Fig. 3: IKAROS regulates CD19 and CD22 surface expression in a dose-dependent and reversible manner.

A–C Relative IKAROS (A, n = 7), CD19 (B, n = 7), and CD22 (C, n = 2) median levels in IKAROS-degron models treated with increasing doses of asunaprevir. Values were measured by flow cytometry and normalized to DMSO-treated (0 µM) condition. N represents independent clones. Dots represent the mean value from two technical replicates. RFI relative fluorescence intensity. D–F IKAROS-degron models were treated with 10 µM asunaprevir for 7 days. Afterward, asunaprevir was withdrawn, and cells were either treated with DMSO (ASU/DMSO) or maintained with 10 µM asunaprevir (ASU/ASU) for an additional 28 days. As a control, IKAROS-degron models were treated with DMSO for 35 days. Relative levels of IKAROS (D), CD19 (E), and CD22 (F) were measured at 9, 14, 21, and 28 days post-asunaprevir withdrawal by flow cytometry and normalized to DMSO-treated condition. The experiment was performed in duplicate using two independent clones (n = 2). RFI relative fluorescence intensity. G IKAROS-degron models were treated with DMSO (D0; n = 6; 2 independent clones in 3 independent experiments) or 10 µM asunaprevir for 3 (n = 2 independent clones), 7 (n = 4; 2 independent clones in 2 independent experiments), and 22 (n = 2 independent clones) days. The protein profile was measured by flow cytometry and CyTOF and normalized to the D0 condition. H Projection of IKAROS-degron models treated with DMSO (left) or 10 µM asunaprevir (right) for 7 days onto the UMAP representation of healthy B cell developmental populations based on developmental classifier protein expressions. I Developmental classification of samples in (G). The frequencies of Pro-BII and Pre-BI populations were statistically different (p-value < 0.001) in the D0 condition against D3, D7, and D22 conditions. J, K Relative IK6 (A, n = 3), CD19 (B, n = 3), and CD22 (C, n = 3) median levels in IK6-degron models treated with increasing doses of asunaprevir. Values were measured by flow cytometry and normalized to DMSO-treated (0 µM) condition. N represents independent clones. Dots represent the mean value from two technical replicates. RFI relative fluorescence intensity.Bar plots in (A–F) and (J–L) show mean ± SEM. Boxplots in (G) extend from the 25th to the 75th percentiles, with a line in the middle representing the median and whiskers extending from the minimum to the maximum values. Curves in (I) show mean ± SEM. Statistical tests used were paired one-way ANOVA followed by Dunnett’s multiple comparisons test (A–C) and (J–L); and two-way ANOVA followed by Dunnett’s multiple comparisons test (D–G) and (I).