Microarray Analysis of Altered Gene Expression and the Role of ATF3 in HK-2 Cells Treated with Hemin

Figures & data

Figure 1. Hemin-induced cell death in HK-2 cells. (A) Cytotoxicity effects of different concentrations of hemin (6 h treatment) on HK-2 cells. (B) Time course of cytotoxic effect of 75 μmol/L hemin on HK-2 cells. Each value is expressed as mean ± SD. *p < 0.05, **p < 0.01 compared to vehicle-treated group. (C) Morphological changes of 75 μmol/L hemin treatment for 6 h in HK-2 cells observed with phase contrast microscope (bar = 25 μm).

Figure 2. QPCR analysis of gene expression after hemin treatment. *p < 0.001 compared to control.

Table 1. Genes up-regulated in expression by hemin.

Description	Fold increase
Testin	2.04
Heme-binding protein 1	2.07
IL-23 subunit alpha	2.27
Inositol-trisphosphate 3-kinase C	2.39
p21	2.58
Regulator of G-protein signaling 17	2.79
B-cell lymphoma 6 protein (BCL-6)	3.48
cAMP-responsive element modulator	3.82
JUN	4.42
Regulator of G-protein signaling 2	7.06
Activating transcription factor 3	9.44
Heat shock 70 kDa protein 1	16.63
Proto-oncogene protein c-fos	17.47
Oxidative stress-induced growth inhibitor 1	22.89
Heme oxygenase 1	23.88
Heat shock 70 kDa protein 6	34.49

Notes: Total RNA was extracted from HK-2 cells treated with or without 75 μmol/L hemin for 6 h. Gene expression profiling was performed using the olgo array (Capital-Bio). Fold changes of up-regulated genes are indicated (≥2 fold change).

Table 2. Genes down-regulated in expression by hemin.

Description	Fold decrease
NF–κB inhibitorα	0.50
G1/S-specific cyclin-D2	0.48
Myc proto-oncogene protein	0.40
Cyclin-A	0.36
Signal transducer and activator of transcription 1	0.35
Transferrin receptor protein 1	0.45
Transforming growth factor beta-2	0.38
Bcl-X	0.28
Interleukin-6	0.09
β-2 adrenergic receptor	0.08

Notes: Total RNA was extracted from HK-2 cells treated with or without 75 μmol/L hemin for 6 h. Gene expression profiling was performed using the olgo array (Capital-Bio). Fold changes of down-regulated genes are indicated (≦0.5 fold change).

Figure 3. Expression of ATF3 in HK-2 cells. (A) ATF3 mRNA expression was determined with RT-PCR. Each value is expressed as mean ± SD. *p < 0.001 compared to vehicle-treated group. (B) The localization of ATF3 in hemin-treated and non-treated cells was determined by immuno- cytochemistry with ATF3 antibody (bar = 50 μm). Each value is expressed as mean ± SD. *p < 0.001 compared to vehicle-treated group.

Figure 4. Effects of ATF3-siRNA on hemin toxicity in HK-2 cells. (A) ATF3-siRNA reduced ATF3 gene expression. (B) ATF3-siRNA reduced cell viability after 75 μmol/L hemin treatment for 6 h. (C) ATF3-siRNA augmented caspase-3 activity after 75 μmol/L hemin treatment for 12 h. Each value is expressed as mean ± SD. *p < 0.05 compared to control siRNA-treated group.