American Society of Hematology

Figure 5.

$PAX5 acts as a negative regulator of cell proliferation and MYC function in pro-B cells. (A) Cell recovery 3 days after seeding of 2000 sorted GFP+CD45+ in vitro expanded FL cells from Ebf1−/− mice exposed to nuclear EBF1 by cultivation in 4-OHT for 4 or 7 days before sorting and reseeding in cultures in the absence of 4-OHT. Mean and standard deviation (SD) are shown; n = 5 to 6, from 2 independent experiments. Total cell recovery (B) and the fraction of CD19+ cells (C) recovered 4 days after seeding 2000 GFP+Ebf1−/− FL cells transduced with either MIG-control, EBF1, or PAX5 encoding virus. Mean and SD are shown; n = 7, from 2 independent experiments. (D) Cell cycle data from Ebf1−/− FL cells transduced with MIG-control, EBF1, or PAX5 encoding virus as determined by FACS analysis. Mean and standard error of the mean are shown; n = 3. (E) Quantitative reverse transcription PCR analysis determining the levels of Myc transcripts in live sorted Wt FL cells or transduced EBF1-deficient cells as in panel B. Mean and SD are shown, n = 4, from 4 samples. (F) The ratio of MYC median fluorescent intensity (MFI) of each stage of the cell cycle compared with G0 in Ebf1-deficient cells transduced with EBF1- or PAX5-encoding vectors. Mean and standard error of the mean are shown; n = 3. (G) Gene set enrichment analysis of genes in HALLMARK_MYC_TARGET_V1 gene set based on RNA-sequencing data normalized per reads per kilobase of transcript, per million mapped reads from Ebf1−/− FL cells transduced with a control GFP or PAX5 encoding virus. (H) Representative histograms of Ebf1−/− BM cells rescued to the CD19+ pro–B-cell stage by transduction with either a conventional or ER-fused EBF1-encoding retrovirus and transduced with a Cas9-encoding virus alone or in combination with a Pax5-targeting gRNA. (I) MFI values for PAX5 levels as determined by flow cytometry. Mean and SD are shown; n = 4, from 4 individual samples. (J) Cell recovery after 3 days of in vitro culture of EBF1- or EBF1-ER–transduced cells expressing Cas9 alone or Cas9 in combination with gRNAs targeted to the Pax5 gene (gPAX5). Mean and SD are shown; n = 4, from 4 individual samples. For panels A-E and I-J, each dot indicates a data point, and the statistical analyses are based on a Student unpaired t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. FDR = false discovery rate; NES = normalized enrichment score.$

PAX5 acts as a negative regulator of cell proliferation and MYC function in pro-B cells. (A) Cell recovery 3 days after seeding of 2000 sorted GFP⁺CD45⁺ in vitro expanded FL cells from Ebf1^−/− mice exposed to nuclear EBF1 by cultivation in 4-OHT for 4 or 7 days before sorting and reseeding in cultures in the absence of 4-OHT. Mean and standard deviation (SD) are shown; n = 5 to 6, from 2 independent experiments. Total cell recovery (B) and the fraction of CD19⁺ cells (C) recovered 4 days after seeding 2000 GFP⁺Ebf1^−/− FL cells transduced with either MIG-control, EBF1, or PAX5 encoding virus. Mean and SD are shown; n = 7, from 2 independent experiments. (D) Cell cycle data from Ebf1^−/− FL cells transduced with MIG-control, EBF1, or PAX5 encoding virus as determined by FACS analysis. Mean and standard error of the mean are shown; n = 3. (E) Quantitative reverse transcription PCR analysis determining the levels of Myc transcripts in live sorted Wt FL cells or transduced EBF1-deficient cells as in panel B. Mean and SD are shown, n = 4, from 4 samples. (F) The ratio of MYC median fluorescent intensity (MFI) of each stage of the cell cycle compared with G₀ in Ebf1-deficient cells transduced with EBF1- or PAX5-encoding vectors. Mean and standard error of the mean are shown; n = 3. (G) Gene set enrichment analysis of genes in HALLMARK_MYC_TARGET_V1 gene set based on RNA-sequencing data normalized per reads per kilobase of transcript, per million mapped reads from Ebf1^−/− FL cells transduced with a control GFP or PAX5 encoding virus. (H) Representative histograms of Ebf1^−/− BM cells rescued to the CD19⁺ pro–B-cell stage by transduction with either a conventional or ER-fused EBF1-encoding retrovirus and transduced with a Cas9-encoding virus alone or in combination with a Pax5-targeting gRNA. (I) MFI values for PAX5 levels as determined by flow cytometry. Mean and SD are shown; n = 4, from 4 individual samples. (J) Cell recovery after 3 days of in vitro culture of EBF1- or EBF1-ER–transduced cells expressing Cas9 alone or Cas9 in combination with gRNAs targeted to the Pax5 gene (gPAX5). Mean and SD are shown; n = 4, from 4 individual samples. For panels A-E and I-J, each dot indicates a data point, and the statistical analyses are based on a Student unpaired t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. FDR = false discovery rate; NES = normalized enrichment score.

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