Table S1. Oligonucleotide primer sequences Primer Name DNA Sequence (5 -> 3 ) Description CD19c CD19d Cre7 Sfpi1lox1 Sfpi1lox2 Sfpi1lox3 5 -AACCAGTCAACACCCTTCC-3 5 -CCAGACTAGATACAGACCAG-3 5 -TCAGCTACACCAGAGACGG-3 5 -CTTCACTGCCCATTCATTGGCTCATCA-3 5 -GCTGGGGACAAGGTTTGATAAGGGAA-3 5 -CAACCGGATCTAGACTCGAGGA-3 CD19 Cre genotyping CD19c+CD19d product size: 452 bp (wild type allele); CD19c + Cre7 product size: ~700 bp (knockin allele) Sfpi1 lox genotyping wild type product 395 bp; lox allele product size: 533 bp. Spib forward Spib reverse PGK reverse sfpi1 exon 34 forward sfpi1 exon 34 reverse 5' -GGGCTCCTTGGCTTATGCTCC -3' 5' CGCGCTGTCAAACTGGTAGGT-3' 5 -CAGAAAGCGAAGGAGCAAAGCTG-3 5 -CGAGTTTGAGAACCTCCCTGAGAACCAC -3 5 - GGACAAGGTTTGATAAGGGAAGCACATCCG - 3 Spib - genotyping Wild type product 950 bp; knockout allele product size: 1100 bp Primer pair to amplify intron between Sfpi1 exons 3 and 4 Product size: 995 bp spfi1 exon 5 5 - AGACAGGCGAGGTGAAGAAAGTCA -3 forward spfi1 exon 5 reverse 5 - GGCACACACATGGCACACATACAT -3 Primer pair to amplify between Sfpi1 exons 5 and 3 UTR Product size: 1667 bp front internal sequencing end internal sequencing 5 - TCAAGAGTTTGCGTGTGCCG -3 Sequencing primer for Sfpi1 exons 3 to 4 PCR product 5 - CCTCAGGAAACACAAACCC -3 Sequencing primer for Sfpi1 exon 5 to 3 UTR product lox-exon4 forward 5 - ATGATCGGAATTCCTCGACGGT -3 qpcr primers to detect intact Sfpi1 lox allele frequency lox-exon4 reverse 5 - TTCCAACTGTGGCCTCTGGTTT -3 Product size: 85 bp cko1 5 - CTTCACTGCCCATTCATTGGCTCATCA -3 cko2 5 - CAACCGGATCTAGACTCGAGGA -3 qpcr primers to detect deleted Sfpi1 lox allele (Sfpi1Δ) frequency Product size: 298 bp
β-actin promoter fwd β-actin promoter rev 5 -CCCCGCGTGTCCCTC-3 5 -AACCACCCCAGGACCCTC-3 qpcr primers to detect intact β- actin promoter gene frequency Product size: 122 bp RT-q PCR primers Gapdh fwd 5 -GAACATCATCCCTGCATCCA-3 Gapdh rev 5 -CCAGTGAGCTTCCCGTTCA-3 Product size: 78 bp IL7αR fwd 5 -ACGATCACTCCTTCTGGTGC -3 IL7αR rev 5 - GCATTTCACTCGTAAAAGAGCC-3 Product size: 140 bp VpreB1fwd 5 -ACGTCTGTCCTGCTCATGCT-3 VpreB1 rev 5 -TGTTATGGTCGTTGCTCAGG-3 Product size: 136 bp CD23 fwd 5 - TGGCTCCATTTCCAACAGAAGTGC-3 CD23 rev 5 - ATTGAGATCCTGGAGGCCAATCCA-3 Product size: 183 bp CD19 fwd 5 -ACCAGTTGGCAGGATGATGGACTT -3 CD19 rev 5 -TTCATGACTGGGACCGGACTGAAT -3 Product size: 160 bp Pax5 fwd 5 -CGGGTCAGCCATGGTTGTG -3 Pax5 rev 5 -GTGCTGTCTCTCAAACACG-3 Product size: 561 bp Btk fwd 5 -GCTCTGTAGGCTCCAAGTTTC -3 Btk rev 5 -ATCTCTCATACGGCATCTTCC-3 Product size: 144 bp Blnk fwd 5 -TCCAAGTCATCTTTGCCTGCC -3 Blnk rev 5 -TGCATTCGGTACGGGAGGAAC -3 Product size: 102 bp E12 fwd 5 -GGGAGGAGAAAGAGGATGA -3 E12 rev 5 -GCTCCGCCTTCTGCTCTG-3 Product size: 138 bp E47 rev 5 -CCGGTCCCTCAGGTCCTTC-3 Combined with E12 fwd. Product size: 138 bp Id2 fwd 5 -CACAGAGTACTTTGCTATCATTCG-3 Id2 rev 5 -CCTGAACACGGACATCAGC-3 Product size: 83 bp Id3 fwd 5 -CACTTACCCTGAACTCAACGCC-3 Id3 rev 5 -CCCATTCTCGGAAAAGCCAG-3 Product size: 160 bp
Figure S1. Analysis of gene expression in sorted follicular B cells (A) Changes in frequencies of mrna transcripts in FO B cells lacking Spi-B or both PU.1 and Spi-B. Cell sorting was used to enrich B220 + CD93 IgM + CD23 + follicular (FO) B cells from the spleen of 9-week old Wild Type, CD19 + + Sfpi1 lox lox Spib, or CD 19+/Cre Sfpi1 lox lox Spib mice. Gating was performed as shown in Fig. 1D, upper panels. RNA was prepared from each sample and RT-qPCR used to measure relative frequencies of steady-state mrna transcript levels for genes indicated on the x-axis. Transcript frequencies were normalized to Gapdh transcript levels. The y-axis represents fold change in transcripts in CD19 + + Sfpi1 lox lox Spib FO B cells or CD19 +/Cre Sfpi1 lox lox Spib FO B cells relative to Wild Type FO B cells. (B) Changes in frequencies of mrna transcripts in Sfpi1 + Spib FO B cells. Cell sorting was used to enrich (FO) B cells from the spleen of 9-week old Wild Type or Sfpi1 + Spib (PUB) mice as described above. The y-axis represents fold change in transcripts in Sfpi1 + Spib FO B cells relative to Wild Type FO B cells. Figure S2. Blastic lymphocytes are present in spleen, lymph node, bone marrow, and blood of moribund CD19 +/Cre Sfpi1 lox lox Spib mice (A) Histology of the spleen in moribund CD19 +/Cre Sfpi1 lox lox Spib mice (original magnification, 60 ). (B) Histology of an enlarged lymph node in moribund CD19 +/Cre Sfpi1 lox lox Spib mice (original magnification, 60 ). (C) Histology of the bone marrow in moribund CD19 +/Cre Sfpi1 lox lox Spib mice (original magnification, 60 ) (D) Elevated lymphocyte counts in the blood of 18-20 week old CD19 +/Cre Sfpi1 lox lox Spib mice. Complete blood cell counts were performed on mice of the indicated genotypes. A legend is provided on the right; EO, eosinophils; MO, monocytes; LY, lymphocytes; NE, neutrophils. Basophils were counted but are not shown (<50/µl).
Figure S3. High frequency of pre-b cells in the thymus, spleen, and lymph node of moribund CD19 +/Cre Sfpi1 lox/lox Spib -/- mice. (A) High frequency of pre-b cells in the thymus of a 24-week old CD19 +/Cre Sfpi1 lox/lox Spib -/- mouse. Flow cytometric analysis was used to analyze expression of cell surface CD4, CD8, CD19, and B220 on thymocytes from a 6-week old C57Bl/6 mouse as a control (left panels) or a 24-week old CD19 +/Cre Sfpi1 lox/lox Spib -/- mouse (right panels). (B) Pre-B cells in the thymus of CD19 +/Cre Sfpi1 lox/lox Spib -/- mice express CD44, BP-1, and low levels of CD45. Flow cytometric analysis was used to analyze expression of cell surface CD19, CD44, BP-1, and CD45.2 on thymocytes from a 20-week old CD19 +/+ Sfpi1 lox/lox Spib -/- mouse (left panels) or a CD19 +/Cre Sfpi1 lox/lox Spib -/- mouse (right panels). (C) High frequency of pre-b cells in the spleen of 24-week old CD19 +/Cre Sfpi1 lox/lox Spib -/- mice. Flow cytometric analysis was used to analyze expression of cell surface CD19, B220, and CD93 on splenocytes from a 6-week old C57Bl/6 mouse (left panels) or a 24-week old CD19 +/Cre Sfpi1 lox/lox Spib -/- mouse (right panels). (D) High frequency of pre-b cells in an enlarged lymph node of a 24-week old CD19 +/Cre Sfpi1 lox/lox Spib -/- mouse. Flow cytometric analysis was used to analyze expression of cell surface CD19, B220, and CD93 on cells from the enlarged lymph node of a 24-week old CD19 +/Cre Sfpi1 lox/lox Spib -/- mouse.