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. 2024 May 7;27(6):109921. doi: 10.1016/j.isci.2024.109921

Figure 4.

Figure 4

PIK3CB/PI3Kβ, but not other PI3K kinases, activates PI3K signaling and regulates TMZ sensitivity in MGMT-deficient GBMs

(A) Combinations of TMZ and knockdown of individual PI3K kinases. PI3Kβ-high SF295 or PI3Kβ-low LN229 cells were transfected with viruses harboring non-silencing (NS) shRNA or shRNA of PIK3CA, PIK3CB, or PIK3CD. Cells were then treated with DMSO or 200 μM TMZ. 4 days after treatment, cell viability was measured using the MTS assay. Cells treated with shNS and DMSO serve as controls (100% of viability).

(B) Knockout of PI3Kα or PI3Kβ. PI3Kα/β-high U87MG cells were transduced with viruses harboring gRNA of non-targeting (NT) or PIK3CA. PI3Kβ-high SF295 cells were transduced with viruses harboring gRNA of NT or PIK3CB. Levels of PI3Kα, PI3Kβ, or PI3K signaling were monitored using immunoblotting. β-actin (ACTB) was the loading control.

(C) Combinations of TMZ and knockout of PI3Kα or PI3Kβ. PI3Kα/β-high U87MG or PI3Kβ-high SF295 cells were transduced with viruses having gRNAs of NT, PIK3CA, or PIK3CB. Cells were then treated with DMSO or 200 μM TMZ. 4 days after treatments, cell viability was measured using the MTS viability assay. Cells treated with NT gRNA and DMSO serve as controls (100% of viability).

(D) Overexpression of active AKT1, AKT2, or AKT3. PI3Kα/β-high U87MG cells were transduced with viruses having pBABE (vector control) or plasmids with active myristoylated AKT isoforms (pBABE-Myr-AKT1, pBABE-Myr-AKT2, or pBABE-Myr-AKT3). Cells were then treated with 20 μM of TGX-221 (PI3Kβ-selective inhibitor) and 200 μM TMZ. Cell viability was measured using the MTS assay. Cells treated with DMSO and pBABE serve as controls (100% of viability). Error bars are standard deviations derived from three to four independent replicates. Student’s t test and One-Way ANOVA were used to determine p values. ns: not significant.