Extended Data Figure 10. Rapamycin rescue of CCM formation is independent of KLF4.

a, A schematic of the experimental approach in which a cranial window is created and AAV-Cre virus injected into the brains of 2 month old mice and along with daily injection of vehicle, 100ug of Rapamycin (Rapa-low), or 400ug of Rapamycin (Rapa-high) started at post-op day 7 continuing through post-op day 21 with serial imaging at post-op day 1, 7, 10, 14, 18, and 21. b, Representative visual images of brains harvested 21 days after injection of AAV-Cre and 2 weeks of daily vehicle or rapamycin (low dose/high dose) treatment in Krit1^fl/fl;iPik3ca^H1047R mice. Dotted circles indicate the site of cranial window and AAV-Cre injection. Arrows indicate detached lesions. Scale bars, 1mm. c, Serial images obtained through the same cranial window of Krit1^fl/fl;iPik3ca^H1047R mice following injection of AAV-Cre and subsequent treatment. Arrows indicate formation and growth of individual cavernous malformations. d, MicroCT quantitation of lesion volumes 21 days after creation of the cranial window and injection of AAV-Cre. Values include duplication of microCT values in Figure 4 (vehicle and low dose treatments). (Vehicle, n=6; Rapa-low, n=7; Rapa-high, n=7). Data are mean ± s.e.m. Unpaired, two-tailed Welch’s t-test. e, Schematic of neonatal endothelial induction of Krit1 deletion and treatment with Rapamyin or vehicle control at P2. f, Immunostaining of hindbrain sections from P6 Krit1^iBECKO animals treated with vehicle or Rapamycin for PECAM1 and p-S6. White arrows indicate p-S6 positive endothelial cells in the control but not the Rapmycin treated animal. Yellow arrowheads indicate p-S6 positive neuronal cells. g, Immunostaining of hindbrain sections from P6 Krit1^iBECKO animals treated with vehicle or Rapamycin for PECAM1 and KLF4. White arrows indicate KLF4-positive endothelial cells detected in the control and in the Rapmycin treated animal. Scale bars, 50 microns.