Defective autophagy is a key feature of cerebral cavernous malformations. (2015)
Figure 2

KRIT1 loss-of-function activates the mTOR-ULK1 pathway

Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.

Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1-KO endothelial cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.

Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.

Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.

Immunoblot analysis of phosphorylated ULK1 (Ser 757), total ULK1, and actin in KRIT1 KO+KRIT1, and KRIT1 KO MEFs. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.

Immunoblot analysis of p62, actin, LC3 I/II in KO+KRIT1 and KRIT1-KO cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.

KRIT1 wt and KRIT1-KO endothelial cells were transiently transfected with mRFP-GFP-LC3. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 5.74e−5 (red dots, WT ctrl vs. WT Tor1); *P = 9.62e−5 (red dots, WT ctrl vs. WT xesto); *P = 0.00727 (red dots, WT ctrl vs. KO ctrl); #P = 0.00046 (red dots, KO ctrl vs. KO Tor1). The data are expressed as the mean ± s.e.m.

KO+KRIT1 and KRIT1-KO MEFs were transiently transfected with the mRFP-GFP-LC3 tandem construct. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 0.00023 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 Tor1); *P = 0.00045 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 xesto); #P = 3.08e−6 (red dots, KO ctrl vs. KO Tor1); ##P = 6.73e−5 (yellow dots, KO+KRIT1 ctrl vs. KO ctrl). The data are expressed as the mean ± s.e.m. of four independent experiments.

Source data are available online for this figure.