Figure 5. Prazosin induces GIC apoptosis in a PKCδ‐dependent manner.

• A
  PKCδ expression analysis in two patient‐derived GICs and NSCs by immunoblotting. GICs express higher levels of PKCδ than NSCs. kDa: kilodaltons.
• B
  PKCδ detection by immunostaining (green). Nuclei were stained with DAPI (blue). Enhanced PKCδ punctate nuclear expression in prazosin‐treated (PRZ, 5 μM) GICs, as compared to vehicle‐treated GICs. PRZ: prazosin. Scale bar: 10 μm.
• C
  Total PKCδ (PKCδ 78 kDa) and cleaved PKCδ (PKCδ 41 kDa) expression analysis in GICs by immunoblotting. Prazosin (PRZ) induces PKCδ cleavage into a 41 kDa active fragment. 2 μM rottlerin (Rott), a PKCδ inhibitor, inhibits prazosin‐induced PKCδ cleavage. V: vehicle. kDa: kilodaltons.
• D
  Pro‐caspase‐3 (Pro‐CASP3) and activated caspase‐3 (CASP3) expression analysis in GICs by immunoblotting. 2 μM rottlerin (Rott) inhibits prazosin (PRZ)‐induced caspase‐3 activation. V: vehicle. kDa: kilodaltons.
• E
  Inhibition of PKCδ by rottlerin prevents, in a concentration‐dependent manner, prazosin‐induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin. *P = 0.0286, n = 4, two‐sided Mann–Whitney U‐test. Error bars mean ± SD.
• F
  Inhibition of PKCδ by the specific anti‐PKCδ RACK peptide δV1.1 (10 μM) counteracts prazosin‐induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1.1. *P < 0.005 for prazosin 1 and 5 µM and P < 0.001 for Prazosin 10 µM by two‐tailed unpaired Student's t‐test, n = 4. Error bars mean ± SD.
• G
  Decreased PKCδ expression using shRNA counteracts prazosin‐induced GIC death. Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h. *P < 0.005 by two‐tailed unpaired Student's t‐test, n = 4. Error bars mean ± SD.
• H
  Prazosin inhibits AKT phosphorylation in GICs. LY294002 (LY, 30 μM), an inhibitor of the PI3K/AKT pathway, was used as a positive control. Terazosin, which does not affect GIC viability, does not alter AKT phosphorylation (see the corresponding cell viability counting in Fig EV4C). Phosphorylated AKT (P‐AKT) and total AKT (AKT) expression analysis in GICs by immunoblotting. V: vehicle. kDa: kilodaltons.
• I
  Prazosin (10 μM) does not inhibit AKT phosphorylation in NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.
• J
  β‐catenin expression, a downstream target of AKT, is decreased in prazosin‐treated GICs, as opposed to NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.
• K, L
  Inhibition of PKCδ using rottlerin (Rott, 2 μM) or δV1.1 (10 μM) counteracts prazosin inhibition of AKT phosphorylation. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.

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