Fig 2: LCN2 protects gastric epithelial cells (AGS, GES1) from palmitic acid-induced apoptosis and inflammatory state.a AGS and CES1 cells groups (NC, LCN2, siRNA-LCN2) were treated with 300 μM PA for 12h, the blank group (Control) was added to the fatty acid free medium, and as assayed via staining with DAPI/PI, significant protection against PA-induced apoptosis was afforded by LCN2. Channels are: DAPI nuclear stain (blue) and apoptosis by PI (red); ×400 magnification. b Cell apoptosis was analyzed by flow cytometry using Annexin V/FITC with the same treatment. Columns (right panel) represent the average percent of Annexin V positive from three independent experiments. c The level of IL6 and TNFα in each groups. The data are expressed as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.005.

Fig 3: SubAB-increased CHOP and C/EBPG regulate SubAB-induced LCN2 expression. (A) HeLa cells were co-cultured for 24 h with the indicated STEC O113:H21 strains as shown in Fig. 1D. 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. (B) Control (NC) or CHOP 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. (C) Control (NC) or CHOP siRNA-transfected cells were incubated for 24 h with mt or wt SubAB (400 ng ml−1). Cell lysates were subjected to immunoblotting with the indicated antibodies. GAPDH served as a loading control. (D) Quantification of cPARP or LCN2 in the transfected cells with mt or wt SubAB was performed by densitometry. Data are presented as mean ± SD of values from three independent experiments and significance is *P < 0.05. (E) Cells were incubated for 24 h with mt or wt SubAB (400 ng ml−1). The mRNA levels of cebpA, cebpB, or cebpG 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. (F) The indicated siRNA-transfected cells were incubated for 24 h with mt or wt 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. (G) Control (NC), C/EBPB, or C/EBPG siRNA-transfected cells were incubated for 24 h with mt or wt SubAB (400 ng ml−1). Cell lysates were subjected to immunoblotting with anti-LCN2 antibodies. GAPDH served as a loading control.

Fig 4: LCN2 protects gastric epithelial cells (AGS, GES1) from ethanol-induced apoptosis and inflammatory state.a AGS and CES1 cells groups (NC, LCN2, siRNA-LCN2) were treated with 6% ethanol for 4h, the blank group (Control) was added to the complete medium, and as assayed via staining with DAPI/PI, significant protection against ethanol-induced apoptosis was afforded by LCN2. Channels are: DAPI nuclear stain (blue) and apoptosis by PI (red); ×400 magnification. b Cell apoptosis was analyzed by flow cytometry using Annexin V/FITC with the same treatment. Columns (right panel) represent the average percent of Annexin V positive from three independent experiments. c The level of IL6 and TNFα in each groups. The data are expressed as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.005.

Fig 5: Effect of CHOP overexpression on LCN2 expression by SubAB. (A) Control or FLAG-tagged CHOP plasmid-transfected HeLa 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. Ns: not significant. (B) Cell lysates from 8 h or 24 h incubation with toxins were subjected to immunoblotting with the indicated antibodies. GAPDH served as a loading control. Experiments were repeated three times with similar results. ND: not detected.