Fig. 2

Proteasome inhibitor enhanced viral replication requires monocytes. (A) Violin-plot showing (σ-NS) signal in 3 MM patients analyzed by CyTOF ∗p ≤ 0.05; (B-C) 34-Ab CyTOF panel used to generate FCS files. Hierarchical clustering and statistical mapping performed algorithmically via the Cytobank© platform. vi-SNE analysis (iterations = 3000, perplexity = 100) displayed in 2D plots using the resultant t-SNE 1 and t-SNE 2 dimensions. High-fidelity FlowSOM (“self-organizing map”) (metacluster = 10 and cluster = 100) based on vi-SNE 2D plots showing 22 different immune-compartments in total PBMCs of MM patients infected or not with RV (10 MOI). Red signal shows RV capsid (σ-NS) in monocytes (B). t-SNE heatmap highlighting density expression of selected RV capsid (σ-NS) signal (C); D-E) CyTOF high-fidelity FlowSOM in MM-PBMCs infected or not with RV (10 MOI) alone or in combination with CFZ (2.5 nM) and t-SNE heatmap, showing increased RV capsid (σ-NS) detection in Classical Monocytes after RV + CFZ co-treatment or RV + CFZ pretreatment (count: RV = 2890, RV + CFZ pretreatment = 4605, RV + CFZ cotreatment = 4702) (D), and heatmap graphical representation of RV absolute count detection in the different monocyte subsets (E); For each experimental condition the same number of events were acquired and analyzed. (F) q-RT-PCR for the viral genome expression of RV-infected HD-PBMCs or isolated HD-CD14+  population after 24 h, normalized to control GAPDH and expressed as the mean ± SEM of triplicates in fold change (FC) compared to RV alone; (G) Western blot analysis of (σ-NS) viral protein in HD CD14 + selected population after 8hrs of PIs (CFZ and BTZ, 2.5 nM) and RV treatments alone or in combination; (H) Offset histograms showing JAM-1 flow cytometry detection in different immune subsets as indicated. The experiment was repeated in n = 3 independent triplicate; (I) CD14+  cells were seeded and incubated with JAM-1 blocking Ab (10-50-100-150 ug/mL) for 1 h, then infected with RV 5 MOI for 24 h. q-RT-PCR for the viral genome expression was normalized to control GAPDH and expressed as the mean ± SEM of triplicates compared to RV alone ∗∗∗∗p ≤ 0.0001; (J) Western blot analysis on THP-1 showing RV (σ-NS) protein detection after specific JAM-1 knockdown; (K) Schematic representation of mice experiment: 12 immune competent myeloma mice (C57BL/KaLwRij) were injected intra-femoral with 1 × 10⁵ 5TGM1 murine MM cells and treated with 150 mg/kg clodronate-liposome for monocytes-macrophages depletion or control, then with or without intravenous injection of RV (5 × 10⁸ TCID₅₀) for 48 h; (L-M) Violin plots showing bone marrow RV (σ-NS) capsid formation (∗∗p ≤ 0.01) (L) of treated mice and in MM-CD138 + cells (∗∗p ≤ 0.01 and ∗p ≤ 0.05) (M), analyzed by flow cytometry