Figure 4. CELSR2 knockdown decreases GR expression and attenuates glucocorticoid modulation of gene expression.

(a.) Volcano plot (n= 3 independent experiments) for untreated CELSR2 knockdown human leukemia cell line (NALM-6) vs. non-target control (NTC). Left side of plot depicts genes with reduced expression in CELSR2 knockdown cells and genes to the right exhibiting increased expression in CELSR2 knockdown cells. Orange and red symbols depict mRNAs with significant changes in expression (linear model p-value); red symbols have a fold change greater than 2. (b.) Volcano plot of gene expression after 24 hours of 10μM prednisolone treatment of CELSR2 knockdown vs. non-target control ALL cells (NALM-6, n=3 independent experiments; linear model p-value). (c.) (left) NR3C1 protein quantification (n= 2 independent experiments) or (right) Prednisolone LC₅₀ (n=3 independent experiments; mean ± SD) in NALM-6 NTC or shCELSR2 cells with either GFP control or re-introduction of NR3C1 (two tailed t-test p-value; * =p < 0.05, ** = p <0.01, *** = p <0.001). (d.) Representative western blot of GR protein expression (n= 2 independent experiments) in NTC or shCELSR2 NALM-6 cells with or without GR re-expression. (e.) The 75 most highly upregulated (top) or downregulated (bottom) genes in human NALM-6 ALL cells after 24 hours treatment with 10μM prednisolone (n= 3 independent experiments). Blue and green bars depict mRNA expression in NALM-6 cells transfected with non-target control vector and gold bars depict blunted induction or repression in cells expressing shRNA for CELSR2 knockdown (mean ± SD). (Inset) Representative western blot (n= 3 independent experiments) showing significantly lower GR levels in CELSR2 knockdown cell lines compared to non-targeting controls, with or without prednisolone treatment for 24hr (cropping performed uncropped image available as source data).