A Commentary on USP50 and NLRP3 Inflammasome Activation: Revisiting Experimental Rigor with ASC-Deficient RAW264.7 Cells

The article "USP50 regulates NLRP3 inflammasome activation in duodenogastric reflux-induced gastric tumorigenesis" published in Frontiers in Immunology, has greatly piqued our curiosity. Zhao et al. describe how bile acids elevate ubiquitin-specific protease 50 (USP50) in macrophages, facilitating NLRP3 inflammasome assembly, HMGB1 release, and activation of PI3K/AKT and MAPK/ERK pathways that may promote gastric cancer progression. In their study, the authors used multiple cell lines for in vitro analyses, including the murine macrophage line RAW264.7, human monocytic U937 cells, and human embryonic kidney 293T cells. They reported detectable ASC protein in RAW264.7 cells and utilized U937 and 293T cells for key ASC-focused mechanistic assays (e.g., ASC-USP50 interaction, ASC specks formation, ASC oligomerization, and ASC ubiquitination); additionally, canonical readouts of inflammasome activation (including cleaved caspase-1 and mature IL-1β) were reported in RAW264.7 cells. However, ample evidence confirms that RAW264.7 cells lack ASC expression due to epigenetic silencing and are widely recommended as a negative control for ASC detection. Thus, the detection of ASC signals and downstream inflammasome activation in RAW264.7 cells is inconsistent with established characteristics of this cell line, and this discrepancy may stem from antibody cross-reactivity, cell contamination, or inaccurate cell line characterization. Notably, this limitation is specific to the RAW264.7-based ASC-dependent inflammasome experiments and does not affect the validity of other key findings in Zhao et al.’s study, including in vivo mouse data, clinical sample analyses, and investigations into the PI3K/AKT and ERK tumor-related pathways. Thus, verification of RAW264.7 cell line identity and ASC expression status is essential solely for consolidating the ASC-related inflammasome conclusions.