Fig 2: NiCl2 induces autophagy and up-regulates HK2 and LCN2 in human bronchial epithelial cellsBEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 in (A) dose- and (B) time-dependent manner. The protein levels of LCN2, HK2 and LC3B-II were measured on western blot. β-actin was used as an internal control. The relative ratios of LCN2/ β-actin, HK2/ β-actin and LC3B-II/ LC3-I are shown. (C) BEAS-2B (1×106 cells/ 6 cm dish) and WI-38 (5×105 cells/ 6 cm dish) were treated with various doses of NiCl2 for 48 h. The mRNA level of HK2 was measured on RT-PCR and real-time PCR. Quantification of HK2 by qPCR was normalized to the level of GAPDH, with the average of three independent readings. (D) Acridine orange (1μg/mL) was used to stain AVOs in NiCl2 (0, 0.06, 0.12, 0.25 mM)-treated BEAS-2B cells (2×105 cells/ 12-well plate) for 48 h. The fluorescence-activated cells were visualized under a red filter fluorescence microscope and quantified by flow cytometry. Scale bar, 20 μm. (E) NiCl2 increased the secretion of LCN2 in BEAS-2B cell culture supernatant as revealed on ELISA. The data are expressed as mean±SD. *p<0.05, ***p<0.001 on two-tailed t test compared with control.

Fig 3: LCN2 is involved in NiCl2-induced autophagy in human bronchial epithelial cells(A) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 (0, 0.25 mM) and metformin (0, 5 mM) for 48 h. The mRNA level of LCN2 was determined on RT-PCR and qPCR. Quantification of LCN2 by qPCR was normalized to the level of GAPDH, with the average of three independent readings. The data are expressed as mean ± S.D. *p<0.05, ***p<0.001 on two-tailed t test. (B) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 (0, 0.25 mM) and metformin (0, 5 mM) or 2-DG (0, 5 mM) for 48 h and subjected to western blotting for LCN2. (C) Metformin or 2-DG decreased NiCl2-induced secretion of LCN2 in BEAS-2B (2×105 cells/12-well plate) cell culture supernatants, as revealed on ELISA. The data shown are the mean ± S.D. of three independent experiments. ***p<0.001 compared to control and ###p<0.001 compared to Ni-treated cells, two-tailed t test. (D) Cells (1×106 cells/6 cm dish) were treated with LCN2 (10 ng/mL) for 0, 6, 12, 24, 48 h. HK2, LC3B levels were analyzed on western blot. β-actin served as the loading control. (E) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with 0, 0.4, 2, 10, 40 ng/mL LCN2 for 48 h. The cells were stained with acridine orange (1 μg/mL) for AVO observation and analyzed using flow cytometry. (F) BEAS-2B cells (1×106 cells/ 6 cm dish) were treated with NiCl2 and metformin for 48 h after infection with lentivirus carrying shLCN2 #89, shLCN2 #90 or vector control (shGFP). The protein levels of LCN2, HK2, LC3B were detected on western blot. β-actin was used as an internal control. (G) Quantitative detection of AVOs by acridine orange staining in cells was performed using flow cytometry.