Figure 4. Effects of metformin on NiCl₂-mediated autophagy in human bronchial epithelial cells.

(A) BEAS-2B cells (2×10⁵ cells/well of 12-well plate) were treated with varying doses of NiCl₂ (0, 0.06, 0.125, 0.25 mM) (top), or with NiCl₂ (0, 0.25 mM) and metformin (0, 1, 2.5, 5 mM), for 48 h (bottom). The cells were stained with Newport Green™ DCF diacetate (1 μM) for green fluorescence observation under a green filter fluorescence microscope. Scale bar, 20 μm. (B) pEGFP-LC3B transfection revealed LC3B puncta (green) in BEAS-2B cells (8×10⁴ cells/well of 24-well plate) treated with NiCl₂ (0, 0.25 mM) and metformin (0, 1, 2.5, 5 mM) for 48 h. Cells were fixed and stained with DAPI for nuclear visualization under a confocal microscope. Dashed lines encircle the enlarge images and arrow heads point to the puncta of GFP-LC3. Scale bars, 20 μm. (C) BEAS-2B cells (1×10⁶ cells/ 6 cm dish) were treated with NiCl₂ (0, 0.25 mM) and metformin (0, 1, 2.5, 5 mM) for 48 h and stained with acridine orange (1 μg/mL) for AVO observation. Cells were visualized under a red filter fluorescence microscope with quantification by flow cytometry. (D) Western blots of p-AKT, p-P70S6K, p-AMPK, LC3B and (E) autophagy-related genes Beclin-1, Atg12-Atg5 and Atg3 expressions in protein lysates from BEAS-2B cells (1×10⁶ cells/ 6 cm dish) treated with NiCl₂ (0, 0.25 mM) and metformin (0, 5 mM) for 48 h. β-actin was used as an internal control. The relative ratios of p-AKT/β-actin, p-P70S6K/β-actin, p-AMPK/β-actin, LC3B-II/β-actin, Beclin-1/β-actin, Atg12-Atg5/β-actin and Atg3/β-actin are shown. (F) p-AMPK and p-P70S6K were determined by western blotting after BEAS-2B shGFP and shHK2 cells (1×10⁶ cells/ 6 cm dish) were treated with NiCl₂ and metformin for 48 h. β-actin was used as an internal control.