Table 1.
Nucleotide sequences of primers used for RT-PCR.
Figure 1.
Cell viability analysis of A549 cells after treatment with ZnSO4.
Cell viability of A549 was assessed using the WST-8 method after treatment with various doses of ZnSO4 for 24 h or 48 h. (data are presented as mean ± SD, *p≤0.05 versus controls, n = 3). A sub-cytotoxic metal concentration (100 µM) and exposure time (24 or 48 h) were selected for 2DE, Western blot and RT-PCR analyses.
Figure 2.
Representative 2DE gel of soluble proteins in A549 cells stained with CBB.
Each protein sample (1 mg) was separated by IEF in a 24 cm long IPG gel strip containing a wide-range linear pH gradient of 3–10, followed by SDS-PAGE on a vertical 12% gel. Proteins differentially expressed in response to excess Zn (100 µM ZnSO4 for 24 h) are illustrated with a diamond symbol. These proteins showed a two-fold or greater difference in abundance (p≤0.05) in A549 cells. Other proteins identified are labeled with a ring symbol.
Figure 3.
PANTHER classification of proteins that showed differential expression in response to extracellular Zn.
(A) Biological process classification; (B) Molecular function classification.
Figure 4.
Time-dependent changes in ZIP-1, ZnT-1, MT-1 and MTF-1 abundance in cells treated with ZnSO4.
(A) Western blot analysis of proteins regulated by MTF-1, including MT-1, ZnT-1 and ZIP-1 in A549 cells after different stimulation periods with exogenous 100 µM ZnSO4; (B) Protein band density was analyzed with Gel Image Analysis software (CLINX, Shanghai, China). Beta-actin was used as the internal control. Data was normalized and mean values ± SD were calculated from at least three independent samples.
Figure 5.
Time-dependent changes in ZIP-1, ZnT-1, MT-1 and MTF-1 transcript abundance in cells treated with ZnSO4.
(A) RT-PCR analysis of ZIP-1, ZnT-1, MT-1 and MTF-1 mRNA in A549 cells after treatment for 0, 6, 8, 10, 12, 24 and 48 h with exogenous 100 µM ZnSO4; (B) Band density was analyzed with Gel Image Analysis software (CLINX, Shanghai, China). GAPDH expression was used as the internal control. Data was normalized and mean values ± SD were calculated from at least three independent samples.