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caspase 3/7 activity assay  (Promega)
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Promega caspase 3/7 activity assay
Inhibition of the unfolded protein response decreases pro-apoptotic UPR signaling. ( A ) Experimental set-up. Organoids were exposed to 12 hours of hypoxia without reoxygenation (0R), 30R, and 120R. ISRIB (0, 10, or 100 nmol/L) was added 2 hours before the start of hypoxia. Control samples were not exposed to hypoxia-reoxygenation (HR) (C). ( B ) Bright field images of human intestinal organoids during hypoxia-reoxygenation (images from the same culture are shown), and in control (magnification, 50×). ( C ) Messenger RNA (mRNA) expression of UPR-related genes CHOP, GADD34, ATF4, XBP1s, BiP, and HSPA1A . n = 3 per organoid line (N = 3). Data are presented as fold change compared with C, and shown in Tukey boxplot ( dots indicate outliers). ( D ) Apoptosis measurement using a <t>caspase</t> 3/7 activity assay (Promega). Results are presented as fold change in luminescence compared with C (Tukey boxplot). n = 4 per organoid line (N = 3). Kruskal–Wallis with the Dunn multiple comparisons test was used to compare HR conditions with C (∗indicates significance), and per time point 10 and 100 nmol/L ISRIB were compared with no ISRIB ( # indicates significance). ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001, # P < .05, ## P < .01, ### P < .001, and #### P < .0001.
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Inhibition of the unfolded protein response decreases pro-apoptotic UPR signaling. ( A ) Experimental set-up. Organoids were exposed to 12 hours of hypoxia without reoxygenation (0R), 30R, and 120R. ISRIB (0, 10, or 100 nmol/L) was added 2 hours before the start of hypoxia. Control samples were not exposed to hypoxia-reoxygenation (HR) (C). ( B ) Bright field images of human intestinal organoids during hypoxia-reoxygenation (images from the same culture are shown), and in control (magnification, 50×). ( C ) Messenger RNA (mRNA) expression of UPR-related genes CHOP, GADD34, ATF4, XBP1s, BiP, and HSPA1A . n = 3 per organoid line (N = 3). Data are presented as fold change compared with C, and shown in Tukey boxplot ( dots indicate outliers). ( D ) Apoptosis measurement using a caspase 3/7 activity assay (Promega). Results are presented as fold change in luminescence compared with C (Tukey boxplot). n = 4 per organoid line (N = 3). Kruskal–Wallis with the Dunn multiple comparisons test was used to compare HR conditions with C (∗indicates significance), and per time point 10 and 100 nmol/L ISRIB were compared with no ISRIB ( # indicates significance). ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001, # P < .05, ## P < .01, ### P < .001, and #### P < .0001.

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Temporal Transcript Profiling Identifies a Role for Unfolded Protein Stress in Human Gut Ischemia-Reperfusion Injury

doi: 10.1016/j.jcmgh.2021.11.001

Figure Lengend Snippet: Inhibition of the unfolded protein response decreases pro-apoptotic UPR signaling. ( A ) Experimental set-up. Organoids were exposed to 12 hours of hypoxia without reoxygenation (0R), 30R, and 120R. ISRIB (0, 10, or 100 nmol/L) was added 2 hours before the start of hypoxia. Control samples were not exposed to hypoxia-reoxygenation (HR) (C). ( B ) Bright field images of human intestinal organoids during hypoxia-reoxygenation (images from the same culture are shown), and in control (magnification, 50×). ( C ) Messenger RNA (mRNA) expression of UPR-related genes CHOP, GADD34, ATF4, XBP1s, BiP, and HSPA1A . n = 3 per organoid line (N = 3). Data are presented as fold change compared with C, and shown in Tukey boxplot ( dots indicate outliers). ( D ) Apoptosis measurement using a caspase 3/7 activity assay (Promega). Results are presented as fold change in luminescence compared with C (Tukey boxplot). n = 4 per organoid line (N = 3). Kruskal–Wallis with the Dunn multiple comparisons test was used to compare HR conditions with C (∗indicates significance), and per time point 10 and 100 nmol/L ISRIB were compared with no ISRIB ( # indicates significance). ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001, # P < .05, ## P < .01, ### P < .001, and #### P < .0001.

Article Snippet: Data are presented as fold change compared with C, and shown in Tukey boxplot ( dots indicate outliers). ( D ) Apoptosis measurement using a caspase 3/7 activity assay (Promega).

Techniques: Inhibition, Control, Expressing, Activity Assay