Figure S1.
Detection of pTIE2 and pVEGFR2 in human brain tissue specimens. (A) Representative images showing immunostaining for CD31, VEGFR2, pVEGFR2, and TIE2, pTIE2, with DAPI counterstaining in freshly resected human CCM specimens and non-CCM human brain tissue. The boxed regions in the lower-magnification images are shown at higher magnification on the right. Arrows indicate pTIE2-positive endothelial cells lining CCM lesions. (B) Representative image of H&E staining revealing the classic histopathological appearance of glioblastoma (GBM), characterized by high cellularity and areas of necrosis. The boxed region represents high cellularity area adjacent to the necrotic tissue and is used for pVEGFR2 and pTIE2 antibodies validation and quantification in the following panels. (C) Representative images showing immunostaining for CD31, VEGFR2, pVEGFR2, TIE2, pTIE2, with DAPI counterstaining in resected human GBM specimen. (D–F) Quantification of MFI of (D) pTIE2Y992, (E) pVEGFR2Y951, and (F) pVEGFR2Y1175, normalized to total TIE2 and VEGFR2, respectively, as compared with endothelium in non-GBM brain tissue. N = 3 human samples in each group were analyzed and quantified. Data shown are means ± SEM. *P <0.05, ****P <0.0001 by unpaired two-tailed Welch t test. (G) Representative images showing immunostaining for CD31, VEGFR2, pVEGFR2, TIE2, and pTIE2, with DAPI counterstaining in recovered human CCM specimen with identified lesional endothelial cell genotypes (PIK3CAH1047R and somatic CCM2 mutation), as previously characterized by Ren et al. (2021). CCM sample was rederived from frozen tissue, sectioned, and processed with methanol fixation, which differs from the processing of all other tissue images (standard histology protocol described in the Materials and methods) reported in this manuscript. White arrows indicate pTIE2-positive endothelial cells lining CCM lesions. Yellow arrows indicate CD31− and VEGFR2-double positive but pVEGFR2-negative endothelial cells lining CCM lesions. Asterisks denote (A) autofluorescence emitted from lumenal red blood cells and (G) tissue border autofluorescence without reliable CD31 or DAPI nuclear staining to identify endothelial cells. Scale bar: 100 μm. Refer to the image caption for details. Panel A shows immunofluorescence microscopy images of C D 31, V E G F R 2, p V E G F R 2, T I E 2, and p T I E 2 with DAPI in human brain vascular tissues; Panel B shows H and E histology image of glioblastoma tissue section; Panel C shows immunofluorescence microscopy images of C D 31, V E G F R 2, p V E G F R 2, and T I E 2 staining in G B M vessels; Panel D shows vertical bar graph of T I E 2 phosphorylation index comparison; Panel E shows vertical bar graph of V E G F R 2 T y r 951 phosphorylation index; Panel F shows vertical bar graph of V E G F R 2 T y r 1175 phosphorylation index; Panel G shows immunofluorescence microscopy images of C D 31, V E G F R 2, p V E G F R 2, T I E 2, and p T I E 2 in C C M lesions.

Detection of pTIE2 and pVEGFR2 in human brain tissue specimens. (A) Representative images showing immunostaining for CD31, VEGFR2, pVEGFR2, and TIE2, pTIE2, with DAPI counterstaining in freshly resected human CCM specimens and non-CCM human brain tissue. The boxed regions in the lower-magnification images are shown at higher magnification on the right. Arrows indicate pTIE2-positive endothelial cells lining CCM lesions. (B) Representative image of H&E staining revealing the classic histopathological appearance of glioblastoma (GBM), characterized by high cellularity and areas of necrosis. The boxed region represents high cellularity area adjacent to the necrotic tissue and is used for pVEGFR2 and pTIE2 antibodies validation and quantification in the following panels. (C) Representative images showing immunostaining for CD31, VEGFR2, pVEGFR2, TIE2, pTIE2, with DAPI counterstaining in resected human GBM specimen. (D–F) Quantification of MFI of (D) pTIE2Y992, (E) pVEGFR2Y951, and (F) pVEGFR2Y1175, normalized to total TIE2 and VEGFR2, respectively, as compared with endothelium in non-GBM brain tissue. N = 3 human samples in each group were analyzed and quantified. Data shown are means ± SEM. *P <0.05, ****P <0.0001 by unpaired two-tailed Welch t test. (G) Representative images showing immunostaining for CD31, VEGFR2, pVEGFR2, TIE2, and pTIE2, with DAPI counterstaining in recovered human CCM specimen with identified lesional endothelial cell genotypes (PIK3CAH1047R and somatic CCM2 mutation), as previously characterized by Ren et al. (2021). CCM sample was rederived from frozen tissue, sectioned, and processed with methanol fixation, which differs from the processing of all other tissue images (standard histology protocol described in the Materials and methods) reported in this manuscript. White arrows indicate pTIE2-positive endothelial cells lining CCM lesions. Yellow arrows indicate CD31− and VEGFR2-double positive but pVEGFR2-negative endothelial cells lining CCM lesions. Asterisks denote (A) autofluorescence emitted from lumenal red blood cells and (G) tissue border autofluorescence without reliable CD31 or DAPI nuclear staining to identify endothelial cells. Scale bar: 100 μm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal