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[Preprint]. 2024 Jun 6:rs.3.rs-4467983. [Version 1] doi: 10.21203/rs.3.rs-4467983/v1

Figure 1. Immunoproteasome inhibition reduces IFNγ-dependent complement gene activation.

Figure 1

Wild-type or β5i knockout BV-2 cells were treated with IFNγ in the absence and presence of immunoproteasome inhibitor, ONX-0914 and levels of C1q genes were analyzed. A. Gene expression analysis revealed a significant difference of C1qa gene expression between treatment groups ([F(5, 20) =9.21], p<.001, n=5). Post hoc analysis revealed that IFNγ resulted in a significant increase of C1qa compared to all other groups (control, p<.001; ONX-0914, p=.001; ONX+IFNγ, p<.01; KO control, p<.001; K.O. IFNγ, p<.001). C1qa levels were not increased by IFNγ, in β5i KO cells (p=.996, n=3). B. Gene expression analysis revealed a significant difference of C1qb gene expression between treatment groups ([F(5, 20) =10.56], p<.001, n=5). Post hoc analysis revealed that IFNγ resulted in a significant increase of C1qb (control, p<.001; KO control, p<.001; KO IFNγ, p<.05. C1qb levels were not increased by IFNγ, in β5i KO cells (p=.974, n=3). C. Gene expression analysis revealed a significant difference of C1qc gene expression between treatment groups ([F(5, 24) =10.56], p<.001, n=6). Post hoc analysis revealed that IFNγ resulted in a significant increase of C1qc (control, p<.001; ONX+IFNγ, p=.012; KO control, p<.001; K.O. IFNγ, p<.01. C1qc levels were not increased by IFNγ, in β5i KO cells (p>.999, n=3). D. Western blot analysis confirms that IFNγ treatment increases C1q protein levels in WT BV2 cells but not in β5i KO BV-2 cells. E. Analysis of C3 gene expression revealed a significant difference between groups (F(3,12)=15.76, p<.001, n=4). Post hoc analysis revealed that ONX-0914 treatment reduced C3 levels compared to control and IFNγ treatments (p<.01 and p≤.001, respectively). Further, ONX-0914 co-treatment with IFNγ reduced C3 levels compared to IFNγ alone, (p=.002). F. Gene expression analysis of C1qa, C1qb and C1qc in iPSC-derived microglia were determined by qRT-PRC (control n=4, SMA n=4; * p<0.05, ** p=0.01, *** p<0.001, **** P<.0001).