Fig 2: Overexpression of SPI1 in CCL7-low cells promotes macrophage migration and M2 polarization. mRNA (a) and protein (b) levels of SPI1 and CCL7 in H125CCL7Low and A549CCL7High cells after SPI1 overexpression plasmid or shRNA transfection detected by qPCR and WB analyses; (c) number of migrated THP-1 cells examined by crystal violet staining; mRNA (d) and protein (e) levels of CD86 and CD206 in cocultured THP-1 cells examined by qPCR and WB analyses; (f) expression of M1 cytokines (IL-6, IL-1ß, and TNF-a) and M2 cytokines (IL-10 and TGF-ß) in the culture medium determined by ELISA kits. Repetition = 3. Data are presented as the mean ± SD. **p < 0.01.

Fig 3: RAG DSB Signals Induce Genome-wide Changes in PU.1 Binding(A) qPCR analysis of Igk genomic DNA from Rag1-/-:Bcl2 (red) and Art-/-:Bcl2 (blue) abl pre-B cells treated with imatinib for 48 h. Schematic shows germline (GL) Igk locus and unrepaired J?1 coding end with location of PCR primers. PCR is normalized toRag1-/-:Bcl2 abl pre-B cells, which do not generate RAG DSBs and have only intact germline Igk DNA. Data are representative of three independent experiments.(B) Dot plot and heatmap of fold changes and signal Intensity for PU.1 peaks Identified by ChIP-seq In Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells treated with Imatinib for 48 h. Data are from common peaks identified in two replicates for each cell.(C) Representative tracks at indicated regions for PU.1 ChIP-seq from (B). ChIP-qPCR validation for PU.1 binding at each locus is also shown. Data are mean and SE for three independent experiments. **p = 0.01 and ****p = 0.0001; ns, not significant.See also Figure S1.

Fig 4: SPI1 promotes CCL7 transcription. (a) Transcription factors that can bind to CCL7 promoter with an over 0.5 correlation coefficient in TCGA-LUAD analyzed using the R TFBS package; (b) binding sites between SPI1 and CCL7 promoter; (c) conservative binding sequence of SPI1; (d) a sketch map for the fragmentation of CCL7 promoter sites using the Crispr-Cas9 system; mRNA (e) and protein (f) levels of CCL7 in A549 cells after knockout of A, B, and C fragments alone or simultaneously; (g) CCL7 promoter fragments enriched by anti-SPI1 examined by the ChIP-qPCR assay; (h) construction of the pGL4-Luc luciferase vector containing the CCL7 promoter sequence for the examination of SPI1-CCL7 binding by luciferase assay. Repetition = 3. Data are presented as the mean ± SD. **p < 0.01.

Fig 5: BCLAF1 Regulates SYK Expression in Primary Pre-B Cells(A-C) Art-/-:µIgh:Bcl2 pre-B cells were transduced with a retrovirus expressing a scrambled shRNA (-) or shBclaf1 (+) and then subsequently withdrawn from IL-7.(A and B) Spic and Syk mRNA expression assessed in indicated small pre-B cells 2 days after IL-7 withdrawal. Data are mean and SE for three independent experiments.(C) Western blot of SYK and BCLAF1 in indicated small pre-B cells 2 days after IL-7 withdrawal. Data are representative of three independent experiments.(D) Flow cytometric analysis showing EGFP (y axis) and FSC (x axis) in bone marrow pre-B cells (B220loCD43-IgM-) from wild-type and Spicigfp/igfp mice. Data are representative of five independent experiments.(E) Percentage of EGFP-positive small pre-B cells in Spicigfp/igfp (circles) and Atm-/-:Spicigfp/igfp (squares) mice was quantified by flow cytometry as in (D). Data are mean and SE from three independent mice of each genotype.(F–H) Syk mRNA expression (F), ChIP-PCR of PU.1 at Syk promoter (G), and ChIP-PCR of BCLAF1 at Syk promoter (H) in EGFP-negative (-) and EGFP-expressing (+) small pre-B cells sorted from Spicigfp/igfp mice. Data in (F) are the mean and SE from three independent experiments. Data in (G) and (H) are representative of two independent experiments.*p = 0.05, **p = 0.01, ***p = 0.001,****p = 0.0001; ns, not significant.