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Supporting Information Figure 1. (A) NAB2 is induced in human pDCs upon CpG, but not upon type I IFN stimulation. Primary human pDCs were activated for 4h with 12.5 μg/ml CpG A or 200 ng/ml IFNα, and NAB2 protein levels were assessed. (B-F) CpG activated CAL-1 cells express CD40, IFNβ and MXA, and kill target cells in a TRAIL-dependent manner. CAL-1 cells were left untreated (-) or activated with Control CpG (Ctrl), CpG or IFNβ for 4h, and CD40 protein levels were measured by flow cytometry and compared with isotype control staining of CpG treated CAL-1 cells (B). mRNA levels of CD40 (C), IFNβ (D) and MXA (E) were assessed by RT-PCR. (F) CAL-1-EV cells were left untreated or CpG-activated for 6h prior to co-culture with DDAO-labeled Jurkat cells for 20h in a ratio 25:1. TRAIL-dependent killing was assessed by adding 10 μg/ml anti-TRAIL antibody to CAL-1 cells 30 min prior to the co-culture (CpG+αTRAIL). Apoptosis induction of DDAO+ Jurkat cells was assessed by AnnexinV or Active Caspase-3 stainings. Numbers represent the percentage of AnnexinV or Active Caspase-3 positive cells. Data are representative of at least 8 (B-D) or 2 (E-F) independent experiments (**p<0.005, ***p<0.001).

Supporting Information Figure 2. Activation of CAL1 cell variants with CpG results in comparable induction of CD40, TNF-α and IRF-7. CAL-1 cell variants were left untreated (Ctrl) or activated for 6h with CpG. (A) CD40 levels were assessed by flow cytometry. Left panel: one representative analysis of CD40 expression of one of 3 independently performed experiments combined in the right panel. (B) TNF-α and IL-6 cytokine expression were measured in the supernatant of 6h untreated or CpG-stimulated CAL-1 cell variants. (C) IRF-7 expression was measured by intracellular flow cytometry staining in CAL-1-EV, – NAB2, -NAB2E51K untreated or stimulated for o/n with CpG. The mean of GeoMFI of IRF-7 minus isotype control are shown. Data are representative of 3 independent experiments (*p<0.05, ***p<0.001). ND: not detectable.

Supporting Information Figure 2. IRF-7 nuclear translocation in CAL-1 cells is not affected by exogenous expression of NAB2 or NAB2E51K. (D-F) CAL-2-EV, -NAB2, or -NAB2E51K were left untreated (Ctrl) or stimulated with CpG for 6h, and IRF-7 translocation was measured with ImageStream technology. Left panel: representative images of Bright field, Hoechst 33258 (Blue), IRF-7 (Orange), and Hoechst 33258/IRF-7 of translocated or non-translocated IRF-7 staining in CAL-1 cells variants. 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 by Western 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 [7], 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) 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. Numbers in the upper right corner represent the percentage of TRAIL+ cells upon activation. (B) CAL-1 cells were activated with 5μg/ml of Imiquimod for indicated time points and TRAIL protein levels were assessed by flow cytometry. Data shown display gating strategy (top row) and time course is representative of 3 independently performed experiments.

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