Figure 5.
Persistent cell swelling leads to caspase-3–dependent and type I IFN-independent cell death. (A and B) WT and VRAC KO BMDMs were incubated in iso-osmotic or hypo-osmotic media for the indicated time points. Cell viability was assessed via an MTT assay (A) and LDH release into the supernatant (B). LLOME (1 mM, 6 h) was used as a positive control of cell death in A (n = 7). (C and D) WT and VRAC KO BMDMs were incubated in iso-osmotic or hypo-osmotic media in the presence of STING inhibitors (H151, 10 µM), pan-caspase inhibitors (ZVAD-FMK, 50 µM), necroptosis inhibitors (RIPK1 inhibitor necrostatin, nec1, 50 µM), caspase-1/11 inhibitors (VX765, 10 µM), or vehicle control (DMSO, 0.5% vol/vol) (n = 6–8). (D) Caspase-Glo 3/7 assay (on combined cells and supernatant) in WT and VRAC KO BMDMs incubated in iso-osmotic or hypo-osmotic media for the indicated time points, or in the presence of ZVAD (50 µM, 6 h) (n = 4). (E) Western blot for caspase-3 and caspase-1 in WT and VRAC KO BMDMs incubated in iso- or hypo-osmotic media (6 h) (n = 3). (F and G) LDH release from WT and IFNAR KO BMDMs incubated in iso- or hypo-osmotic media (6 h) (F) (n = 3), and in the presence of ZVAD (50 µM) or vehicle control (DMSO, 0.5% vol/vol) (G) (n = 3). (H) Caspase-Glo 3/7 assay (on combined cells and supernatant) in WT and IFNAR KO BMDMs incubated in iso-osmotic or hypo-osmotic media in the presence of ZVAD (50 µM) or vehicle control (DMSO, 0.5% vol/vol) (6 h) (n = 3). (I) Western blot for caspase-3 in WT and IFNAR KO BMDMs incubated in iso- or hypo-osmotic media (6 h) (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, determined by a two-way ANOVA with Sidak’s post hoc analysis (5C and 5E), uncorrected Fisher’s LSD (5D), or Tukey’s post hoc analysis (5G and 5H). Values shown are the mean ± the SEM. Source data are available for this figure: SourceData F5. Refer to the image caption for details. Panel A shows a line graph depicting relative MTT values over time for WT and VRAC KO BMDMs in iso-osmotic and hypo-osmotic media. The x-axis represents time in hours, and the y-axis represents relative MTT values. Panel B shows a line graph depicting LDH release over time for the same conditions. The x-axis represents time in hours, and the y-axis represents LDH release as a percent of whole well lysis. Panel C shows a scatter bar plot comparing LDH release in WT and VRAC KO BMDMs under various treatment conditions. The x-axis lists the treatment conditions, and the y-axis represents LDH release as a percent of whole well lysis. Panel D shows a line graph depicting relative Caspase 3/7 activity over time for WT and VRAC KO BMDMs in iso-osmotic and hypo-osmotic media, with and without ZVAD treatment. The x-axis represents time in hours, and the y-axis represents relative Caspase 3/7 activity. Panel E shows Western blots for caspase-3 and caspase-1 in WT and VRAC KO BMDMs under iso-osmotic and hypo-osmotic conditions. Panel F shows a scatter bar plot comparing LDH release in WT and IFNAR KO BMDMs under iso-osmotic and hypo-osmotic conditions. The x-axis lists the conditions, and the y-axis represents LDH release as a percent of whole well lysis. Panel G shows a scatter bar plot comparing LDH release in WT and IFNAR KO BMDMs under iso-osmotic and hypo-osmotic conditions with and without ZVAD treatment. The x-axis lists the conditions, and the y-axis represents LDH release as a percent of whole well lysis. Panel H shows a scatter bar plot depicting relative Caspase 3/7 activity in WT and IFNAR KO BMDMs under iso-osmotic and hypo-osmotic conditions with and without ZVAD treatment. The x-axis lists the conditions, and the y-axis represents relative Caspase 3/7 activity. Panel I shows Western blots for caspase-3 in WT and IFNAR KO BMDMs under iso-osmotic and hypo-osmotic conditions.

Persistent cell swelling leads to caspase-3–dependent and type I IFN-independent cell death. (A and B) WT and VRAC KO BMDMs were incubated in iso-osmotic or hypo-osmotic media for the indicated time points. Cell viability was assessed via an MTT assay (A) and LDH release into the supernatant (B). LLOME (1 mM, 6 h) was used as a positive control of cell death in A (n = 7). (C and D) WT and VRAC KO BMDMs were incubated in iso-osmotic or hypo-osmotic media in the presence of STING inhibitors (H151, 10 µM), pan-caspase inhibitors (ZVAD-FMK, 50 µM), necroptosis inhibitors (RIPK1 inhibitor necrostatin, nec1, 50 µM), caspase-1/11 inhibitors (VX765, 10 µM), or vehicle control (DMSO, 0.5% vol/vol) (n = 6–8). (D) Caspase-Glo 3/7 assay (on combined cells and supernatant) in WT and VRAC KO BMDMs incubated in iso-osmotic or hypo-osmotic media for the indicated time points, or in the presence of ZVAD (50 µM, 6 h) (n = 4). (E) Western blot for caspase-3 and caspase-1 in WT and VRAC KO BMDMs incubated in iso- or hypo-osmotic media (6 h) (n = 3). (F and G) LDH release from WT and IFNAR KO BMDMs incubated in iso- or hypo-osmotic media (6 h) (F) (n = 3), and in the presence of ZVAD (50 µM) or vehicle control (DMSO, 0.5% vol/vol) (G) (n = 3). (H) Caspase-Glo 3/7 assay (on combined cells and supernatant) in WT and IFNAR KO BMDMs incubated in iso-osmotic or hypo-osmotic media in the presence of ZVAD (50 µM) or vehicle control (DMSO, 0.5% vol/vol) (6 h) (n = 3). (I) Western blot for caspase-3 in WT and IFNAR KO BMDMs incubated in iso- or hypo-osmotic media (6 h) (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, determined by a two-way ANOVA with Sidak’s post hoc analysis (5C and 5E), uncorrected Fisher’s LSD (5D), or Tukey’s post hoc analysis (5G and 5H). Values shown are the mean ± the SEM. Source data are available for this figure: SourceData F5.

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