Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 1 FAK inhibition reduces Skp2 expression by promoting nuclear localization of FAK. FAK inhibitor (VS-4718) was used at 2.5 mM for the indicated times. (A, C, D, and F) Representative blots (n = 3). (A) Skp2 levels reduced upon pharmacological FAK inhibition monitored by immunoblotting with mouse VSMC lysates. Skp2 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. (B) Mouse VSMCs were treated with FAK inhibitor for 2 days, and Skp2 mRNA levels were measured via RT-qPCR (± SEM, n = 6, Student t-test). (C) Human coronary artery SMCs (hCASMCs) were treated with FAK inhibitor. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK was quantified relative to total FAK. (D) Skp2 lev- els in FAK-WT and FAK-KD mouse VSMCs. Skp2 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. (E) Skp2 mRNA levels were measured in FAK-WT and FAK-KD mouse VSMCs via RT-qPCR (± SEM, n = 6, Student t-test). (F) Mouse VSMCs were treated with FAK inhibitor with or without proteasome inhibitor (MG132, 20mM) for 6 h. Skp2 blots were quantified relative to actin. pY397 FAK was quantified relative to total FAK. (G) Mouse VSMCs were treated with or without FAK inhibitor for 24h. FAK, Skp2, pY397 FAK, p27, and p21 immunostainings are shown. PCNA was used as a proliferation marker. Representative images (n = 4). Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Marker

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 2 FAK interacts with Skp2 and the APC/C E3 ligase activator Fzr1 through FAK FERM domain. FAK inhibitor (VS-4718) was used at 2.5 mM for the indicated times. (A–E) Representative blots (n = 3). (A) FAK interacts with Skp2 and Fzr1 by immunoprecipitation (IP) with VSMC lysates. Mouse VSMCs were treated with FAK inhibitor together with proteasome inhibitor (MG132, 20lM) for 6 h. FAK and Fzr1 blots within Skp2-IP were quantified relative to untreated controls. The arrow head indicates the heavy chain of IgG. (B) Immunoblots of mouse VSMC cytosolic (C) and nuclear (N) fractionated lysates for FAK, Fzr1, and Skp2. PARP and GAPDH as nuclear and cytosolic markers. Cytosolic or nuclear FAK, Skp2, and Fzr1 blots were quantified relative to GAPDH or PARP.. (C) FAK FERM binds to both Skp2 and Fzr1 in 293T cells transfected with GFP-fused FAK and its subdomains. Skp2 and Fzr1 blots were quantified relative to GFP. (D) FAK FERM F1 lobe binds Skp2 and F3 lobe binds Fzr1 in 293T cells co-transfected with Myc-Skp2, HA-Fzr1, and FAK FERM F1, F2, and F3 lobes as GST fusion proteins. Skp2 and Fzr1 blots were quantified relative to GST. (E) Mouse VSMCs were treated with FAK inhibitor. pY397 FAK and Fzr1 blots were quantified relative to GAPDH. (F) Fzr1 mRNA levels were measured in mouse VSMCs treated with FAK inhibitor for 2 days or in genetic FAK inhibition via RT-qPCR (± SEM, n = 6, Student t-test). (G) Nuclear localization of FAK plays a key role in Fzr1 and Skp2 degradation. FAK-/-

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Immunoprecipitation, Western Blot, Transfection, Inhibition, Quantitative RT-PCR

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 3 Skp2 is increased after wire injury and pharmacological FAK catalytic inhibition significantly reduces Skp2 and neointimal hyperplasia. Mice were treated with vehicle or VS-4718 (50 mg/kg) twice daily following wire injury. Representative H&E staining of femoral artery cross sections 14 days after wire injury for vehicle or VS-4718-treated (A, n = 5), Scale bar: 50mm. Intima/media (I/M) ratios were quantified (± SD, n = 5, **P < 0.005 vs. vehicle injury, two- way ANOVA followed by Sidak multiple comparisons test). (B) Representative immunoblots of femoral arteries 14days postinjury for pY397 FAK, FAK, Skp2, p27, p21, and GAPDH as loading control. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n= 4). (C) Representative immunofluorescence staining of femoral arteries 14 days postinjury for pY397 FAK, FAK, p27, p21, Skp2, Fzr1, and a-SMA. Red, green, and blue (DAPI) were merged (n = 5). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Inhibition, Staining, Western Blot, Control, Immunofluorescence

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 4 VSMC-specific FAK-KD mice development reduces hyperplasia and exhibits less Skp2 and more p27, p21 expression after wire injury. (A) Representative H&E staining of femoral artery cross sections 2 weeks after wire injury for genetic FAK-WT or FAK-KD mice (n = 5). Scale bar: 50mm. Intima/media (I/M) ratios were quantified (± SD, n = 5, **P< 0.005 vs. FAK-WT injury, two-way ANOVA followed by Sidak multiple comparisons test). (B) Representative immunoblots of femoral arteries 14days postinjury for pY397 FAK, FAK, Skp2, p27, p21 GAPDH as loading control. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 4). (C) Representative immunofluores- cence staining of FAK-WT and FAK-KD femoral arteries 2 weeks postinjury for pY397 FAK, FAK, p27, p21, Skp2, Fzr1, and a-SMA. Red, green, and blue (DAPI) were merged (n = 4). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Expressing, Staining, Western Blot, Control

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 5 Skp2 directly regulates CDKI expression and functions independently of cyclin D1 in the cell cycle progression. (A) Mouse VSMCs were trans- duced to overexpress Skp2. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (B) Skp2 overexpression slightly increased mouse VSMC proliferation (± SD, n = 3, *P < 0.01, **P < 0.005 vs. control, two-way ANOVA followed by Sidak multiple comparisons test). (C) p27 or p21 mRNA levels were measured in mouse VSMCs overexpressing Skp2 via RT-qPCR (± SEM, n = 3, paired t-test). (D) shRNA-mediated knockdown of Skp2 increased p27 and p21 protein. Skp2, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (E) Skp2, p27, and p21 mRNA levels were measured in Skp2 knockdown mouse VSMCs via RT-qPCR (± SEM, n = 3, **P < 0.005 vs. scramble, Student t-test). (F) Skp2 knockdown reduced mouse VSMC proliferation (± SD, n = 3, **P < 0.005 vs. scramble, two-way ANOVA followed by Sidak multiple comparisons test). (G) Cyclin D1 and Skp2 were overexpressed in FAK-WT and FAK-KD mouse VSMCs. Skp2, cyclin D1, p27, and p21 blots were quantified relative to GAPDH. pY397 FAK blots were quantified relative to total FAK. Representative blots (n = 3). (H) Skp2 overexpression rescues defect of mouse VSMCs proliferation in cyclin D1-overexpressing FAK-KD VSMCs (± SD, n = 3, **P < 0.005 vs. FAK-KD, two-way ANOVA followed by Sidak multiple comparisons test).

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Expressing, Over Expression, Control, Quantitative RT-PCR, shRNA, Knockdown

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 6 Knockdown of Skp2 reduces wire injury-induced neointi- mal hyperplasia. Femoral arteries were coated with either lentivirus encoding mCherry , scramble shRNA (shScr), or Skp2 shRNA (shSkp2) immediately following wire injury. After 2 week wire injury, immunos- tainings for a-SMA, Skp2, and p27 were shown. SMA (green), mCherry (red), and DAPI (blue) were merged (n = 4). mCherry was used to ver- ify viral infection. Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Knockdown, shRNA, Infection

Journal: Cardiovascular research

Article Title: FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

doi: 10.1093/cvr/cvab132

Figure Lengend Snippet: Figure 7 FAK inhibition blocks neointimal hyperplasia in overex- pressing Skp2 mice upon wire injury. Femoral arteries were coated with Myc-Skp2 overexpressing lentivirus immediately following wire in- jury. Mice were treated with vehicle or VS-4718 (50 mg/kg) twice daily. After 2 week wire injury, immunostainings for a-SMA, Myc, FAK, pY397 FAK, p27, and Skp2 were shown. SMA (green), Myc-Skp2 or pY397 (red), and DAPI (blue) were merged (n = 4). Dotted line in image marks the external or internal elastic lamina. White line shows endothelial layer. Scale bar: 20mm.

Article Snippet: GFP-tagged FAK WT, FERM, kinase, and C-terminal domain plasmid were used as previously described.34,35 FAK FERM F1 (33-132), FERM F2 (128-253), and FERM F3 (253-353) lobes were amplified by PCR and cloned into the pEBG mammalian GST fusion expression vector as described.35 Myc-tagged Skp2 (#19947) and HA-tagged Fzr1 (#11596) plasmid constructs were from Addgene.

Techniques: Inhibition