Journal: International Journal of Molecular Sciences

Article Title: Cereulide Exposure Caused Cytopathogenic Damages of Liver and Kidney in Mice

doi: 10.3390/ijms22179148

Figure Lengend Snippet: Long-time exposure to cereulide triggers ER stress and induces apoptosis of liver and kidney cells in mice. ( A , B ) The mRNA levels of XBP1 , XBP1s , IRE1α , ATF4 and BiP were detected in liver and kidney tissues by RT-qPCR. Data are shown as the mean ± SD ( n = 5). ( C , D ) Western blot analysis for IRE1α, p-IRE1α, XBP1s, PERK, p-PERK, p-eIF2α, and CHOP in the liver and kidney tissues of mice exposed to cereulide (50 and 200 μg/kg). ( E , F ) TUNEL analysis of apoptosis level in liver and kidney tissue of mice exposed to cereulide (200 μg/kg). Arrows indicated apoptotic cells. Image magnification: ×400; scale bar = 50 µm. Data are presented as the mean ± SD ( n = 6). For all panels, * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the control group.

Article Snippet: Antibodies against CHOP (AC532-L63F7), p-eIF2α (AF1237), XBP1s (AF8366), Phospho-PERK (AF5902) was purchased from Beyotime Biotech (Shanghai, China), anti-ATF6 (D262665) were purchased from BBI (Shanghai, China).

Techniques: Quantitative RT-PCR, Western Blot, TUNEL Assay

Journal: International Journal of Molecular Sciences

Article Title: Cereulide Exposure Caused Cytopathogenic Damages of Liver and Kidney in Mice

doi: 10.3390/ijms22179148

Figure Lengend Snippet: Cereulide triggers apoptosis by mediating ER stress in HEK293T cells and HepaRG cells. ( A , B ) Cytotoxicity of cereulide in HepaRG and HEK293T cells. HepaRG and HEK293T cells were treated with different concentrations of cereulide for 24 h. Cell viability was then determined with CCK-8 ( n = 5). ( C ) Cell apoptosis resulting from cereulide treatment in HepaRG and HEK293T cells. HepaRG and HEK293T cells were evaluated with 0.3 and 1 ng/mL cereulide by flow cytometry. The data were analyzed using FlowJo software ( n = 3). ( D , E ) Representative transmission electron microscopy images of HepaRG and HEK293T cells treated with cereulide toxin. Cereulide treated HepaRG and HEK293T cells for 24 h, and observed by transmission electron microscope. M indicates mitochondria. Scale bars: left panel, 2 µm; right panel, 500 nm. ( F , G ) ER labeling. HepaRG and HEK293T cells were treated with 0, 0.3, 1 ng/mL cereulide for 24 h. Image magnification: ×200; scale bar = 100 µm. The cells were stained with ER-Tracker and observed under a fluorescence microscope at 587 nm excitation and 615 nm emission. ( H , I ) ER stress-related protein levels during cereulide exposure were evaluated in HepaRG and HEK293T cells. HepaRG and HEK293T cells were exposed to cereulide toxin (0, 0.3, 1 ng/mL) for 3, 6 and 24 h and Western blot analysis for BiP, ATF6, IRE1α, p-IRE1α, XBP1(s), PERK, p-PERK, p-eIF2α, CHOP. ( J ) IRE1α knock-down blocked cereulide to down-regulate XBP1s. HepaRG cells were transfected with scramble siRNA as negative control (NC) or IRE1α siRNA for 24 h and then exposed to cereulide (1 ng/mL) for 6 h. ( K ) IRE1α knock-down blocked cereulide to increase in apoptosis. HepaRG cells were transfected with scramble siRNA as negative control (NC) or IRE1α siRNA for 24 h and then evaluated with 1 ng/mL cereulide for 24 h by flow cytometry ( n = 3).

Article Snippet: Antibodies against CHOP (AC532-L63F7), p-eIF2α (AF1237), XBP1s (AF8366), Phospho-PERK (AF5902) was purchased from Beyotime Biotech (Shanghai, China), anti-ATF6 (D262665) were purchased from BBI (Shanghai, China).

Techniques: CCK-8 Assay, Flow Cytometry, Software, Transmission Assay, Electron Microscopy, Microscopy, Labeling, Staining, Fluorescence, Western Blot, Transfection, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Cereulide Exposure Caused Cytopathogenic Damages of Liver and Kidney in Mice

doi: 10.3390/ijms22179148

Figure Lengend Snippet: Cereulide induces ER Stress response by ROS production. ( A , B ) Cereulide causes an increase in ROS production. HepaRG and HEK293T cells were treated with 0, 0.3, 1 ng/mL cereulide for 24 h, the fluorescence of dichlorofluorescindiacetate (DCF) were detected at 488 nm excitation and 525 nm emission; n = 5. ( C , D ) Effects of ROS inhibitor on ROS production after cereulide treatment. HEK293T cells were pretreated with 5 mM NAC followed by treatment of 0.5 ng/mL cereulide for 2, 4, 24 h. Cytosolic ROS production measured by DHE labeling, n = 5. ( E , F ) HepaRG cells were pretreated with 7 mM NAC followed by treatment of 1 ng/mL cereulide for 2, 4, 24 h; n = 5. For C and E panels, image magnification: ×400; scale bar = 50 µm. ( G , H ) ROS inhibitor relieves ER stress caused by cereulide. According to the dosage described above, HepaRG and HEK293T cells were treated for 4 h and Western blot analysis for p-eIF2α, CHOP. ( I ) NAC treatment reduce the apoptosis caused by cereulide. HepaRG and HEK293T cells were treated as described above and then evaluated by flow cytometry; n = 3. For all panels, * p < 0.05 and ** p < 0.01 compared to the control cells; # p < 0.05 and ## p < 0.01 compared to NAC treated cells.

Article Snippet: Antibodies against CHOP (AC532-L63F7), p-eIF2α (AF1237), XBP1s (AF8366), Phospho-PERK (AF5902) was purchased from Beyotime Biotech (Shanghai, China), anti-ATF6 (D262665) were purchased from BBI (Shanghai, China).

Techniques: Fluorescence, Labeling, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Cereulide Exposure Caused Cytopathogenic Damages of Liver and Kidney in Mice

doi: 10.3390/ijms22179148

Figure Lengend Snippet: NaB relieves cereulide-induced liver and kidney damage by inhibiting ER stress. ( A ) Representative images of H&E-stained liver and kidney tissues from cereulide + NaB-treated mice and cereulide-treated mice (×200 magnification). ( B , C ) The mRNA levels of TNF-α, IL-6 and IL-10 were detected in liver and kidney tissues by RT-qPCR, n = 5. ( D , E ) Western blot analysis for XBP1(s), p-eIF2α, CHOP and Caspase3 in the liver and kidney tissues of mice that exposed to cereulide (50 μg/kg) were treated with NaB (100 mg/kg) for 28 days. ( F , G ) TUNEL analysis of apoptosis level in liver and kidney tissue of mice exposed to cereulide (50 μg/kg) and cereulide + NaB (100 mg/kg) (×400 magnification). Arrows indicated apoptotic cells. Data are presented as the mean ± SD ( n = 6). For all panels, * p < 0.05 and ** p < 0.01 compared to the control group; # p < 0.05 and ## p < 0.01 compared to cereulide + NaB group.

Article Snippet: Antibodies against CHOP (AC532-L63F7), p-eIF2α (AF1237), XBP1s (AF8366), Phospho-PERK (AF5902) was purchased from Beyotime Biotech (Shanghai, China), anti-ATF6 (D262665) were purchased from BBI (Shanghai, China).

Techniques: Staining, Quantitative RT-PCR, Western Blot, TUNEL Assay