Hemizygous expression of Uba1^M41L in a myeloid cell line models the biochemical, morphological, and inflammatory features of VEXAS syndrome. (A) Schematic depiction of the knock-in strategy used for generation of isogenic 32D cell lines with hemizygous expression of Uba1^WT and Uba1^M41L. (B) Proliferation of 32D cells with hemizygous expression of Uba1^WT and Uba1^M41L after 3-day stimulation with IL-3. A single representative experiment (left, mean ± standard deviation [SD] of 3 technical replicates) and calculated 50% effective concentration (EC₅₀) values (right, mean ± SD of 3 biological replicates) are shown. Statistical significance was determined using a 2-tailed unpaired t test with Welch correction. (C) Immunoblot for UBA1, ubiquitin (Poly-Ub), and UBA6 proteins in 32D cells with hemizygous expression of Uba1^WT and Uba1^M41L. Relative positions of UBA1a, UBA1b, and UBA1c protein isoforms are indicated in red lettering. Immunoblot for vinculin (VINC) was performed as a protein loading control. (D) Images of Hema 3–stained cytospin preparations of Uba1^WT and Uba1^M41L 32D cells. Scale bar, 50 μm. (E) Transmission electron microscopy images of Uba1^WT (left) and Uba1^M41L (middle and right) 32D cells. Arrow indicates vacuole depicted in right image. Scale bar, 500 nm. (F) Volcano plot showing differential cytokine secretion by unstimulated 32D Uba1^WT and Uba1^M41L cells. Three biological replicates of 2 single-cell clones per genotype were analyzed by multiplex cytokine assay in duplicate. Statistical significance was determined using multiple Mann-Whitney tests corrected by the Benjamini, Krieger, and Yekutieli false discover rate method. FC, fold change; WT, wild-type.