Fig 1.
PAX2/5/8 domains share high levels of homology.
Schematic of full-length PAX5 protein. Equivalent domains of PAX2 and PAX8, indicated in key, are shown above. Distance from PAX5 represents level of homology to PAX5, scale at right. See also S1 Fig.
Fig 2.
PAX2 and PAX8 compensate for PAX5 loss-of-function by modulating developmental gene expression in pre-B ALL.
A) qRT-PCR of RNA/cDNA preparation from FACS of ZsGreen-positive Reh cells transduced with lentivirus containing transgenes indicated in key. B) 697 cells treated/harvested similarly. PAX2 and PAX8 levels are presented relative to baseline PAX5 due to the lack of detectable endogenous PAX2 or PAX8 (see Methods). Both A and B are representative of 3 separate experimental replicates. Error bars = standard deviation. Statistical significance derived using one sample t-test vs. empty vector, assuming unequal variation (EV = 1), p-values * <0.05, ** <0.005, *** <0.0005.
Fig 3.
PAX2 and PAX8 rescue immunophenotypic advancement of B cell differentiation in PAX5 loss-of-function pre-B ALL cells.
A) Representative histogram comparisons of developmental marker antibody staining intensity for ZsGreen-positive Reh cells transduced with lentivirus containing either empty vector (black outlines) or indicated PAX mutant or wild type transgenes (red-dotted outlines). Antibodies used for flow cytometry denoted beneath each panel. B) Relative mean fluorescence intensity for each antibody, from A, average of 3 separate experimental replicates each for Reh cells, and C) for 697 cells. Values relative to empty vector transduced cells. Error bars = standard deviation. Statistical significance derived using one sample t-test vs. empty vector (EV = 100%), p-values * <0.05, ** <0.005, *** <0.0005.
Fig 4.
PAX2 and PAX8 promote developmentally characteristic large-to-small B cell transition and exit from the cell cycle in PAX5-deficient pre-B ALL cells.
A) Representative histogram of FSC-A intensity for empty vector (black outlines) vs. PAX transduced (red-dotted outlines) Reh cells. B) Percent deviation from empty vector (set to 0) of mean FSC-A values for cells expressing indicated PAX mutant or wild type transgenes (see key) for an averaged 6 and 3 experimental replicates for Reh and 697 cells, respectively. C) Reh (3 experimental replicates) and D) 697 (2 experimental replicates) cell culture density vs. time, following sorting (day 4 post transduction) for ZsGreen-positive cells expressing indicated transgenes. Data points for all replicates are shown, along with lines fitting the mean values for each treatment. (-) ZsGreen cells represent unsuccessfully transduced cells sorted from the PAX5 lentivirus exposed cell suspension. E) Percentage of ZsGreen positive vs. negative cells at 11–14 days post sort for ZsGreen for 2 experimental replicates for per cell line. Error bars = standard deviation. Statistical significance derived using one sample t-test vs. empty vector, p-values * < .05, ** < .005, *** < .0005. See also S3, S4 and S5 Figs.
Fig 5.
Extracellular hyperosmolarity induces endogenous PAX2 and PAX5 expression in pre-B ALL cells.
A) Fold-expression of PAX2, PAX8 (none detected), and PAX5 mRNA in response to 24 hour exposure to 80mM treatments of indicated compounds in Reh cells. B) Fold-expression of downstream markers in response to treatments in A. C) Dose-response curve in Reh cells showing PAX2 and D) PAX5 mRNA expression in response to varying K-gluconate and CaCl2 concentrations. E) Relative PAX2, F) relative PAX5, and G) relative downstream gene levels following pulse chase, where x-axis represents the incubation time in normal media following 24 hours incubation in 80mM K-gluconate or CaCl2 and flow sorting for live cells via FSC-A/SSC-A. PAX2 values shown are 2-ΔΔCT, relative to vehicle-treated PAX5 levels (as there is no detectable baseline PAX2 expression). All other gene expression values are 2-ΔΔCT, relative to corresponding vehicle expression values. Error bars = standard deviation. Statistical significance derived using one sample t-test vs. vehicle treated, assuming unequal variation (vehicle = 1), p-values * <0.05, ** <0.005, *** <0.0005. A and B are each 3 averaged experimental replicates while C-G are each 2. All values shown are relative to ACTB as endogenous reference gene. See also S6A–S6C Fig for PAX amplification curves, S7A and S7B Fig for GAPDH normalized dose-response curves, and S7C and S7D Fig for 697 dose response to K-gluconate.
Fig 6.
RNA-seq data shows that PAX2, PAX8, K-gluconate, and CaCl2 affect pathways modulated by the restoration of PAX5.
A) Venn diagram of enriched gene sets in Reh samples transfected with PAX5, PAX2, or PAX8. B) Venn diagram of enriched gene sets in Reh cells transfected with PAX5 or exposed to either CaCl2 or K-gluconate (80mM). C) Venn diagram of common PAX2∩PAX5∩PAX8 and common PAX5∩CaCl2∩K-gluconate enriched gene sets. D) Gene set enrichment plots for the PAX5_PROB gene set adapted from [13]. E) Changes in expression of genes regulated by PAX5 in Reh cells and that are related to remission of B-ALL in mice. A black bar indicates the median gene log2 fold change for each sample. Liu et al. samples were first reported in [18]. See also S8–S11 Figs.
Fig 7.
Increasing PAX expression or osmolarity changes expression of genes related to B cell development.
A) Fold change heatmap of PAX5 related pro-B cell genes with PAX5 binding sites in the promoter. B) Fold change heatmap of PAX5 related mature B cell genes with PAX5 binding sites in the promoter. C) Fold change heatmap of genes involved in the PAX5 dependent transition of pro-B cells to mature B cells. Average gene expression across samples is illustrated to the left of each heatmap. The pro-to-mature B cell heatmap has been cut in half and displayed side-by-side due limited space. See also S8–S11 Figs.
Fig 8.
NFAT5 plays a role in hypertonicity mediated PAX2 expression.
A) qRT-PCR validation of RNA-seq data for NFAT5, BAFF-R, and BAFF. Graphs represent the average of two separate experimental replicates. Fold change values are 2-ΔΔCT, relative to each samples’ respective control (i.e., empty vector or untreated), with ACTB used as endogenous reference gene. B) Representative western blot of PAX5 and NFAT5 protein knockdown by siRNA. C) Fold expression of PAX2 (left scale), PAX5, NFAT5, and downstream genes (right scale) after treatment with (+/-) 80mM K-gluconate and (+/-) siRNA knockdown of PAX2, PAX5, PAX2/5, or NFAT5, for 3 experimental replicates. PAX2 values are 2-ΔΔCT, relative to vehicle-treated PAX5 levels. All other gene expression values are 2-ΔΔCT, relative to corresponding vehicle expression values. Protein lysates in part B were bulk harvested from treated cells while RNA in part C was isolated from live cells first flow sorted by FSC-A/SSC-A. Error bars = standard deviation. Statistical significance derived using one sample t-test vs. control treated, assuming unequal variance (i.e., vehicle = 1), p-values * <0.05, ** <0.005, *** <0.0005. See also S9 Fig for TonE elements at PAX2 and PAX5 loci, as well as S8 Fig for NFAT5 target solute channels in response to NFAT5 siRNA knockdown.
Fig 9.
PAX5 upregulation and downstream gene modulation in response to 80mM K-gluconate in a PAX5 mutant primary ALL sample, and cellular response to near clinical dosing of mannitol, support the potential of targeting hypertonicity response pathways in vivo.
A) qRT-PCR analysis of PAX2, PAX5, and several downstream genes for NSGS mouse passaged aliquots of primary patient sample in response to 24 hour treatment with 80mM K-gluconate. Shown are two experimental replicates from separately thawed aliquots. Cells were sorted by FSC-A/SSC-A for live cells prior to isolation/harvest of RNA. B and C) qRT-PCR analysis of gene expression in Reh cells in response to 24 hour treatment with 80 or 160mM mannitol, compared with 80mM K-gluconate. Shown are 3 experimental replicates. Values are 2-ΔΔCT, relative to vehicle-treated. Statistical significance derived using one sample t-test vs. vehicle treated, assuming unequal variance (vehicle = 1), p-values * <0.05, ** <0.005, *** <0.0005. See also S14A Fig for a single replicate of a non-NSG passaged primary cell sample and S14B Fig for Reh viability in response to 80mM and 160mM mannitol vs. 80mM K-gluconate or CaCl2.