Figure 3. MARCH5 is essential for activating the NLRP3 inflammasome.

• A
  March5 ^fl/fl and March5 ^{fl/fl;Lyz‐Cre} BMDMs were untreated or treated with 200 ng/ml LPS for 4 h followed by 5 mM ATP for 45 min. The indicated proteins in the cell lysate and supernatants were immunoblotted.
• B
  The activity of caspase‐1 was detected using ELISA. March5 ^fl/fl and March5 ^{fl/fl;Lyz‐Cre} BMDMs were treated with 200 ng/ml LPS alone for 4 h or with an additional 5 mM ATP for 45 min. Supernatants of stimulated cells were collected and analyzed by ELISA. Each experiment was carried out in triplicate three times.
• C, D
  BMDMs from March5 ^fl/fl and March5 ^{fl/fl;Lyz‐cre} mice were primed with LPS (200 ng/ml) for 4 h. Cells were subsequently treated with 5 mM ATP (C) or with 15 μM nigericin (D). (C) Cell lysates were then immunoprecipitated with ASC antibody. Proteins were analyzed by immunoblotting with the indicated antibodies. The immunoprecipitated NLRP3 levels were quantified by using image J. (D) Triton X‐100 insoluble pellets were cross‐linked with 2 mM DSS and immunoblotted for ASC oligomerization. Analysis of Triton X‐100 soluble and insoluble proteins was performed by immunoblotting.
• E
  March5 ^fl/fl and March5 ^{fl/fl;Lyz‐cre} BMDMs were either transfected with 2 μg/ml poly(dA:dT) for 6 h or treated stimulated with 200 ng/ml LPS for 4 h along with 5 mM ATP or 15 μM nigericin for 30 min subsequently. Representative confocal images show ASC speck formation in BMDMs. The right graph shows the percentage of cells containing ASC specks. At least 100 BMDMs were analyzed in three independent experiments, showing representative data. White arrows indicate ASC speck.

Data information: Values are the mean ± SD. ***P < 0.001 (two‐tailed Student's t‐test). Bars, 10 μm. See also Fig EV4.

Source data are available online for this figure.