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( A <t>)</t> <t>hCMEC/D3</t> cells treated with Stx1a or Stx2a exhibited prominent morphological alterations when observed under a fluorescence microscope at 20X magnification, whereas cells treated with Stx1a mut or Stx2a mut did not show such changes. Images were collected from cells incubated with or without Stxs for 24, 48, and 72 h; ( B ) hCMEC/D3 cells were seeded in 6-well plates (5.0 ×10 5 cells/well) and incubated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) or Stx2a mut (10 ng/ml) for 24, 48, 72 h. Cell viability was determined using WST-8 assays, expressed as percentage viability and fold change relative to untreated controls ( left panel ). hCMEC/D3 cells (1.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and caspase-3/7 activity was measured using the caspase Glo-3/7 assay. Under the same conditions, hCMEC/D3 cells (5.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and then protein samples were subjected to Western blotting using an anti-cleaved caspase-3 antibody ( right panel ). β-Actin was used as a control for equal protein loading. The results are a representative experiment obtained from three independent experiments; ( C ) Gb3 expression in hCMEC/D3 cells was detected by staining with Alexa Fluor 647-conjugated anti-CD77/Gb3 antibody or isotype control (mouse IgM-Alexa Fluor 647) for 1 h at 4oC. Representative results from three independent experiments are shown. Statistical significance. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panel B). * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

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1) Product Images from "Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture"

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

Journal: Journal of Microbiology and Biotechnology

doi: 10.4014/jmb.2512.12006

( A ) hCMEC/D3 cells treated with Stx1a or Stx2a exhibited prominent morphological alterations when observed under a fluorescence microscope at 20X magnification, whereas cells treated with Stx1a mut or Stx2a mut did not show such changes. Images were collected from cells incubated with or without Stxs for 24, 48, and 72 h; ( B ) hCMEC/D3 cells were seeded in 6-well plates (5.0 ×10 5 cells/well) and incubated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) or Stx2a mut (10 ng/ml) for 24, 48, 72 h. Cell viability was determined using WST-8 assays, expressed as percentage viability and fold change relative to untreated controls ( left panel ). hCMEC/D3 cells (1.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and caspase-3/7 activity was measured using the caspase Glo-3/7 assay. Under the same conditions, hCMEC/D3 cells (5.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and then protein samples were subjected to Western blotting using an anti-cleaved caspase-3 antibody ( right panel ). β-Actin was used as a control for equal protein loading. The results are a representative experiment obtained from three independent experiments; ( C ) Gb3 expression in hCMEC/D3 cells was detected by staining with Alexa Fluor 647-conjugated anti-CD77/Gb3 antibody or isotype control (mouse IgM-Alexa Fluor 647) for 1 h at 4oC. Representative results from three independent experiments are shown. Statistical significance. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panel B). * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

Figure Legend Snippet: ( A ) hCMEC/D3 cells treated with Stx1a or Stx2a exhibited prominent morphological alterations when observed under a fluorescence microscope at 20X magnification, whereas cells treated with Stx1a mut or Stx2a mut did not show such changes. Images were collected from cells incubated with or without Stxs for 24, 48, and 72 h; ( B ) hCMEC/D3 cells were seeded in 6-well plates (5.0 ×10 5 cells/well) and incubated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) or Stx2a mut (10 ng/ml) for 24, 48, 72 h. Cell viability was determined using WST-8 assays, expressed as percentage viability and fold change relative to untreated controls ( left panel ). hCMEC/D3 cells (1.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and caspase-3/7 activity was measured using the caspase Glo-3/7 assay. Under the same conditions, hCMEC/D3 cells (5.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and then protein samples were subjected to Western blotting using an anti-cleaved caspase-3 antibody ( right panel ). β-Actin was used as a control for equal protein loading. The results are a representative experiment obtained from three independent experiments; ( C ) Gb3 expression in hCMEC/D3 cells was detected by staining with Alexa Fluor 647-conjugated anti-CD77/Gb3 antibody or isotype control (mouse IgM-Alexa Fluor 647) for 1 h at 4oC. Representative results from three independent experiments are shown. Statistical significance. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panel B). * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

Techniques Used: Fluorescence, Microscopy, Incubation, Activity Assay, Caspase-Glo Assay, Western Blot, Control, Expressing, Staining

( A ) hCMEC/D3 cells were seeded in 6-well plates (5.0 × 10 5 cells/well). After washing with culture medium, cells were fixed and nuclei were stained with DAPI reagent. Representative DAPI-positive cells were visualized by fluorescence microscopy. To detect Stx translocation to the ER, cells were stimulated with complete growth medium containing 50 nM ER-tracker (red) live cell staining dye 2 h after treatment with Alexa Fluor 488-conjugated Stx1a (100 ng/ml). After washing, cells were captured using a fluorescence microscope EVOS M5000. Yellow fluorescence indicates co-localization of Stx1a with the ER marker. The scale bars represent 150 μm. The bar graph represents the mean ± SEM of the Pearson's correlation coefficient for the co-localization of Stx1-Alexa Fluor 488/ER tracker. At least 30 cells per condition were analyzed. Asterisks indicate statistically significant differences between the control and Stx1-treated groups. *** = p < 0.001; ( B ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 3, 6, 9, 12 and 24 h. After washing, cells were lysed at the indicated time points, and the presence of activated ER stress sensors and downstream targets in the cell lysates was determined by Western blotting. Untreated cells served as controls, and β-actin was used as a loading control; ( C ) hCMEC/D3 cells were treated as described above, and CHOP and DR5, spliced XBP1/unspliced XBP1 mRNA expression was measured by RT-qPCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); ( D ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 24, 48, and 72 h. At the indicated time points, cells were lysed, and the expression of ROS-related markers (Nrf-2, HO-1, SOD-2, NO2) in cell lysates was measured by RT-qPCR and normalized using GAPDH. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panels C and D) at each time point. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data are shown as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Figure Legend Snippet: ( A ) hCMEC/D3 cells were seeded in 6-well plates (5.0 × 10 5 cells/well). After washing with culture medium, cells were fixed and nuclei were stained with DAPI reagent. Representative DAPI-positive cells were visualized by fluorescence microscopy. To detect Stx translocation to the ER, cells were stimulated with complete growth medium containing 50 nM ER-tracker (red) live cell staining dye 2 h after treatment with Alexa Fluor 488-conjugated Stx1a (100 ng/ml). After washing, cells were captured using a fluorescence microscope EVOS M5000. Yellow fluorescence indicates co-localization of Stx1a with the ER marker. The scale bars represent 150 μm. The bar graph represents the mean ± SEM of the Pearson's correlation coefficient for the co-localization of Stx1-Alexa Fluor 488/ER tracker. At least 30 cells per condition were analyzed. Asterisks indicate statistically significant differences between the control and Stx1-treated groups. *** = p < 0.001; ( B ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 3, 6, 9, 12 and 24 h. After washing, cells were lysed at the indicated time points, and the presence of activated ER stress sensors and downstream targets in the cell lysates was determined by Western blotting. Untreated cells served as controls, and β-actin was used as a loading control; ( C ) hCMEC/D3 cells were treated as described above, and CHOP and DR5, spliced XBP1/unspliced XBP1 mRNA expression was measured by RT-qPCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); ( D ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 24, 48, and 72 h. At the indicated time points, cells were lysed, and the expression of ROS-related markers (Nrf-2, HO-1, SOD-2, NO2) in cell lysates was measured by RT-qPCR and normalized using GAPDH. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panels C and D) at each time point. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data are shown as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Techniques Used: Staining, Fluorescence, Microscopy, Translocation Assay, Marker, Control, Western Blot, Expressing, Quantitative RT-PCR

( A ) hCMEC/D cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx1B (100 ng/ml), Stx2a (10 ng/ml), Stx2a mut (10 ng/ml), or Stx2B (10 ng/ml) for 3 h. After 3 h, cells were washed and cell lysates were collected. Phosphorylation of p38, JNK, and ERK were assessed by Western blotting. β-Actin was used as a loading control. The graphs show the mean ± SEM of band densities normalized by the division of β-Actin band densities and compared to untreated control cell values ( right panel ). Statistical analyses of densitometric scans from at least three independent experiments are shown; ( B ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 24, 48, and 72 h. At each time point, cells were lysed and mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2) were measured by RT-qPCR and normalized to GAPDH; ( C ) Under the same conditions, supernatants were collected and protein levels of IL-1β, IL-6, IL-8, TNF-α and CCL2 were quantified by ELISA using kit standards. Data are presented as mean ± SEM from three independent experiments. Asterisks indicate significant differences between control and Stx-treated cells (Panels B, C) at the indicated time points. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure Legend Snippet: ( A ) hCMEC/D cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx1B (100 ng/ml), Stx2a (10 ng/ml), Stx2a mut (10 ng/ml), or Stx2B (10 ng/ml) for 3 h. After 3 h, cells were washed and cell lysates were collected. Phosphorylation of p38, JNK, and ERK were assessed by Western blotting. β-Actin was used as a loading control. The graphs show the mean ± SEM of band densities normalized by the division of β-Actin band densities and compared to untreated control cell values ( right panel ). Statistical analyses of densitometric scans from at least three independent experiments are shown; ( B ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 24, 48, and 72 h. At each time point, cells were lysed and mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2) were measured by RT-qPCR and normalized to GAPDH; ( C ) Under the same conditions, supernatants were collected and protein levels of IL-1β, IL-6, IL-8, TNF-α and CCL2 were quantified by ELISA using kit standards. Data are presented as mean ± SEM from three independent experiments. Asterisks indicate significant differences between control and Stx-treated cells (Panels B, C) at the indicated time points. * p < 0.05; ** p < 0.01; *** p < 0.001.

Techniques Used: Phospho-proteomics, Western Blot, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

( A ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 3, 6, 9, 12, and 24 h. At each time point, cells were lysed, and mRNA expression of ZO-1, CLDN3, Occludin and JAM2 was measured by RT-qPCR. Expression levels were normalized to GAPDH; ( B ) hCMEC/D3 cells were seeded as above and treated with Stx2a for 0, 6, 12, 24, and 48 h. At each time point, cells were lysed, and the tight junction proteins CLDN1, ZO-1, and Ecadherin were analyzed by Western blotting. β-Actin was used as a loading control; ( C ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well. Cells were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml), or Stx2a mut (10 ng/ml) for 24 h. Fluorescein-conjugated 45kDa ovalbumin was added to the toxin-treated insert wells, and supernatants were collected from the lower chamber after 1 h. Cell permeability was evaluated by measuring the fluorescence of ovalbumin translocated from the insert wells to the lower chamber using a microplate reader. Asterisks indicate significant differences between the control cell values and the Stxs-treated cells (Panels A, C) at each time point. * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Figure Legend Snippet: ( A ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 3, 6, 9, 12, and 24 h. At each time point, cells were lysed, and mRNA expression of ZO-1, CLDN3, Occludin and JAM2 was measured by RT-qPCR. Expression levels were normalized to GAPDH; ( B ) hCMEC/D3 cells were seeded as above and treated with Stx2a for 0, 6, 12, 24, and 48 h. At each time point, cells were lysed, and the tight junction proteins CLDN1, ZO-1, and Ecadherin were analyzed by Western blotting. β-Actin was used as a loading control; ( C ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well. Cells were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml), or Stx2a mut (10 ng/ml) for 24 h. Fluorescein-conjugated 45kDa ovalbumin was added to the toxin-treated insert wells, and supernatants were collected from the lower chamber after 1 h. Cell permeability was evaluated by measuring the fluorescence of ovalbumin translocated from the insert wells to the lower chamber using a microplate reader. Asterisks indicate significant differences between the control cell values and the Stxs-treated cells (Panels A, C) at each time point. * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Control, Permeability, Fluorescence

( A ) hCMEC/ D cells were seeded in 6-well plates. Cells were pretreated with z-VAD (20 μM) for 1 h before Stx2a exposure and divided into the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. After 24 h of Stx2a treatment, cell viability was assessed using the WST-8 assay ( left panel ). Subsequently, Western blot analysis showed that the total levels of caspase-3/7 in the cell lysates were decreased only in the Stx2a-treated group. Protein samples were prepared using anti-caspase-3, anti-cleaved caspase-3, and anti-β-Actin antibodies ( right panel ). β-Actin was used as a control for equal protein loading; ( B ) hCMEC/D3 cells were seeded as described above and treated with z-VAD 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. Culture supernatants were collected, and the levels of secreted IL-1β, IL-6, IL-8, TNF-α and CCL2 were measured using ELISA ( left panel ). Total RNA was isolated from cell lysates, and mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2 were quantified using RT-qPCR ( right panel ). RNA expression levels were normalized using GAPDH; ( C ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of 5.0 × 10 5 cells/well and treated with NAC (5μM) 1 h before Stx2a exposure. The experimental groups included control, Stx2a-treated, NAC only, and Stx2a + NAC pre-treated. After 24 h of Stx2a treatment, the cytotoxicity level was measured by LDH assay using the cell culture supernatant. mRNA expression levels of Nrf-2, HO-1, and SOD-2 were measured by RT-qPCR and normalized to GAPDH, and the degree of NO expression was measured by NO assay; ( D ) hCMEC/D3 cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with z-VAD and NAC 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: control, Stx2a-treated, z-VAD + Stx2a, and NAC + Stx2a groups. After 24 h of Stx2 treatment, washed cell lysates were harvested, and the mRNA expression of ZO-1, CLDN3, Occludin and JAM2 were measured using RT-qPCR. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences compared to the control group, Stx-treated group, or between monotherapy vs. combination therapy (e.g., z-VAD alone vs. z-VAD+Stx2a) (Panels A, B) and compared to the control group, Stx-treated group, or between monotherapy vs. pre-treatment therapy ( e.g. , NAC alone vs. NAC+Stx2a) (Panel C). Significant differences are indicated between the control and cells treated with Stx, Stx+z-VAD, or Stx+NAC (Panel D).* = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure Legend Snippet: ( A ) hCMEC/ D cells were seeded in 6-well plates. Cells were pretreated with z-VAD (20 μM) for 1 h before Stx2a exposure and divided into the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. After 24 h of Stx2a treatment, cell viability was assessed using the WST-8 assay ( left panel ). Subsequently, Western blot analysis showed that the total levels of caspase-3/7 in the cell lysates were decreased only in the Stx2a-treated group. Protein samples were prepared using anti-caspase-3, anti-cleaved caspase-3, and anti-β-Actin antibodies ( right panel ). β-Actin was used as a control for equal protein loading; ( B ) hCMEC/D3 cells were seeded as described above and treated with z-VAD 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. Culture supernatants were collected, and the levels of secreted IL-1β, IL-6, IL-8, TNF-α and CCL2 were measured using ELISA ( left panel ). Total RNA was isolated from cell lysates, and mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2 were quantified using RT-qPCR ( right panel ). RNA expression levels were normalized using GAPDH; ( C ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of 5.0 × 10 5 cells/well and treated with NAC (5μM) 1 h before Stx2a exposure. The experimental groups included control, Stx2a-treated, NAC only, and Stx2a + NAC pre-treated. After 24 h of Stx2a treatment, the cytotoxicity level was measured by LDH assay using the cell culture supernatant. mRNA expression levels of Nrf-2, HO-1, and SOD-2 were measured by RT-qPCR and normalized to GAPDH, and the degree of NO expression was measured by NO assay; ( D ) hCMEC/D3 cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with z-VAD and NAC 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: control, Stx2a-treated, z-VAD + Stx2a, and NAC + Stx2a groups. After 24 h of Stx2 treatment, washed cell lysates were harvested, and the mRNA expression of ZO-1, CLDN3, Occludin and JAM2 were measured using RT-qPCR. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences compared to the control group, Stx-treated group, or between monotherapy vs. combination therapy (e.g., z-VAD alone vs. z-VAD+Stx2a) (Panels A, B) and compared to the control group, Stx-treated group, or between monotherapy vs. pre-treatment therapy ( e.g. , NAC alone vs. NAC+Stx2a) (Panel C). Significant differences are indicated between the control and cells treated with Stx, Stx+z-VAD, or Stx+NAC (Panel D).* = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Techniques Used: Control, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Quantitative RT-PCR, RNA Expression, Lactate Dehydrogenase Assay, Cell Culture

( A ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well, and A172 cells were seeded in the bottom wells of Trans-well plates at 5.0 × 10 5 cells/well. The insert wells containing hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 24 h. After 24 h, the supernatants were collected and cytotoxicity in A172 cells was assessed by LDH assay ( left panel ). A representative flow-cytometry readout of Gb3 in A172 astrocytes ( right panel ). ( B ) Subsequently, the A172 cells in the bottom wells were washed, and the cell lysates were harvested and the protein levels of caspase-3 and cleaved caspase-3 were determined by Western blotting. Anti-caspase-3, anticleaved caspase-3, and anti-β-Actin antibodies were used for protein samples. β-Actin served as a loading control ( C-D ) Culture supernatants of A172 cells seeded in the same manner as above were harvested, and the secretion levels of IL-1β, IL-6, IL-8, TNF-α, and CCL2 were determined using ELISA. Subsequently, the mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1 and CCL2 were assessed by using RT-qPCR in cell lysates. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences between the control group and cells treated with Stx, Stx+z-VAD, and Stx+NAC (Panels A, C, D). * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Figure Legend Snippet: ( A ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well, and A172 cells were seeded in the bottom wells of Trans-well plates at 5.0 × 10 5 cells/well. The insert wells containing hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 24 h. After 24 h, the supernatants were collected and cytotoxicity in A172 cells was assessed by LDH assay ( left panel ). A representative flow-cytometry readout of Gb3 in A172 astrocytes ( right panel ). ( B ) Subsequently, the A172 cells in the bottom wells were washed, and the cell lysates were harvested and the protein levels of caspase-3 and cleaved caspase-3 were determined by Western blotting. Anti-caspase-3, anticleaved caspase-3, and anti-β-Actin antibodies were used for protein samples. β-Actin served as a loading control ( C-D ) Culture supernatants of A172 cells seeded in the same manner as above were harvested, and the secretion levels of IL-1β, IL-6, IL-8, TNF-α, and CCL2 were determined using ELISA. Subsequently, the mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1 and CCL2 were assessed by using RT-qPCR in cell lysates. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences between the control group and cells treated with Stx, Stx+z-VAD, and Stx+NAC (Panels A, C, D). * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Techniques Used: Lactate Dehydrogenase Assay, Flow Cytometry, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, RNA Expression

Image Search Results

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells treated with Stx1a or Stx2a exhibited prominent morphological alterations when observed under a fluorescence microscope at 20X magnification, whereas cells treated with Stx1a mut or Stx2a mut did not show such changes. Images were collected from cells incubated with or without Stxs for 24, 48, and 72 h; ( B ) hCMEC/D3 cells were seeded in 6-well plates (5.0 ×10 5 cells/well) and incubated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) or Stx2a mut (10 ng/ml) for 24, 48, 72 h. Cell viability was determined using WST-8 assays, expressed as percentage viability and fold change relative to untreated controls ( left panel ). hCMEC/D3 cells (1.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and caspase-3/7 activity was measured using the caspase Glo-3/7 assay. Under the same conditions, hCMEC/D3 cells (5.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and then protein samples were subjected to Western blotting using an anti-cleaved caspase-3 antibody ( right panel ). β-Actin was used as a control for equal protein loading. The results are a representative experiment obtained from three independent experiments; ( C ) Gb3 expression in hCMEC/D3 cells was detected by staining with Alexa Fluor 647-conjugated anti-CD77/Gb3 antibody or isotype control (mouse IgM-Alexa Fluor 647) for 1 h at 4oC. Representative results from three independent experiments are shown. Statistical significance. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panel B). * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Fluorescence, Microscopy, Incubation, Activity Assay, Caspase-Glo Assay, Western Blot, Control, Expressing, Staining

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in 6-well plates (5.0 × 10 5 cells/well). After washing with culture medium, cells were fixed and nuclei were stained with DAPI reagent. Representative DAPI-positive cells were visualized by fluorescence microscopy. To detect Stx translocation to the ER, cells were stimulated with complete growth medium containing 50 nM ER-tracker (red) live cell staining dye 2 h after treatment with Alexa Fluor 488-conjugated Stx1a (100 ng/ml). After washing, cells were captured using a fluorescence microscope EVOS M5000. Yellow fluorescence indicates co-localization of Stx1a with the ER marker. The scale bars represent 150 μm. The bar graph represents the mean ± SEM of the Pearson's correlation coefficient for the co-localization of Stx1-Alexa Fluor 488/ER tracker. At least 30 cells per condition were analyzed. Asterisks indicate statistically significant differences between the control and Stx1-treated groups. *** = p < 0.001; ( B ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 3, 6, 9, 12 and 24 h. After washing, cells were lysed at the indicated time points, and the presence of activated ER stress sensors and downstream targets in the cell lysates was determined by Western blotting. Untreated cells served as controls, and β-actin was used as a loading control; ( C ) hCMEC/D3 cells were treated as described above, and CHOP and DR5, spliced XBP1/unspliced XBP1 mRNA expression was measured by RT-qPCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); ( D ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 24, 48, and 72 h. At the indicated time points, cells were lysed, and the expression of ROS-related markers (Nrf-2, HO-1, SOD-2, NO2) in cell lysates was measured by RT-qPCR and normalized using GAPDH. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panels C and D) at each time point. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data are shown as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Staining, Fluorescence, Microscopy, Translocation Assay, Marker, Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx1B (100 ng/ml), Stx2a (10 ng/ml), Stx2a mut (10 ng/ml), or Stx2B (10 ng/ml) for 3 h. After 3 h, cells were washed and cell lysates were collected. Phosphorylation of p38, JNK, and ERK were assessed by Western blotting. β-Actin was used as a loading control. The graphs show the mean ± SEM of band densities normalized by the division of β-Actin band densities and compared to untreated control cell values ( right panel ). Statistical analyses of densitometric scans from at least three independent experiments are shown; ( B ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 24, 48, and 72 h. At each time point, cells were lysed and mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2) were measured by RT-qPCR and normalized to GAPDH; ( C ) Under the same conditions, supernatants were collected and protein levels of IL-1β, IL-6, IL-8, TNF-α and CCL2 were quantified by ELISA using kit standards. Data are presented as mean ± SEM from three independent experiments. Asterisks indicate significant differences between control and Stx-treated cells (Panels B, C) at the indicated time points. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Phospho-proteomics, Western Blot, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 3, 6, 9, 12, and 24 h. At each time point, cells were lysed, and mRNA expression of ZO-1, CLDN3, Occludin and JAM2 was measured by RT-qPCR. Expression levels were normalized to GAPDH; ( B ) hCMEC/D3 cells were seeded as above and treated with Stx2a for 0, 6, 12, 24, and 48 h. At each time point, cells were lysed, and the tight junction proteins CLDN1, ZO-1, and Ecadherin were analyzed by Western blotting. β-Actin was used as a loading control; ( C ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well. Cells were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml), or Stx2a mut (10 ng/ml) for 24 h. Fluorescein-conjugated 45kDa ovalbumin was added to the toxin-treated insert wells, and supernatants were collected from the lower chamber after 1 h. Cell permeability was evaluated by measuring the fluorescence of ovalbumin translocated from the insert wells to the lower chamber using a microplate reader. Asterisks indicate significant differences between the control cell values and the Stxs-treated cells (Panels A, C) at each time point. * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Permeability, Fluorescence

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/ D cells were seeded in 6-well plates. Cells were pretreated with z-VAD (20 μM) for 1 h before Stx2a exposure and divided into the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. After 24 h of Stx2a treatment, cell viability was assessed using the WST-8 assay ( left panel ). Subsequently, Western blot analysis showed that the total levels of caspase-3/7 in the cell lysates were decreased only in the Stx2a-treated group. Protein samples were prepared using anti-caspase-3, anti-cleaved caspase-3, and anti-β-Actin antibodies ( right panel ). β-Actin was used as a control for equal protein loading; ( B ) hCMEC/D3 cells were seeded as described above and treated with z-VAD 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. Culture supernatants were collected, and the levels of secreted IL-1β, IL-6, IL-8, TNF-α and CCL2 were measured using ELISA ( left panel ). Total RNA was isolated from cell lysates, and mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2 were quantified using RT-qPCR ( right panel ). RNA expression levels were normalized using GAPDH; ( C ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of 5.0 × 10 5 cells/well and treated with NAC (5μM) 1 h before Stx2a exposure. The experimental groups included control, Stx2a-treated, NAC only, and Stx2a + NAC pre-treated. After 24 h of Stx2a treatment, the cytotoxicity level was measured by LDH assay using the cell culture supernatant. mRNA expression levels of Nrf-2, HO-1, and SOD-2 were measured by RT-qPCR and normalized to GAPDH, and the degree of NO expression was measured by NO assay; ( D ) hCMEC/D3 cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with z-VAD and NAC 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: control, Stx2a-treated, z-VAD + Stx2a, and NAC + Stx2a groups. After 24 h of Stx2 treatment, washed cell lysates were harvested, and the mRNA expression of ZO-1, CLDN3, Occludin and JAM2 were measured using RT-qPCR. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences compared to the control group, Stx-treated group, or between monotherapy vs. combination therapy (e.g., z-VAD alone vs. z-VAD+Stx2a) (Panels A, B) and compared to the control group, Stx-treated group, or between monotherapy vs. pre-treatment therapy ( e.g. , NAC alone vs. NAC+Stx2a) (Panel C). Significant differences are indicated between the control and cells treated with Stx, Stx+z-VAD, or Stx+NAC (Panel D).* = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Control, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Quantitative RT-PCR, RNA Expression, Lactate Dehydrogenase Assay, Cell Culture

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well, and A172 cells were seeded in the bottom wells of Trans-well plates at 5.0 × 10 5 cells/well. The insert wells containing hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 24 h. After 24 h, the supernatants were collected and cytotoxicity in A172 cells was assessed by LDH assay ( left panel ). A representative flow-cytometry readout of Gb3 in A172 astrocytes ( right panel ). ( B ) Subsequently, the A172 cells in the bottom wells were washed, and the cell lysates were harvested and the protein levels of caspase-3 and cleaved caspase-3 were determined by Western blotting. Anti-caspase-3, anticleaved caspase-3, and anti-β-Actin antibodies were used for protein samples. β-Actin served as a loading control ( C-D ) Culture supernatants of A172 cells seeded in the same manner as above were harvested, and the secretion levels of IL-1β, IL-6, IL-8, TNF-α, and CCL2 were determined using ELISA. Subsequently, the mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1 and CCL2 were assessed by using RT-qPCR in cell lysates. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences between the control group and cells treated with Stx, Stx+z-VAD, and Stx+NAC (Panels A, C, D). * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Lactate Dehydrogenase Assay, Flow Cytometry, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, RNA Expression

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