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Blood, Vol. 110, Issue 9, 3387-3390, November 1, 2007
Somatic hypermutation of SOCS1 in lymphocyte-predominant Hodgkin lymphoma is accompanied by high JAK2 expression and activation of STAT6
Blood Mottok et al.
110: 3387
Supplemental materials for: Mottok et al
Files in this Data Supplement:
- Table S1. Primer for SOCS1 and JAK2 PCR (PDF, 11.2 KB) -
Single micromanipulated cells were digested with Proteinase K (0.05 U) in 20 µl 1× PCR buffer (Roche, Mannheim, Germany) without MgCl2 for 3 hours at 55°C. Proteinase K was subsequently inactivated by incubation for 10 min at 95°C. First round PCRs were performed in 50 µl in 1× PCR buffer with 2 mM MgCl2 (Roche), 2 mM dNTPs (Amersham, Freiburg, Germany), Betain (1 M) and 0.02 µM of each of the 6 first round primers (SOCS1-NTR-US1, -NTR-LS1, -CR-US1, -CR-LS1, -CTR-US1, -CTR-LS1), or 0.125 mM of SOCS1-NTR-US1 and -NTR-LS1 when the N-terminal SOCS1 fragment was amplified alone and JAK2-Ex12-US1 and JAK2-Ex12-LS1 when JAK2 was amplified. After an initial cycle of 2 min at 95°C, 30 sec at 61°C and 1 min at 72°C, 34 cycles of 30 sec 95°C, 30 sec 61°C and 45 sec 72°C were performed. One µl aliquots of the first rounds were used as templates for second rounds in which the 3 fragments were separately amplified. Second round PCR was performed as above but with 0.125 µM each primer and 40 PCR cycles.
- Table S2. PCR results and sequence analysis of SOCS1 (PDF, 46.8 KB) -
NTR: N-terminal region PCR fragment; CR: central region PCR fragment; CTR: C-terminal region PCR fragment; del: deletion; dup: duplication
Only cases with mutations are shown. For cases bearing mutations in more than 1 of the different SOCS1 fragments amplified (cases 4, 5 and 6) the cells from which the PCR products were amplified are specified by their numbers (assigned during the micromanipulation procedure). For the 6 cases without mutations each SOCS1 fragment was at least amplified and sequenced from 4 different cells. For each 10 cells 4 aliquots of buffer covering the sections were used as negative controls. From 2 of 10 buffer controls of case 5 CR fragments were amplified which were unmutated. For the lpHL derived cell line DEV single round PCRs with 100 ng genomic DNA as templates were performed. The mutations in the NTR and CTR fragments were obtained as single peaks in the electropherogramms while for the CR fragment the unmutated sequence was amplified in addition to the mutated sequence. This indicates that in DEV 2 alleles of SOCS1 are present, both containing all NTR and CTR mutations, but only one containing the 12 bp deletion in the CR fragment. The sequences of the NTR, CR and CTR of case 4 and the CR of case 5 contained frequently double peaks at sites of mutations due to presence of also the non-mutated germline sequence. Numbering of nucleotides and amino acids refers to GenBank NM_003745. The coding sequence encompasses nucleotides 155-790. For replacement mutations the amino acid changes are shown in brackets.
Among the somatic mutations 32 were replacement and 7 silent mutations (ratio 4.6) and 3 del/dup were observed. Of the 39 mutations 10 (25.6%) affected the G of the RGYW somatic hypermutation hotspot motif (on both strands). Guanins in RGWY hotspot motifs represent 39 residues of the 699 nucleotides amplified of SOCS1 (5.6%)(on both strands) and guanins in hotspots were thus 4.5 times more frequently affected than expected if mutations were randomly distributed. Nineteen mutations were transitions and 20 were transversions.
- Figure S1. Specificity test for the anti-JAK2 antibody (JPG, 25 KB)
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Immunohistochemistry (IHC) and Western blot analysis (WB) was performed with 2 breast cancer cases, from which formalin-fixed paraffin-embedded tissues for IHC (A) and frozen material for WB analysis were available (B). A band of expected length in WB analysis was only observed with the lysate of the case positive in JAK2 IHC.
- Figure S2. Specificity test for the anti-p-STAT6 antibody (JPG, 42.3 KB)
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(A) Formalin-fixed paraffin-embedded pellets of cell lines L428, L1236, NCEB1 and RAJI were used for IHC with the anti-p-STAT6 antibody and a signal amplification system was used for visualisation of bound antibody. In line with previous reports, positivity was observed with both Hodgkin lymphoma cell lines L428 and L1236. RAJI and NCEB1 showed no positivity in IHC. (B) In WB analysis bands of expected length were only observed with lysates from L428 and L1236.
- Figure S3. Specificity test for p-JAK2 antibodies (JPG, 118 KB)
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A lung adenocarcinoma was stained for JAK2. As shown in (A) only the tumor iniltrate showed positivity for JAK2. Using two different anti-p-JAK2 antibodies ((B) and (C) from Cell Signaling; (D) and (E) from Chemicon) the same staining pattern restricted to parts of the JAK2 positive region was observed. Pretreatment of the sections with T cell phosphatase abolished p-JAK2 staining completely ((C) and (E)) while JAK2 staining was not affected (F). In Western blot analysis with both antibodies bands of expected length were only observed with the cell lines DEV and Karpas 1106P, which carry SOCS1 mutations while no band was detected with lysates from KMH2, which does not carry SOCS1 mutations (Figure 1E and data not shown). All 5 JAK2 negative lpHL cases stained with p-JAK2 antibodies were negative.
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