American Society of Hematology

Figure 2.

$Figure 2. PMA induces κ germline transcription and gene rearrangement in the pre-B-cell lines. (A) BKO84 cells were cultured with PMA (10 ng/mL) or solvent alone (-) for 2 days and then stained simultaneously for κ and λ chains and pre-BCR and analyzed by flow cytometry for the expression of κ and λ chains (top). Numbers indicate percentages of the total live cells gated by light scatters. The dose-response analysis showed that the induction of κ-positive cells reached plateau with 1 ng/mL PMA (data not shown). Also shown are the forward scatter (FSC) profile (lower left) and the pre-BCR expression (lower right) of untreated (dotted line) and PMA-treated (thick line) cells. (B) (Left) BKO84 cells were cultured with PMA (10 ng/mL) or solvent alone (-) for 2 days, and then the number of live cells was counted by trypan blue dye exclusion. Bar graph shows the mean ratio (± SD) of the cell number on day 0 to that on day 2 in 10 independent experiments. *Determined by Student unpaired t test. (Right) BKO84 cells were labeled with CFDA-SE, cultured with PMA (10 ng/mL) or solvent alone (-) for 60 hours, and then stained for κ and λ chain and analyzed by flow cytometry. Numbers indicate percentages of the total live cells gated by light scatters. (C) Schematic representation of Ig κ gene locus (top), an example of the rearranged locus (middle), and an example of aJκ-associated break ligated with the linker (bottom), with relative positions of the primers and the probes used in this study. Eκi and Eκ3′ indicate κ intronic and 3′ enhancers, respectively. (D) Purified genomic DNA from indicated cell lines treated with PMA for the indicated time (in hours) was analyzed for κ gene rearrangements by PCR (top) and for Jκ-associated DNA double strand breaks by LM-PCR (middle) followed by Southern blot hybridization. Primers and probes indicated in panel C were used for these assays. The estimated locations of the amplified bands corresponding to each VκJκ rearrangement (top) or each linker-ligated Jκ break (middle) are indicated on the right of the panels. The amount of genomic DNA used for these assays was monitored by PCR amplifying the 3′ region of RSS that is not compromised after the κ gene rearrangements. (E) Reverse transcriptase-PCR analyses of the indicated transcripts and mRNA. The PCR products were analyzed by agarose gel electrophoresis followed by EtBr staining. (F) BKO84 cells were cultured with PMA for 2 days, then sorted for κ positivity (κ+) or κ negativity (κ-) or cultured without PMA (-). Genomic DNAs from these fractions, or DNAs from WEHI279 and ES cells mixed at the indicated ratio, were treated with (+) or without (-) HhaI, together with EcoRI, and subjected to PCR with primers HhaI-5′ and HhaI-3′ indicated at the top of panel C. The amount of genomic DNA used for this assay was monitored as in the bottom of panel D.$

PMA induces κ germline transcription and gene rearrangement in the pre-B-cell lines. (A) BKO84 cells were cultured with PMA (10 ng/mL) or solvent alone (-) for 2 days and then stained simultaneously for κ and λ chains and pre-BCR and analyzed by flow cytometry for the expression of κ and λ chains (top). Numbers indicate percentages of the total live cells gated by light scatters. The dose-response analysis showed that the induction of κ-positive cells reached plateau with 1 ng/mL PMA (data not shown). Also shown are the forward scatter (FSC) profile (lower left) and the pre-BCR expression (lower right) of untreated (dotted line) and PMA-treated (thick line) cells. (B) (Left) BKO84 cells were cultured with PMA (10 ng/mL) or solvent alone (-) for 2 days, and then the number of live cells was counted by trypan blue dye exclusion. Bar graph shows the mean ratio (± SD) of the cell number on day 0 to that on day 2 in 10 independent experiments. ^*Determined by Student unpaired t test. (Right) BKO84 cells were labeled with CFDA-SE, cultured with PMA (10 ng/mL) or solvent alone (-) for 60 hours, and then stained for κ and λ chain and analyzed by flow cytometry. Numbers indicate percentages of the total live cells gated by light scatters. (C) Schematic representation of Ig κ gene locus (top), an example of the rearranged locus (middle), and an example of aJκ-associated break ligated with the linker (bottom), with relative positions of the primers and the probes used in this study. Eκi and Eκ3′ indicate κ intronic and 3′ enhancers, respectively. (D) Purified genomic DNA from indicated cell lines treated with PMA for the indicated time (in hours) was analyzed for κ gene rearrangements by PCR (top) and for Jκ-associated DNA double strand breaks by LM-PCR (middle) followed by Southern blot hybridization. Primers and probes indicated in panel C were used for these assays. The estimated locations of the amplified bands corresponding to each VκJκ rearrangement (top) or each linker-ligated Jκ break (middle) are indicated on the right of the panels. The amount of genomic DNA used for these assays was monitored by PCR amplifying the 3′ region of RSS that is not compromised after the κ gene rearrangements. (E) Reverse transcriptase-PCR analyses of the indicated transcripts and mRNA. The PCR products were analyzed by agarose gel electrophoresis followed by EtBr staining. (F) BKO84 cells were cultured with PMA for 2 days, then sorted for κ positivity (κ⁺) or κ negativity (κ^-) or cultured without PMA (-). Genomic DNAs from these fractions, or DNAs from WEHI279 and ES cells mixed at the indicated ratio, were treated with (+) or without (-) HhaI, together with EcoRI, and subjected to PCR with primers HhaI-5′ and HhaI-3′ indicated at the top of panel C. The amount of genomic DNA used for this assay was monitored as in the bottom of panel D.

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