Fig. 7

SHQ1 regulation of MYC gene splicing and expression is important for T-ALL cell survival. a MYC minigene splicing was analyzed in 293T cells expressing control (Ctrl) or SHQ1 shRNA (sh1 or sh2) (top), and also in 293T cells expressing SHQ1 or empty vector in the presence of DMSO or GSI (Compound E, 1 μM) (bottom). Semi-quantitative RT-PCR was performed to analyze unspliced and spliced RNA. Splicing efficiency was calculated and shown as the ratio of spliced verse total RNA. b Schematic of MYC gene with three exons and two introns. Specific primer sets were designed to amplify pre-mRNA (E1–I1 and E2–I2) and mature mRNA (E1–E2 and E2–E3) (left). Endogenous MYC pre-mRNA and mature mRNA in HPB-ALL were analyzed by qPCR (right). Blue, red, or purple bars represent indicated RNA quantifications in control shRNA, SHQ1 shRNA-1, or -2-expressing cells. c Representative immunohistological images of MYC in spleen sections from mice bearing sgSHQ1-JURKAT cells injected with or without doxycycline (Fig. 4d). Scale bar, 50 μm. Histological stain was quantified using ImageJ and plotted on the right. d Immunoblots of SHQ1 and MYC in GFP⁺ cells from 3 fetal liver HSPC transplant samples in Fig. 5d. e HPB-ALL cells were infected with lentiviruses expressing control (Ctrl, blue) or SHQ1 shRNA (sh2, purple). Glucose uptake and lactate secretion were examined 4 days post infection, normalized to the same number of live cells. f Analysis of MYC target genes implicated in glycolysis by qPCR and immunoblots in control or SHQ1-deficient HPB-ALL cells 4 days post infection. Blue or purple bars represent indicated RNA quantifications in control shRNA, SHQ1 shRNA-2-expressing cells. g SHQ1 inducible-knockout JURKAT cells were overexpressed with MYC or blank control. Cell death was analyzed and quantified with (purple) or without (blue) doxycycline (Dox, 1 μg ml⁻¹). h Cell growth was assessed using murine T-ALL T6E and 8946 cells. T6E cells were infected with MSCV-shControl (Ctrl, black), MSCV-shmSHQ1 (shmSHQ1, blue), MSCV-shControl-MYC (MYC OE+Ctrl, red), or MSCV-shmSHQ1-MYC (MYC OE+shmSHQ1, purple) as denoted. Sorted GFP⁺ cells were used for cell growth analysis (left). In all, 8946 cells were infected with MSCV-shControl (Ctrl) or MSCV-shmSHQ1 (shmSHQ1). Sorted GFP⁺ cells were subjected to doxycycline treatment (1 μg ml⁻¹) to suppress human MYC transgene expression (MYC off+Ctrl, red; MYC off+shmSHQ1, purple), with PBS treatment as a control (Ctrl, black; shmSHQ1, blue), followed by cell growth analysis (right). Data shown represent the means (±SEM) of three biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001; n.s. non-significant, unpaired t-test (b, c, e, f, g, h)