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Blood, Vol. 112, Issue 7, 2927-2934, October 1, 2008
Overexpression of B cell–activating factor of TNF family (BAFF) is associated with Helicobacter pylori–independent growth of gastric diffuse large B-cell lymphoma with histologic evidence of MALT lymphoma
Blood Kuo et al.
112: 2927
Supplemental materials for: Kuo et al
Supplemental methods
For detection of mRNA of BAFF, total RNA was extracted from patients’ frozen specimens or NHL cell lines using the TRIzol (Invitrogen, Carlsbad, CA). For reverse transcription–polymerase chain reaction (RT-PCR), RNA was converted into cDNA using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia, Little Chalfont, Buckinghamshire, England) according to the manufacturer’s instructions. BAFF, and β-actin cDNAs were detected by PCR amplification with HotStarTaq (Qiagen, Valencia, CA) in steps of 1 minute each at 94°C, 60°C and 72°C for 35 cycles, using primers previously described as being specific for BAFF (5′ GGA GAA GGC AAC TCC AGT CAG AAC and 3′ CAA TTC ATC CCC AAA GAC ATG GAC).
Files in this Data Supplement:
- Figure S1. Expression of BAFF mRNA was analyzed by RT-PCR (JPG, 84.8 KB)
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Lane 1: H. pylori-dependent case without BAFF expression (immunohistochemical staining showed no BAFF expression). Lane 2: H. pylori-independent case with BAFF expression (immunohistochemical staining showed BAFF expression). Lane 3: H. pylori-independent case without BAFF expression (immunohistochemical staining showed no BAFF expression). Lane 4: Raji cell served as a negative control (no BAFF mRNA expression).
- Figure S2. Immunocytochemical and immunofluroscence staining for expression of pAKT, TNF-α, BCL3, BCL10, and NF-κB in Pfeiffer cell lines (JPG, 491 KB)
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Low expression of pAKT (A1, A3), TNF-α (B1, B3), and cytoplasmic expression of BCL3 (C1, C3), BCL10 (D1, D3), and NF-κB (E1, E3) were found in the tumor cells of pre-treated Pfeiffer cell lines. After 24 hours of 20ng/ml BAFF treatment, the increased expression of pAKT (A2, A4), TNF-α (B2, B4), and nuclear localization of BCL3 (C2, C4), BCL10 (D2, D4), and NF-κB (E2, E4) were found in tumor cells of post-treated Pfeiffer cell lines.
- Figure S3 (JPG, 132 KB)
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(A) BAFF induces a time course dependent pAKT expression, and nuclear translocation of BCL10, and BCL3.Following BAFF (20 ng/ml) treatment, the protein level of pAKT, BCL10, and BCL3 of Ramos lymphoma cells was determined by immunoblotting (IB). Staining with NF-κB p65, NF-κB p52 and β-actin served as loading control. (B) Inhibition of NF-κB activation decreases pAKT activation, c-myc activation, BCL3 nuclear translocation, and BCL10 nuclear translocation. Ramos cells were treated with 20ng/ml BAFF for 24hr with or without LY294002 1hr-pretreatment as indicated. The nuclear and cytoplasmic lysates were subjected to immunoblotting with anti-pAKT antibody, anti–c-myc, anti-BCL3 antibody, anti-BCL10 antibody, and anti–NF-κB p65 antibody. The membrane was reprobed with anti–β-actin antibody as a loading control.
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