Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells

doi: 10.1038/jcbfm.2012.123

Figure Lengend Snippet: Subcellular localization of NOTCH3 in CADASIL vascular smooth muscle cells (VSMCs). For detection of cell surface NOTCH3, VSMCs were fixed with 3.5% phosphate-buffered paraformaldehyde. NOTCH3 forms more aggregates on the cell surface in CADASIL than in control VSMCs ( A – I ). For intracellular NOTCH3 detection, VSMCs were fixed with methanol. Permeabilized VSMCs show that NOTCH3 does not aggregate inside the cell in either CADASIL or control VSMCs ( J – L ). Immunostainings were performed with antibody (1E4) directed against N3ECD recognizing full-length NOTCH3 and N3ECD. Number of cell lines analyzed: HUmbVSMC: CADASIL n =4, control n =4, HPlaVSMC CADASIL n =4, control n =4, HArtVSMC: CADASIL n =3, HCerVSMC: CADASIL n =2, CADASIL-hmz n =1, control n =3.

Article Snippet: Primary antibodies used in Western blot and immunocytochemistry were NOTCH3 clone 1E4, NOTCH3 clone 5E1, both directed against N3ECD (both kind gifts from Dr A Joutel), NOTCH3 ICD (D11B8, Cell Signaling, Beverly, MA, USA), α -SMA (Sigma), HES5 (M-104, Santa Cruz Biotechnology, Heidelberg, Germany) β -Actin (Sigma), vinculin (Biohit, Helsinki, Finland), integrin β 1, tensin (BD Transduction Laboratories, Mississauga, Canada).

Techniques: Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells

doi: 10.1038/jcbfm.2012.123

Figure Lengend Snippet: Total NOTCH3 and α -SMA expression in CADASIL and control vascular smooth muscle cells (VSMCs). ( A ) Western blot analysis of the expression of NOTCH3 detected with 5E1 directed against N3ECD in CADASIL and control VSMCs. In all, 40 μ g of cell lysate was loaded on each lane. The 280-kDa full-length NOTCH3 (N3FL) and 220 kDa N3ECD are indicated with arrowheads. The 245-kDa band in between (white arrowhead) is unknown. ( B ) The expression of NOTCH3 is significantly higher in HCerVSMCs and HArtVSMCs than in HUmbVSMCs or HPlaVSMCs. There are no statistically significant differences in the total expression level of NOTCH3 or N3FL/N3ECD ratio between CADASIL versus the corresponding control or between control versus heterozygous or homozygous VSMCs. ( C ) Western blot analysis of α -SMA expression in CADASIL and control VSMCs. In all, 20 μ g of cell lysate was loaded on each lane. ( D ) Even though, CADASIL cells have altered actin filament organization, the total amount of α -SMA in CADASIL VSMCs is not significantly different from that in corresponding control VSMCs. The bar graphs show the average intensities and ±s.e.m. from two independent experiments. Both experiments were repeated two times with cells at passage 5.

Article Snippet: Primary antibodies used in Western blot and immunocytochemistry were NOTCH3 clone 1E4, NOTCH3 clone 5E1, both directed against N3ECD (both kind gifts from Dr A Joutel), NOTCH3 ICD (D11B8, Cell Signaling, Beverly, MA, USA), α -SMA (Sigma), HES5 (M-104, Santa Cruz Biotechnology, Heidelberg, Germany) β -Actin (Sigma), vinculin (Biohit, Helsinki, Finland), integrin β 1, tensin (BD Transduction Laboratories, Mississauga, Canada).

Techniques: Expressing, Control, Western Blot

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells

doi: 10.1038/jcbfm.2012.123

Figure Lengend Snippet: Actin cytoskeleton in CADASIL vascular smooth muscle cells (VSMCs). VSMCs fixed with methanol were immunostained for smooth muscle cell α -actin ( α -SMA). α -SMA filament have altered organization in CADASIL VSMCs. In all CADASIL VSMCs the α -SMA positive actin filaments are short, poorly organized and form nodes (clusters of short haphazardly oriented filaments, selected five marked with arrowheads) ( A : HUmbVSMC, B : HCerVSMC, C : HCerVSMC-G528C, F : HCerVSMC-hmz, and G : HArtVSMC). Actin filament nodes in CADASIL VSMCs are shown in higher magnification in an inset ( I : HCerVSMC-hmz). Control VSMCs have robust cell spanning actin filaments ( D : HUmbVSMC and E : HCerVSMC and an inset H : HCerVSMC). Number of cell lines analyzed: HUmbVSMC: CADASIL n =4, control n =4, HPlaVSMC CADASIL n =4, control n =4, HArtVSMC: CADASIL n =3, HCerVSMC: CADASIL n =2, CADASIL-hmz n =1, control n =3. Control HUmbVSMCs were transduced with nontarget short hairpin RNA (shRNA) ( J ) or NOTCH3 -targeted shRNA2 ( K ). Silencing of NOTCH3 expression induced similar alterations to organization of actin filaments as observed in CADASIL VSMCs. Silencing of NOTCH3 expression was verified by Western blot ( L ). NOTCH3-targeted shRNA1 and shRNA2 reduced relative intensity of NOTCH3 expression by 36 and 63% when compared with nontarget shRNA. NOTCH3 signaling activity is also reduced as indicated by lower expression level of NOTCH3 target gene HES5 (shRNA1 5% and shRNA2 30%). Relative intensity is calculated from an average of combined N3FL and N3ICD intensities. Intensities are averages from two independent experiments normalized to β -actin. Average intensities are shown in proportion to nontarget shRNA sample (relative intensity). N3FL and N3ICD were detected by antibody directed against N3ICD.

Article Snippet: Primary antibodies used in Western blot and immunocytochemistry were NOTCH3 clone 1E4, NOTCH3 clone 5E1, both directed against N3ECD (both kind gifts from Dr A Joutel), NOTCH3 ICD (D11B8, Cell Signaling, Beverly, MA, USA), α -SMA (Sigma), HES5 (M-104, Santa Cruz Biotechnology, Heidelberg, Germany) β -Actin (Sigma), vinculin (Biohit, Helsinki, Finland), integrin β 1, tensin (BD Transduction Laboratories, Mississauga, Canada).

Techniques: Control, Transduction, shRNA, Expressing, Western Blot, Activity Assay