of MET (rep. images and quant.; Scale bar, 50 μm). (d-f) IF of Beclin-1, LC3B, p62: the MET-associated pattern (higher Beclin-1 and LC3B, lower p62) is attenuated by EX-527 (Scale bar, 50 μm). (g) WB with densitometry: TBHP decreases Beclin-1 and LC3B-II/I and increases p62; MET reverses these changes; EX-527 blunts the MET effects. Data are mean ± SD (n = 3). Stats: one-way ANOVA (Tukey). Exact P values are shown in panels.

Article Snippet: Cells were fixed with 4% paraformaldehyde (Servicebio, China; Cat# G1101) for 15 min and permeabilized with 0.2% Triton X-100 (Servicebio, China; Cat# GC204003 ) for 10 min. After blocking with 2.5% normal goat serum (Invitrogen, USA; Cat# R37624 ) for 1 h at room temperature, cells were incubated overnight at 4 °C with primary antibodies against Beclin 1 (Affinity Biosciences, China; Cat# AF5128), LC3B (Affinity Biosciences, China; Cat# AF4650), p62/SQSTM1 (Abcam, UK; Cat# ab109012), SIRT1 (Abcam, UK; Cat# ab189494), and FOXO1 (Abcam, UK; Cat# ab179450) (all 1:200).

Techniques: TUNEL Assay

Journal: PLOS One

Article Title: Metformin attenuates TBHP-induced oxidative injury in human lens epithelial cells and is associated with SIRT1/FOXO1-related autophagy

doi: 10.1371/journal.pone.0346822

Figure Lengend Snippet: (a) MDC puncta: TBHP reduces puncta; MET restores/increases puncta (rep. images and quant.; Scale bar, 50 μm). (b-d) IF for Beclin-1, LC3B, p62: TBHP lowers Beclin-1/LC3B and elevates p62; MET reverses these trends; 3-MA attenuates MET-induced changes (rep. images and quant.; Scale bar, 50 μm). (e) WB with densitometry (to GAPDH): TBHP decreases Beclin-1 and LC3B-II/I and increases p62; MET increases Beclin-1 and LC3B-II/I and decreases p62; 3-MA blunts these effects. (f) Flux assay with lysosomal blockade: vs TBHP, MET increases LC3B-II and reduces p62; with CQ, LC3B-II remains high and p62 accumulates; with BafA1, p62 accumulates while LC3B-II shows no further rise under short exposure—consistent with degradation blockade during active autophagosome formation. Data are mean ± SD (n = 3). Stats: one-way ANOVA (Tukey). Exact P values are shown in panels.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST (Servicebio, China; Cat# G0004) for 1 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against Beclin 1 (1:1000), LC3B (1:1000), p62/SQSTM1 (1:10,000), SIRT1 (1:1000), FOXO1 (1:1000), acetyl-FOXO1A (Lys294) (Affinity Biosciences, China; Cat# AF2305; 1:1000), and GAPDH (Affinity Biosciences, China; Cat# T0004).

Techniques: Flux Assay

Journal: PLOS One

Article Title: Metformin attenuates TBHP-induced oxidative injury in human lens epithelial cells and is associated with SIRT1/FOXO1-related autophagy

doi: 10.1371/journal.pone.0346822

Figure Lengend Snippet: (a) ROS: EX-527 partially abrogates the MET-induced reduction (rep. images and quant.; Scale bar, 50 μm). (b) Biochemistry: under TBHP, MET lowers MDA/MPO; EX-527 increases both vs MET (SOD/GSH show no significant difference between MET and MET + EX-527). (c) TUNEL (DAPI): EX-527 diminishes the anti-apoptotic effect of MET (rep. images and quant.; Scale bar, 50 μm). (d-f) IF of Beclin-1, LC3B, p62: the MET-associated pattern (higher Beclin-1 and LC3B, lower p62) is attenuated by EX-527 (Scale bar, 50 μm). (g) WB with densitometry: TBHP decreases Beclin-1 and LC3B-II/I and increases p62; MET reverses these changes; EX-527 blunts the MET effects. Data are mean ± SD (n = 3). Stats: one-way ANOVA (Tukey). Exact P values are shown in panels.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST (Servicebio, China; Cat# G0004) for 1 h at room temperature and incubated overnight at 4 °C with rabbit primary antibodies against Beclin 1 (1:1000), LC3B (1:1000), p62/SQSTM1 (1:10,000), SIRT1 (1:1000), FOXO1 (1:1000), acetyl-FOXO1A (Lys294) (Affinity Biosciences, China; Cat# AF2305; 1:1000), and GAPDH (Affinity Biosciences, China; Cat# T0004).

Techniques: TUNEL Assay