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Blood, Vol. 113, Issue 11, 2416-2425, March 12, 2009
PARP-14, a member of the B aggressive lymphoma family, transduces survival signals in primary B cells
Blood Cho et al.
113: 2416
Supplemental materials for: Cho et al
BrdU assay
Splenocytes were cultured (16 h) in the presence of anti-IgM or anti-IgM + IL-4, and then pulsed 16 h with 5-bromo-2′-deoxyuridine (BrdU; 10 µM). After staining with anti-B220, cells were fixed with paraformaldehyde (4%), permeabilized with 0.2% Saponin, 1% FBS in PBS, re-fixed (4% paraformaldehyde), treated (30 min at 37°C) with 50 U DNase I (Roche), stained with Alexa647–anti-BrdU Ab (Invitrogen), and analyzed by flow cytometry. TUNEL assays
Cells were fixed with 4% paraformaldehyde after surface staining with anti-B220, CD4, and CD8, permeabilized with 0.1% Triton-X-100–containing PBS, followed by standard reactions (30 min at 37°C) containing biotin-14-dCTP. Cells were then washed twice, stained with avidin-FITC (30 min on ice), washed twice, and analyzed. The extent of protection against apoptosis provided by IL-4 was analyzed both by the formula “% decrease in death” = {( % TUNEL+medium-only minus % TUNEL+IL-4–treated) divided by % TUNEL+medium-only and by calculation of “% rescued” = ( % TUNEL+medium-only) − (% TUNEL+IL-4–treated), yielding comparable and statistically significant results by each approach. Micro-array screening for PARP-14–dependent IL-4–regulated gene expression
WT and PARP-14 KO B cells were purified by depleting Thy1+ cells and cultured 20 h in the presence or absence of IL-4 (4 ng/ml). RNA was isolated with TriZol reagent. Total RNA was amplified with Illumina RNA TotalPrep Amplification kit (Ambion), hybridized to Illumina’s Sentrix Mouse-6 WholeGenome BeadChip Arrays at 55°C for 18 h, and detected with 1 µg/ml Cyanine3-streptavidin (Amersham Biosciences). Chips were scanned with Illumina BeadArray Reader. REFERENCES Youn J, Chen J, Goenka S, et al. In vivo function of an interleukin 2 receptor beta chain (IL-2Rbeta)/IL-4Ralpha cytokine receptor chimera potentiates allergic airway disease. J Exp Med. 1998;188:1803–1816. Stephenson L, Johns M H, Woodward E, Mora A L, Boothby M. An IL-4R alpha allelic variant, I50, acts as a gain-of-function variant relative to V50 for Stat6, but not Th2 differentiation. J Immunol. 2004;173:4523–4528.
Files in this Data Supplement:
- Table S1. List of PCR primers (PDF, 15.6 KB) -
PCR primers used for genotyping PARP-14 null and WT alleles, and RT-PCR analyses.
- Table S2. Percentage and number of TCRhi and CD44hi T cells (PDF, 46.7 KB)
- Table S3. Principal micro-array results for PARP-14–dependent gene regulation by IL-4 (PDF, 26.7 KB)
- Figure S1. PARP-14 is highly expressed in B lymphoma cell lines (JPG, 50 KB)
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Protein extracts of normal B cells and culture-adapted B lymphoma cell lines were analyzed by immunoblots using anti–PARP-14 and anti-actin Ab. Shown to the bottom is a bar graph of the results after quantitating the fluorescence intensity of the Western bands and normalizing each of these values for PARP-14 to the respective actin loading control.
- Figure S2. Equivalence of IL-4R expression and regulation (JPG, 50.3 KB)
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Freshly isolated samples of the indicated genotype were stained for B220, CD21, CD23, and M1–anti–IL-4Rα (left panels), or cultured overnight in the indicated conditions followed by staining for B220 and IL-4Rα (tetrad of panels to right). Shown in each panel is a representative histogram for M1 staining in the appropriate gate, with an unfilled line toward left indicating the signal for isotype control stains, and two virtually superimposed histograms (filled vs thick-edged) for anti–IL-4Rα on WT or PARP-14-null B cells as indicated. Insets indicate the MFI of each sample.
- Figure S3. IL-4–induced rescue of CD4 and CD8 T cells from apoptosis is PARP-14–independent (JPG, 50.7 KB)
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Splenocytes from WT and PARP-14 KO mice were cultured (20 h) in the presence or absence of IL-4 and analyzed by TUNEL after ‘death by neglect’ or irradiation (2 Gy). FACS profiles show TUNEL data (Avidin-FITC) from the CD4+ and CD8+ gates in one experiment representative of three independent replicates. Inset numbers indicate both the % of events TUNEL-positive in the samples, and MFI for the overall population.
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