Fig 1: PGK1 and IGFBP2 are secreted miR-10b targets(A) The levels of PGK1 and IGFBP2 proteins are increased in CM of miR-10b-edited GSCs and glioma cells. Naive GBM8, GBM4, and LN229 cells were transduced with either EV (Cas9 only), G1, or G3 (miR-10b-editing) lentivirus, or UT, for 5 days, and the CM samples collected and analyzed by western blotting. Ponceau S staining was used as loading control. (B) miR-10b mimic regulates luciferase reporter containing a single miR-10b complementary site (mean ± SD, n = 5 samples/group, two-tailed unpaired t test). Scramble mimic (sc-mimic) was used as a control. (C) miR-10b mimic reduces the activity of luciferase reporters of PGK1 and IGFBP2 with several putative miR-10b-binding sites (mean ± SD, n = 5 samples/group, two-tailed unpaired t test). The detailed description of specific reporter constructs is in the materials and methods. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns-not significant.
Fig 2: PGK1 and IGFPB2 promote glioma cell death(A) Recombinant human PGK1 (rhPGK1) inhibits the growth of glioma cells. Sequentially diluted rhPGK1 was added to LN229 cultures, and cell viability was evaluated after 7 days by WST-1 assays (mean ± SEM, n = 5 samples/group, one-way ANOVA). (B) Recombinant hPGK1 inhibits the growth of GSCs. Sequentially diluted rhPGK1 was added to GBM8 and GBM4 cells and their growth monitored over 7 days and the resulting spheroids analyzed. Representative bright-field images and data quantification are demonstrated (mean ± SEM, n = 3 samples/group, one-way ANOVA). (C and D) LN229 and GBM8 cells were treated with recombinant hPGK1 for 5 days, followed by annexin V and 7-AAD staining (C) and cell-cycle (D) quantitative flow cytometry analysis (mean ± SEM, n = 3 samples/group, two-tailed unpaired t test). (E) Recombinant hIGFBP2 inhibits the growth of glioma cells. Sequentially diluted rhIGFBP2 was added to LN229 cultures, and cell viability was evaluated after 7 days by WST-1 assays (mean ± SD, n = 3 samples/group, two-tailed unpaired t test). (F) Recombinant hIGFBP2 inhibits the growth of GSCs. Sequentially diluted rhIGFBP2 was added to GBM8 and GBM4 cells and their growth monitored over 7 days and the resulting spheroids analyzed. Representative bright-field images and data quantification are demonstrated (mean ± SEM, n = 3 samples/group, two-tailed unpaired t test). (G and H) LN229 and GBM8 cells were treated with recombinant hIGFBP2 for 5 days, followed by annexin V and 7-AAD staining (G) and cell-cycle (H) quantitative flow cytometry analysis (mean ± SEM, n = 3 samples/group, two-tailed unpaired t test). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, ns, not significant.
Fig 3: PGK1 and IGFBP2 mediate bystander effects of miR-10b editing(A) CM samples derived from miR-10b-edited GBM8 cultures (G1 and G3) and the corresponding EV control cells were incubated with PGK1 inhibitor CBR-470-1 (10 μM) or DMSO and used for culturing naive GBM8 for 7 days. Representative microscopy images are shown. The effects on GSC growth were quantified (right panel, mean ± SEM, n = 3 samples/group, two-tailed unpaired t test). (B) CM derived from miR-10b-edited GBM8 cultures (G1 and G3) and the corresponding EV control cells were incubated with 10 μg/mL anti-IGFBP2 neutralizing antibody or control goat IgG and used for culturing naive GBM8 for 7 days (mean ± SEM, n = 3 samples/group, two-tailed unpaired t test). ∗∗∗∗ p < 0.0001.
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