Figure 4. Proteasomal activity of PBMCs.
(A) Proteasome activity of PBMCs from CANDLE/PRAAS patients (turquoise, Pt.1; orange, Pt.2; purple, Pt.4 and Pt.5; red, CANDLE patients with PSMB8 mutations), patient family members or heterozygous carriers (gray), healthy controls (HC, black), patients with an undifferentiated interferonopathy disease who are negative for proteasome gene mutations (UID, green), and active pretreatment NOMID patients (blue) was analyzed and expressed as percentage relative to the average of healthy controls. The dashed lines indicate 100% activity. Whole blood RNA samples from the same date for patient and patient family members were analyzed by qRT-PCR for IFN-regulated gene expression and an IFN score was calculated. For patient 2, we used control samples from nonrelated heterozygous carriers for the PSMB8 T75M (HZMa, HZMb, and HZMc) for comparison. Measurements for patient 4, patient 5, and their parents were performed at only 1 time point before treatment. Patients with PSMB8 mutations, C1 (T75M/T75M), C2 (C135X/C135X), and C3 (T75M/A92T), were assessed as CANDLE controls. Two-sample, 2-tailed t tests were performed, and P values were stated. Error bars indicate technical triplicates. (B) Patient proteasome activity was normalized to the UID mean for each of the proteasome activities. Paired t test was performed. (C) PBMCs from 3 healthy donors were stimulated with the indicated cytokines or none for 6, 12, or 24 hours. The cell numbers were counted and a proteasome activity assay was done. Fold changes of activity against no-treatment control was calculated. The dashed lines on the graph indicate the activity of no-treatment controls. The numbers on top of the data points are IFN scores. Data represent mean ± SEM from n = 3 samples. Two-sample, 2-tailed t tests were performed. *P < 0.05.