Fig. 2: Uba1M41T/y and Uba1∆Spl/y macrophages undergo aberrant inflammatory cell death.
From: Independent mechanisms of inflammation and myeloid bias in VEXAS syndrome
a, Cell death was measured over 24 h among BMDMs of the indicated genotypes treated with the indicated stimuli 9 days after electroporation, quantified as the percentage of YOYO-1+ cells among total cells, averaged across 3 images taken per well. Data are mean ± s.e.m. of triplicate wells per genotype and condition. P values were calculated using one-way ANOVA with Tukey’s multiple-comparison test. The timepoints used for statistical tests were as follows: t = 24 h (TNF, TNF + emricasan (Emr), TNF + IFNγ, LPS, LPS + IFNγ); t = 16 h (TNF + IAPi); t = 7 h (LPS + emricasan). b, Crispresso227 analysis of amplicon sequencingof the genomic region surrounding exon 3 splice (spl.) acceptor site of Uba1 (DNA) and of a Uba1 reverse transcription–PCR (RT–PCR) product generated from RNA, both isolated from BMDMs 5 days after electroporation with Cas9 ribonucleoprotein. The deletion frequency is shown in black at each nucleotide (nt) position with the reference sequence and gene structure depicted above. Bottom left, the relative frequency of reads containing deletions of the indicated lengths. Values are expressed as the percentage of all deletion-containing reads. Middle, schematic of the most commonly detected deletion events. Right, immunoblots of Uba1∆Spl/y BMDMs 8 days after electroporation. c, Cell death of BMDMs of the indicated genotypes over 24 h after indicated treatments 9 days after electroporation, quantified as the percentage YOYO-1+ cells among total cells. Data are mean ± s.e.m. of triplicate wells, with three images taken per well for quantification. d,e, Immunoblots of BMDMs treated with TNF plus emricasan (TE) (d) or either TNF or LPS alone or in combination with IAPi, IFNγ or emricasan (e) as indicated for 6 h. The results are representative of 2–3 independent experiments. Cl., cleaved; Unstim., unstimulated.
