Right panel: Similarity analysis between Hoechst 33258 and IRF-7 in untreated or CpG-stimulated CAL-1 cell variants. Values depicted in the histograms represent the percentage of cells with similarity values above an arbitrary value of 1.7 over a total of approximately 20.000 cells. Supporting Information Figure 3. NAB2 knowdown by siRNA reduces TRAIL induction in CpG treated CAL1 cells but does not affect CD40 expression. CAL-1 cells were transfected with siGLO transfection indicator
together with Ctrl siRNA or siRNA targeting NAB2 in a ratio of 1:3. (A) 48h post-transfection TRAIL expression of unstimulated, or CpG-stimulated CAL-1 cells was measured by flow cytrometry in the siGLO+ and total transfected cell populations. Numbers in the upper right corner represent TRAIL GeoMFI of CpG stimulated cells. (B) The knock-down of NAB2 protein of the total transfected cell population was assessed Doramapimod clinical trial by Western LY2157299 clinical trial blot analysis. (C) CD40 expression was measured by flow cytometry in siGLO+ (left panel) or in the total cell population (right panel). Numbers depict the percentage of CD40+ cells. Data are representative of 2 independent experiments. Supporting Information Figure 4. Activated CAL-1 NAB2E51K cells are less potent in inducing
apoptosis in Jurkat cells. (A) DDAO-labeled Jurkat cells were co-cultured for 20h with unstimulated or CpG stimulated CAL-1-EV, -NAB2, or -NAB2E51K cells. Active Caspase-3 was measured in Jurkat cells by CaspGLOW Red Active Caspase-3 Staining Kit. Data are representative of 2 independent experiments. Supporting Information Figure 5. Analysis of the specificity of inhibition of PI3K , p38MAPK, NF-kB and effects of
mTOR and PI3K pathways. (A-B) CAL-1 cells were pre-incubated for 30 min with PI-103 (PI), SB203580 (SB), and BAY11–7082 (Bay), DMSO (Ctrl) or left untreated (-), before being activated with CpG for 30min (A) or 1h (B). Protein expression of Akt, p38MAPK, NF-kB p65 and the respective phosphorylated forms (p-) were assessed by Western blot analysis. NAB2 induction is independent on mTOR. (C) CAL-1 cells were incubated for 30 min with PI-103 (PI) Montelukast Sodium or Rapamycin (Rap) followed by 4h activation with CpG. NAB2 mRNA levels were measured by RT-PCR. (D) CAL-1 cells were stimulated for 4h with CpG in the absence or presence of PI-103, and IFNβ mRNA levels were measured. Supporting Information Figure 6. Differential TRAIL levels in CAL-1-NAB2E51K cells are not correlated with NAB2E51K expression levels, but rather a consequence of not fully activated CAL-1 cells. (A) CAL-1- NAB2E51K cells were activated for 6h with CpG, and TRAIL expression levels were assessed by flow cytometry of the top GFP-expressing cells (GFP high) the bottom GFP-expressing cells (GFP low). Shaded plots represent unstimulated CAL-1-NAB2E51K cells.