Fig 2: mRNA and protein levels of NGAL in NC-HK2 cells and NGAL KD-HK2 cells. NGAL, neutrophil gelatinase-associated lipocalin; HK2, human kidney 2; NC, negative control; KD, knockdown.

Fig 3: Oxidative stress measurement under glycemia in vitro and in vivo. (A) ROS test by flow cytometry in HK2 cells in vitro. (B) Quantitation of ROS levels in HK2 cells. (C) MDA, (D) GSH, (E) SOD and (F) catalase concentration or IU in kidney tissues in normal control, DN-wild and DN NGAL-/- mice in vivo. NGAL, neutrophil gelatinase-associated lipocalin; HK2, human kidney 2; ROS, reactive oxygen species; MDA, malonaldehyde; GSH, glutathione; SOD, superoxide dismutase; DN, diabetic nephropathy; KD, knockdown; NC, negative control; HG, high glucose; NG, normal glucose.

Fig 4: Controlled reoxygenation reduces the expression of LCN2 in both gene and protein levels. (A) Quantitative PCR analysis of LCN2 gene transcript. These data were normalized with an amount of β-actin as an internal control. (B) The expression of LCN2 in the hippocampus region (upper panel) and in the cortex region (lower panel) was detected by western blotting. Intensities of protein bands were standardized to those of β-actin and expressed as relative band intensities. Data represents three independent experiments. Error bars reflect±s.e.m. *P<0.05, **P<0.01.

Fig 5: Knockdown of LCN2 increases SubAB-induced apoptosis. (A) Control (NC) or LCN2 siRNA-transfected cells were incubated for 24 h with mt SubAB or SubAB (400 ng ml−1). The lcn2 mRNA levels were measured by RT-qPCR as described in “Methods”. GAPDH was used as an internal control. Data are mean ± SD (n = 3). *P < 0.05, versus mt SubAB treated control cells. (B) Cell lysates were subjected to immunoblotting with the indicated antibodies. GAPDH served as a loading control. Quantification of cPARP in HeLa cells was performed by densitometry (right panel). Data are presented as mean ± SD of values from three independent experiments and significance is *P < 0.05. (C) Control (NC) or LCN2 siRNA-transfected cells were incubated for 24 h with mt or wt SubAB (400 ng ml−1). The chop mRNA levels were measured by RT-qPCR as described in “Methods”. GAPDH was used as an internal control. Data are mean ± SD (n = 3). *P < 0.05, versus mt SubAB-treated control cells. (D) Cell lysates were subjected to immunoblotting with the indicated antibodies. GAPDH served as a loading control. Experiments were repeated three times with similar results.