Figure 4. Prazosin induces GIC apoptosis independently from α‐AR .

1. Immunoblotting for pro‐caspase‐3 (pro‐CASP3) and active caspase‐3 (CASP3) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin activates caspase‐3. ZVAD, a caspase inhibitor, prevents prazosin‐induced caspase‐3 activation. kDa: kilodaltons.
2. Immunoblotting for pro‐caspase‐9 (pro‐CASP9) and active caspase‐9 (CASP9) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin does not activate caspase‐9.
3. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of ZVAD, a caspase inhibitor. ZVAD counteracts prazosin‐induced GIC death. *P = 0.0286, n = 4, two‐sided Mann–Whitney U‐test. Results are mean ± SD in biological quadruplicates from three independent experiments.
4. Prazosin induces glioblastoma cell apoptosis in vivo. GFP⁺ and GFP⁻ cells were analyzed from tumors in vehicle (upper panel) or prazosin‐treated (lower panel) mice. Annexin V/DAPI staining was used to identify apoptotic cells by FACS. Tumors were initiated by GBM44 GICs implantation.
5. Dose–response curve of prazosin on GIC survival (24 h treatment). EC₅₀ was determined with curve fit using nonlinear regression. Results are mean ± SD in biological triplicates from one experiment out of three independent experiments giving similar results.
6. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of cirazoline, a subtype agonist of the α‐ARs. Cirazoline did not alter GIC survival and counteracted only poorly prazosin‐induced GIC death. *P < 0.05, n = 4, two‐sided Mann–Whitney U‐test. Results are mean ± SD in biological quadruplicates from three independent experiments.
7. Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations. Positive control shows that prazosin binds to membrane preparations of yeast expressing α1‐AR. PRZ: prazosin. CRZ: cirazoline.
8. Immunoblotting for phosphorylated ERK1/2 (P‐ERK1/2) and total ERK1/2 following prazosin treatment for 30 min. Prazosin induces ERK1/2 phosphorylation in GICs.
9. SRE‐luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK‐activating kinase MEK. U0126 prevented prazosin‐induced SRE‐luciferase activation in GICs. Results are from three independent experiments.
10. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 (10 μM), an inhibitor of the ERK‐activating kinase MEK. U0126 did not counteract prazosin‐induced GIC death. Blockade of prazosin‐induced ERK1/2 phosphorylation by U0126 was confirmed by immunoblotting (insert). PRZ: prazosin. Results are presented as mean ± SD in biological quadruplicates from three independent experiments.

Source data are available online for this figure.