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: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

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: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

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: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

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: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

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: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

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: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

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: Two different shRNAs targeting mouse Skp2 were cloned into pSicoR-mCherry (Addgene #21907) using XhoI and HpaI restriction site. shRNA sequences for Skp2-1 and -2 are listed in Online Table I.

Techniques: Inhibition

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

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet:

Article Snippet: Antibodies against ATG5 (mouse), USP28, SKP2, TRIM32, CHIP, and the His tag were purchased from Proteintech (China).

Techniques: Virus, shRNA, Recombinant, Plasmid Preparation, Software, Luciferase, Reporter Gene Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 1. RNA interference-mediated silencing of SKP1A in SN4741 cell line. A, SN4741 cells were infected for 1 week with LVs plasmid vectors encoding shRNAs targeting SKP1A, shRNA-1, and shRNA-2 or a scrambled sequence.RNAwasextractedandconvertedtosingle-strandedforanalyzingbyQ-PCR.Therelativeexpression level was assessed by normalizing to the housekeeping genes 18S-rRNA and -actin. The values are the means S.E. from two independent experiments conducted in two to five replicates. *, p 0.01 versus control (scrambled). B, after lentiviral infection cells homogenates were analyzed by Western blotting using Skp1 specific antibody. The bands were quantified by densitometry and normalized to -actin. C and D, scram- bled or shRNA-1 infected cells were grown with 10% FCS at 33 °C, and after fixation and permeabilization, Skp1 protein was detected by fluorescence microscopy. The images are representative fields from two independent experiments. The chart represents the mean immunoreactive densities of six to nine sepa- rate fields from two independent experiments normalized to number of cells in each field. *, p 0.01 versus control (scrambled).

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Infection, Plasmid Preparation, shRNA, Sequencing, Control, Western Blot, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE2.EffectofSKP1Asilencingoncellcycleprogression.A,lentivirus-infectedSN4741cellswereseeded inDulbecco’smodifiedEagle’smediumwith10%FCSandpuromycinforselection.SKP1A-infectedcellsappear less dense than their corresponding control (scrambled vector). Pictures were acquired after 24 h, using an inverted microscope connected to a digital camera (20 objective). B, shRNA-1, shRNA-2, and scrambled vector infected cells were gently suspended in a hypotonic fluorochrome solution, incubated in the dark at 37 °C for 30 min, and analyzed for DNA content on a logarithmic scale by FACSCalibur flow cytometer with Cell Quest research software; 3 104 events/sample were acquired. Both shRNAs decreased the number of cells in G0/G1 and increased the number of cells in S phase, with a delay in completion the cell cycle. The values are the means S.E. from three independent experiments conducted in three replicates. *, p 0.05; **, p 0.01 versus control scrambled vector infected cells.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Control, Plasmid Preparation, Inverted Microscopy, shRNA, Infection, Incubation, Flow Cytometry, Software

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 3. Effect of RNA interference-mediated SKP1A inhibition on the expression of dopaminergic markers. Following infection with SKP1A shRNA-1 or scrambled vector, the mRNA levels of ALDH1A1, HSPA8 (encoding Hsc-70), TH, DAT, and VMAT2 were quantified by Q-PCR. The relative expres- sion was assessed by normalizing to the housekeeping gene 18S-rRNA. The valuesarethemeansS.E.fromtwoindependentexperimentsconductedin two to five replicates. *, p 0.05; **, p 0.01 versus scrambled vector.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Inhibition, Expressing, Infection, shRNA, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 4. SKP1A overexpression decreases the susceptibility to MPP and proteasomal inhibition injury. SN4741 cells were stably transfected with expression vector pcDNA3.1hygro-SKP1A or empty expression vector, pcDNA(selectionwithhygromycin).A,representativegelmicrographofSkp1 protein levels evaluated by Western blot analysis. The autoradiogram-de- rived bands were quantified by densitometry, and the values of Skp1 were normalized to -actin and expressed as relative expression of control (pcDNA3.1hygro vector). B and C, transfected cells were injured with MPP (B, 150 or 250 M) for 48 h or with the proteasome inhibitor MG-132 (C, 12.5 and 25 M, dissolved in Me2SO) for 6 h. Cell viability was assessed by the MTT test. The results are the means S.E. of three to five independent experi- ments and expressed as percentages of control (pcDNA3.1hygro empty vec- tor). *, p 0.01 versus empty vector; #, p 0.01 versus respective control (without MPP or MG-132).

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Over Expression, Inhibition, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 5. Differentiative features of naïve SN4741 cells. A, SN4741 cells carrying the temperature-sensitive mutant from the oncogene, SV40Tag-tsA58, have a fibroblast-like flat morphology at 33 °C. Under differentia- tion conditions (i.e. nonpermissive temperature of 39 °C and 0.5% FCS), the SN4741 cell line ceased prolifera- tion and after 48 h started to display a neuronal morphology with extensive neurite outgrowth. B, represent- ativehistogramsandpercentageofcellsatdifferentphasesofthecellcycleasanalyzedbyFACS.Thevaluesare the means S.E. from three independent experiments conducted in three replicates. *, p 0.01 versus control (33 °C, FCS 10%). C, SKP1A and DA neuron-specific markers were analyzed by Q-PCR in naïve SN4741. RNA was extracted from nondifferentiated and differentiated cells, and the expression levels of various DA neuron genes were assessed. The relative expression level was assessed by normalizing to -actin. The values are the means S.E. from two independent experiments conducted in two to five replicates. *, p 0.01 versus SN4741 cells treated under permissive conditions (10% FCS and 33 °C). D, after cell fixation and permeabilization, Skp1 protein was detected by fluorescence microscopy using a specific Skp1 primary antibody. The chart represents mean immunoreactive density of six to nine separate fields from two independent experiments normalized to number of cells in each field. *, p 0.05; **, p 0.01 versus SN4741 cells under permissive conditions.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Mutagenesis, Control, Expressing, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 6. Differentiation-induced lethality of SKP1A-silenced cells. A, SKP1A silenced (shRNA-1 infected) and nonsilenced cells (scrambled) seeded in 10% FCS-containing medium were induced to differentiate for up to 48 h. There is a robust lethality in cultures of cells deficient in SKP1A that were induced to differentiate at restrictive temperature, compared with the viability and differentiative phenotype of scrambled vector-infected cells. The pictures were acquired using an inverted microscope connected to a digital camera (10 objective). B, cells were analyzed for DNA content by FACS. The percentage of cells in the G0/G1, S, and G2/M fractions was calcu- lated. The values are the means S.E. from three independent experiments conducted in three replicates. *, p 0.01 versus scrambled. C, differentiation with retinoic acid (10 M).

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: shRNA, Infection, Plasmid Preparation, Inverted Microscopy

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 7. SKP1A-silencing causes inclusion body formation in differentiating cells. Scrambled and lentivirus-stable infected SN4741 cells grown on coverslips were induced to differentiate under restrictive conditions for 24 h (pre-lethal stage), fixed, and subjected to double immunostaining with anti--synuclein (red) and anti-TH(green)antibodies(AandB),anti--synuclein(red)andanti-ubiquitin(green)antibodies(CandD),anti--synuclein(red)andanti-PSMC4(green)antibodies(E), anti--tubulin (red) and anti-ubiquitin (green) antibodies (F), and anti--tubulin (red) and anti-PSMC4 (green) antibodies (G). Scrambled infected (control) cells show a diffusestainingpatternanddonotshowanyinclusionsatbothpermissiveandrestrictivetemperatures(A–D,upperimages).SKP1A-deficientcellsdevelopedmultiple and perinuclear (B, D, and E, bottom images, see arrowsand insets) inclusion bodies 24 h after induction of differentiation, with characteristics of aggresomes, staining for-tubulin(FandG).Thereactivityoftheaggregatestothedifferentantibodiesdemonstratedasimilarpatternasindicatedbyco-localizedyellowimmunostaining in the overlaid right-hand panel, with the addition of Topro staining (blue) to identify the nucleus. No fluorescence was detected when the primary antibody was omitted. Each panel shows a representative picture of 10–15 views in two independent experiments.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Infection, Double Immunostaining, Ubiquitin Proteomics, Control, Staining, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

doi: 10.1074/jbc.m109.034223

Figure Lengend Snippet: FIGURE 8. Expression of Skp1 protein in MPTP-treated mouse midbrain and its abundance in brain tissue. C57/Bl mice (n 6–9) were treated with the parkinsonism-inducing neurotoxin MPTP (20 mg/kg/day) for 4 days followed by an additional 4-day resting period. A, mice midbrains homogenates were resolved by SDS-PAGE (4–15%gradient)andimmunoblottedwithspecificrabbitanti-Skp1andmouseanti-THantibodies.Representative blotimagesareshown.B,analysisofthebands,giveninarbitraryunits,isrepresentedgraphically.Thevaluesarethe meansS.E.fromtwoindependentexperiments.*,p0.01versusMPTP.C,therelativeabundanceofSkp1protein in mouse brain from naïve, untreated mice (n 5) was assessed by Western analysis in tissue lysates from different brain areas. FC, frontal cortex; Hip, hippocampus; St, striatum; Mb, midbrain.

Article Snippet: The cells were blocked with PBS containing 10% donkey serum at 37 °C for 1 h and incubated at 4 °C overnight with mouse monoclonal antibody against Skp1 p19 (H-6, antibody sc-5281, 1:25; Santa Cruz, CA), rabbit polyclonal antibody against ubiquitin (antibody ab19247, 1:100; Abcam, Cambridge, MA), mouse monoclonal anti-Hsc-70 antibody (antibody SM5072, 1:25; Acris Antibodies, Herford, Germany), mouse monoclonal -synuclein ( -synuclein) antibody (antibody 610786, 1:25; BDBiosciences Transduction Laboratories, Heidelberg, Germany), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (antibody AB152, 1:150; Millipore, Billerica, MA), mouse monoclonal anti- -tubulin antibody (antibody T6557, 1:10,000; Sigma), rabbit polyclonal antibody to 19 S regulator ATPase subunit Rpt3 (antibody PSMC4/S6b/ TBP7; PW8175, 1:150; Enzo Life Sciences, formerly Biomol, Plymouth, PA), all diluted in PBS containing 1% donkey serum and 0.05% Triton X-100.

Techniques: Expressing, SDS Page, Western Blot

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 binds to SKP2 in vivo : Lysates from H1299 cells were pulled down with anti-FBXW2 (left panel) or anti-SKP2 (right panel), followed by IB. ( b ) FBXW2 failed to bind to a SKP2 mutant: H1299 cells were tranfected with indicated plasmids, followed by FLAG IP and HA IB or direct IB. ( c , d ) Ectopically expressed FBXW2 reduces the endogenous levels of SKP2 protein, but not mRNA: Cells were transfected with HA-FBXW2, followed by IB ( c ) or qRT-PCR for SKP2 ( d ). Error bars indicate mean +s.d. of three repeats. ( e , f ) FBXW2 overexpression decreases the levels of the exogenous and endogenous SKP2 proteins: Cells were co-transfected with indicated plasmids, followed by IB 48 h post transfection. ( g ) FBXW2 depletion increases the levels of SKP2 protein, but not mRNA: Cells were transfected with FBXW2 siRNA and scramble siRNA, followed by IB or qRT-PCR for SKP2. Error bars indicate mean+s.d. of three repeats. ( h ) FBXW2-induced SKP2 degradation is independent of CDH1: Cells were transfected with FLAG-FBXW2 and/or siRNA against CDH1, and then harvested for IB. ( i ) FBXW2 shortens SKP2 half-life: Cells were transfected with HA-FBXW2, and switched 48 h post transfection to fresh medium containing CHX for indicated periods with or without MG132 treatment for last 2 h, and then harvested for IB. The band density was quantified. ( j ) FBXW2 knockdown extends SKP2 half-life: Cells were transfected with siRNA targeting FBXW2. Cells were switched 48 h later to fresh medium containing CHX for indicated periods and harvested for IB. The band density was quantified. ( k , l ) FBXW2 promotes ubiquitylation of SKP2 ( k ), but not SKP2-3A mutant ( l ): Cells were transfected with indicated plasmids, followed by Ni-beads pull-down and IB for SKP2. ( m ) FBXW2 promotes SKP2 ubiquitylation by in vitro assay: H1299 cells were transfected with indicated plasmids. Pull-down purified FBXW2 and FBXW2ΔF (E3s), Pull-down purified SKP2 (substrate), were added into a reaction mixture containing ATP, ubiquitin, E1 and E2, followed by IB using anti-FLAG Ab. ( n ) FBXW2 promotes SKP2 ubiquitylation via K48 linkage: Cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down and IB for SKP2.

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: In Vivo, Mutagenesis, Transfection, Quantitative RT-PCR, Over Expression, Knockdown, In Vitro, Purification, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) Fluctuation of the levels of F-box proteins and VRK2 kinase during cell cycle progression: H358 cells were serum starved for 48 h, followed by serum addition. Cells were harvested at indicated time points and subjected to FACS and IB analyses using indicated Abs. ( b – f ) FBXW2-3A mutant rescues growth-promoting phenotype induced by β-TrCP1 overexpression ( b – d ), and wt FBXW2 rescues growth-promoting phenotype induced by SKP2 overexpression ( b , e , f ): H1299 cells were co-transfected with the indicated plasmids, followed by IB ( b ), ATP-lite proliferation assay ( n =3) ( c , e ) and clonogenic survival assay ( n =3) ( d , f ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01. ( g – i ) FBXW2 depletion stimulates cell growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, and then harvested for IB ( g ), ATP-lite proliferation assay ( n =3) ( h ) and clonogenic survival assay ( n =3) ( i ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( j , k ) FBXW2 depletion stimulates tumour growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, followed by injection (1 × 10 6 cells) into nude mice. Tumour growth was observed for 33 days ( j ). Tumours were then harvested, photographed and weighted ( k ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: Mutagenesis, Over Expression, Transfection, Proliferation Assay, Clonogenic Cell Survival Assay, shRNA, Control, Injection

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a – c ) Expression levels among three F-box proteins in lung cancer cell lines and tissues: Cell lysates from eight lung cancer cell lines and one immortalized line (BEAS-2B) were subjected to IB ( a ); SE: Short exposure; LE: Long exposure. Lung cancer tissue microarrays were stained with indicated Abs and photographed ( b , Scale bars, 100 μm), and data were then analysed using SPSS software to obtain coefficient ( c ; P <0.001, Pearson's test). ( d – f ) Protein expression of three F-box proteins in lung cancer and their relationship with patient survival: Continuous protein expression values were classified into the low and high groups with equal number of patients, and 5-year survival time was used for Kaplan-Meier survival analysis ( d – f ). Kaplan-Meier survival analysis indicated that patient with higher expression of FBXW2 was related to a better overall survival (log-rank test, P =0.032) ( e ); Higher expression of β-TrCP1 and SKP2 were related to a worse overall patient survival (log-rank test, P <0.001 and 0.001, respectively) ( d , f ).

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: Expressing, Staining, Software

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 mutants have reduced binding with SCF components: H1299 cells were transfected with indicated plasmids, followed by G418 selection. Resistant clones were pooled and subjected to FLAG-bead IP and IB with indicated Abs. ( b , c ) FBXW2 mutants MU-S84C ( b ) and MU-E269K ( c ) are unable to shorten SKP2 protein half-life: Stable H1299 cells were incubated with CHX for indicated time periods and harvested for IB. The band density was quantified. ( d , e ) Loss- or gain-of-function of FBXW2 mutants: H1299 cells stably expressing MU-S84C and MU-E269K mutants were subjected to ATP-lite proliferation assay ( n =3) ( d ), and clonogenic survival assay ( n =3) ( e ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( f , g ) FBXW2 mutants either lose the tumour suppressor function or gain the oncogenic function in vivo : H1299 cells stably expressing MU-S84C and MU-E269K mutants (1 × 10 6 cells) were inoculated s.c. in both flanks of nude mice. The tumour growth was monitored twice a week for up to 28 days and growth curve plotted ( f ). Tumour tissues were harvested, photographed and weighed at 28 days ( g ). Student's t -test was used to compare each experimental group with the control group. Shown are mean±s.e.m., * P <0.05; ** P <0.01; *** P <0.001. ( h ) Immunohistochemical staining of xenograft tumour tissues. Tumour tissues from four groups of mice were fixed, sectioned and stained with indicated antibodies. Scale bars: 100 μm. Shown are mean±s.e.m., * P <0.05; ** P <0.01. ( i ) The oncogene-tumour suppressor-oncogene axis—a working model: During tumorigenesis, oncogenic β-TrCP1 is activated to promote ubiquitylation and degradation of FBXW2 tumour suppressor, resulting in abrogation of FBXW2-induced SKP2 degradation. Accumulated oncogenic SKP2 promotes ubiquitylation and degradation of tumour suppressors such as p21, p27, p130, FOXO1, and leading to activation of CDKs, E2F and mTOR, eventually to uncontrolled proliferation of cancer cells.

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: Binding Assay, Transfection, Selection, Clone Assay, Incubation, Stable Transfection, Expressing, Proliferation Assay, Clonogenic Cell Survival Assay, In Vivo, Control, Immunohistochemical staining, Staining, Activation Assay

Journal: iScience

Article Title: A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response

doi: 10.1016/j.isci.2023.106808

Figure Lengend Snippet: TRIM21 is the primary E3 ligase recruited by PHB1 to participate in the LY6E-meidated ubiquitination of CD14 (A) PHB1-mediated connection between CD14 and a ubiquitin E3 ligase. HEK-293T cells stably expressing non-tagged LY6E or control cells described in Figure 2 C were infected with control shRNA or PHB1-specific shRNA (sh2, denoted as shPHB1) encoding lentiviral vector, prior to transfection with a plasmid expressing FLAG-CD14 or an empty plasmid. The cells were used for preparation of cell extracts that were subjected to selection by anti-FLAG affinity gel, and the resultant immunoprecipitaes were analyzed for the presence of the indicated proteins, including two known PHB1-interacting E3 ligases, SKP2 and TRIM21, by immunoblotting. (B) Determination of the role of TRIM21 in LY6E-mediated CD14 ubiquitination. The effect of the expression of TRIM21-specific shRNA (shTRIM21) on the ubiquitination of transfected FLAG-CD14 was evaluated in the HEK-293T cells stably expressing non-tagged LY6E, using the procedures detailed in Figure 2 D. (C) Examination of TRIM21 dependency of LY6E-mediated CD14 suppression in THP-1 cells. LY6E-overexpressing THP-1 cells were enforced to express control shRNA (shCtrl) or TRIM21-specific shRNA (shTRIM21) by lentiviral vector infection, and analyzed for CD14 surface expression by flow cytometry. Shown are representative flow cytometric plots ( top ) and aggregate data ( bottom ) on the percentage of CD14 positive cells and MFI (n = 3; ∗∗∗∗, p < 0.0001; one-way ANOVA). (D) Dissection of the relationship of LY6E and PHB1 in interacting with CD14. Wild-type HEK-293T were infected with an empty lentiviral vector or vectors encoding either a LY6E-targeting shRNA (shLY6E) or non-tagged LY6E, and subsequently mock transfected or transfected with an FLAG-CD14 expression plasmid. The cells were analyzed for the association between CD14 and LY6E, PHB1 by anti-FLAG co-immunoprecipitations followed by immunoblotting with the indicated antibodies. Cells under LY6E overexpression were also treated with MG132 to stabilize the co-expressed CD14 proteins.

Article Snippet: SKP2 Rabbit pAb , ABclonal , Cat# A7728; AB_2772237.

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Control, Infection, shRNA, Plasmid Preparation, Transfection, Selection, Western Blot, Flow Cytometry, Dissection, Over Expression

Journal: iScience

Article Title: A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response

doi: 10.1016/j.isci.2023.106808

Figure Lengend Snippet:

Article Snippet: SKP2 Rabbit pAb , ABclonal , Cat# A7728; AB_2772237.

Techniques: Recombinant, Software, Membrane, Protein Extraction

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet:

Article Snippet: Antibodies against ATG5 (mouse), USP28, SKP2, TRIM32, CHIP, and the His tag were purchased from Proteintech (China).

Techniques: Virus, shRNA, Recombinant, Plasmid Preparation, Software, Luciferase, Reporter Gene Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet:

Article Snippet: Antibodies against ATG5 (mouse), USP28, SKP2, TRIM32, CHIP, and the His tag were purchased from Proteintech (China).

Techniques: Virus, shRNA, Recombinant, Plasmid Preparation, Software, Luciferase, Reporter Gene Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription

Journal: Regenerative Therapy

Article Title: Circ_0087960 stabilizes KDM5B by reducing SKP2 mediated ubiquitination degradation and promotes osteogenic differentiation in periodontal ligament stem cells

doi: 10.1016/j.reth.2022.01.003

Figure Lengend Snippet: Knocking down of circ_0087960 inhibited osteogenic differentiation. (A) Validation of circ_0087960 knocking down in PDLSCs at different time points post transfection. (B) Represented images of ALP and ARS staining of PDLSCs after indicated treatment. (C) mRNA and (D) protein levels of KDM5B, Runx2, ALP and OCN after indicated treatment. n = 3, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Primary mouse or rabbit monoclonal antibodies (diluted at 1:1000) against GAPDH (#5174), Runx2 (#8486), ALP (#8681), KDM5B (#15327), SKP2 (#2652), Flag-tag (#14793), Myc-tag (#2276) and HA-tag (#3724) were purchased from Cell Signaling Technology (MA, USA), and OCN (ab93876) was purchased from Abcam (MA, USA).

Techniques: Biomarker Discovery, Transfection, Staining

Journal: Regenerative Therapy

Article Title: Circ_0087960 stabilizes KDM5B by reducing SKP2 mediated ubiquitination degradation and promotes osteogenic differentiation in periodontal ligament stem cells

doi: 10.1016/j.reth.2022.01.003

Figure Lengend Snippet: Suppression of KDM5B inhibited osteogenic induction. (A) Validation of KDM5B inhibition by real-time PCR (at different time points post transfection) and western blot (4 days post transfection). (B) Images of ALP and ARS staining of PDLSCs after indicated treatment. (C) mRNA and (D) protein levels of KDM5B, Runx2, ALP and OCN after indicated treatment. n = 3, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Primary mouse or rabbit monoclonal antibodies (diluted at 1:1000) against GAPDH (#5174), Runx2 (#8486), ALP (#8681), KDM5B (#15327), SKP2 (#2652), Flag-tag (#14793), Myc-tag (#2276) and HA-tag (#3724) were purchased from Cell Signaling Technology (MA, USA), and OCN (ab93876) was purchased from Abcam (MA, USA).

Techniques: Biomarker Discovery, Inhibition, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Staining

Journal: Regenerative Therapy

Article Title: Circ_0087960 stabilizes KDM5B by reducing SKP2 mediated ubiquitination degradation and promotes osteogenic differentiation in periodontal ligament stem cells

doi: 10.1016/j.reth.2022.01.003

Figure Lengend Snippet: KDM5B promoted osteogenic genes expression. (A–C) Illustration of predicted binding sites for KDM5B in the promoter regions of ALP, Runx2 and OCN gene locus. (D–F) Fold enrichment of ChIP assay in PDLSCs demonstrated the direct binding of KDM5B to indicated gene's promoter. (G) Representative images of EMSA assay showed the shift of indicated probes after incubation with KDM5B protein. (H) Representative images and statistic results showed the interaction of KDM5B and target genes promoters in PDLSCs by ChIP assay after transfection with scramble or KDM5B shRNA. n = 3, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Primary mouse or rabbit monoclonal antibodies (diluted at 1:1000) against GAPDH (#5174), Runx2 (#8486), ALP (#8681), KDM5B (#15327), SKP2 (#2652), Flag-tag (#14793), Myc-tag (#2276) and HA-tag (#3724) were purchased from Cell Signaling Technology (MA, USA), and OCN (ab93876) was purchased from Abcam (MA, USA).

Techniques: Expressing, Binding Assay, Incubation, Transfection, shRNA

Journal: Regenerative Therapy

Article Title: Circ_0087960 stabilizes KDM5B by reducing SKP2 mediated ubiquitination degradation and promotes osteogenic differentiation in periodontal ligament stem cells

doi: 10.1016/j.reth.2022.01.003

Figure Lengend Snippet: Circ_0087960 bind to KDM5B and protected from SKP2-induced degradation. (A) Prediction of the interaction between circ_0087960 and KDM5B by RPIseq database. (B) RNA pull-down assay to determine the interaction between circ_0087960 and KDM5B, lncRNA H19 was used as a positive control. (C) Validation of circ_0087960 overexpression in 293T cells and PDLSCs. (D) Determination of KDM5B protein degradation after indicated treatment in 293T cells and PDLSCs. (E) Protein levels of KDM5B, ALP, Runx2 and OCN in PDLSCs after indicated treatment. n = 3, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Primary mouse or rabbit monoclonal antibodies (diluted at 1:1000) against GAPDH (#5174), Runx2 (#8486), ALP (#8681), KDM5B (#15327), SKP2 (#2652), Flag-tag (#14793), Myc-tag (#2276) and HA-tag (#3724) were purchased from Cell Signaling Technology (MA, USA), and OCN (ab93876) was purchased from Abcam (MA, USA).

Techniques: Pull Down Assay, Positive Control, Biomarker Discovery, Over Expression

Journal: Cancer research

Article Title: Targeted inhibition of the E3 ligase SCF Skp2/Cks1 has antitumor activity in RB1 -deficient human and mouse small cell lung cancer (SCLC)

doi: 10.1158/0008-5472.CAN-19-2400

Figure Lengend Snippet: (A) In situ neuroendocrine airway lesion (arrow) in RP Skp2-WT lung 4.6 months post Adeno-Cre inhalation. Sections were stained with H&E, Synaptophysin (Syp), or Ki67, as indicated. (B) Representative lung and liver from a RP Skp2-wild type (WT) mouse at 9.6 months post Adeno-Cre inhalation. SCLC lung tumors (black arrows), NSCLC (box) and liver metastasis (arrow heads) are indicated. (C&D) Lack of primary SCLC tumor or liver metastasis in a RP mouse in which Skp2 has also been deleted (RP Skp2-KO), 14.3 months after Adeno-Cre (C), or in a RP p27-AA mouse at 11 months, NSCLC is indicated in the box (D). (E) NSCLC (left) and SCLC (right) in the lung of a RP Skp2-WT mouse, showing positive staining for neuroendocrine markers (Syp and chromogranin A) only in the SCLC. Scale bar = 50 μm (20 μm in inserts). (F) IHC of liver metastasis from a RP Skp2-WT mouse showing positive staining for SCLC Syp, chromogranin A and Ki67. (G) Immunostaining of lung tumors from RP Skp2-KO and RP p27-AA mice had positive staining for pan-cytokeratin (Pan-CK), an epithelial marker, but not for synaptophysin.

Article Snippet: Proteins were transferred to PVDF membrane (IPVH00010, Millipore) and probed with the following antibodies: Skp2 (sc-7164), p21 (sc-397), and α-tubulin (sc-8035), from Santa Cruz Biotechnology; cleaved caspase-3 (#9661) and p53 (#2524) from Cell Signaling Technology; cullin-1 (ab75812) and p73 (ab40658) from abcam; p27 (#610242, BD Bioscience), and pRb (554136, BD Pharmingen).

Techniques: In Situ, Staining, Immunostaining, Marker

Journal: Cancer research

Article Title: Targeted inhibition of the E3 ligase SCF Skp2/Cks1 has antitumor activity in RB1 -deficient human and mouse small cell lung cancer (SCLC)

doi: 10.1158/0008-5472.CAN-19-2400

Figure Lengend Snippet: Lung and liver tumor incidence in RP Skp2-WT, RP Skp2-KO, and RP p27-AA mice from tissues obtained after Adeno-Cre administration

Article Snippet: Proteins were transferred to PVDF membrane (IPVH00010, Millipore) and probed with the following antibodies: Skp2 (sc-7164), p21 (sc-397), and α-tubulin (sc-8035), from Santa Cruz Biotechnology; cleaved caspase-3 (#9661) and p53 (#2524) from Cell Signaling Technology; cullin-1 (ab75812) and p73 (ab40658) from abcam; p27 (#610242, BD Bioscience), and pRb (554136, BD Pharmingen).

Techniques:

Journal: Cancer research

Article Title: Targeted inhibition of the E3 ligase SCF Skp2/Cks1 has antitumor activity in RB1 -deficient human and mouse small cell lung cancer (SCLC)

doi: 10.1158/0008-5472.CAN-19-2400

Figure Lengend Snippet: (A) Kaplan-Meier survival curves for the three mice groups described in Fig. 1. Survival was significantly different between the groups by Log-rank (Mantel-Cox) test. (B&C) Primary tumor cells from liver metastasis from a RP mouse (RP-LvMet) were transduced with a lenti-shRNA lentivirus vector for inducible Skp2 knockdown (pTripZ-shSkp2) or a control virus. (B) Cell growth in vitro with (■,▲,▼) and without (○, open triangle) doxycycline (Dox) treatment for control (▼,open triangle) and shSkp2 (○,■,▲) vectors. (C) Western blot showing treatment of the cells with 2 μg/ml Dox for 24 or 48 hours in vitro caused decreased expression of Skp2 and increased expression of p27 and cleaved caspase 3. (D&E) H520 NSCLC cells were infected with the pTripZ-shSkp2 or a control virus. (D) RP-LvMet cells with the pTripZ-shSkp2 vector were inoculated subcutaneously (SC) in nude mice and tumors allowed to grow for 10 days, at which time tumors were 100-300 mm3. Half of the mice were treated with doxycycline (■) (+DOX; 5 μg/ml DOX in their drinking water plus daily oral gavage of 1 mg DOX). Tumor sizes were measured every other day. Control mice (□) (−DOX) did not receive doxycycline. (E) Skp2 mRNA levels in lung tumors from mice treated with and without doxycycline, measure by RT-PCR (n= 5 mice). (F) H&E stained lung and liver from mice inoculated orthotopically in the lung with 106 Skp2-shRNA RP-LvMet SCLC cells. Treatment of the mice with DOX to knockdown Skp2 began on day 15 after inoculation, and the mice were sacrificed when moribund. (G) Cell growth with (black bars) and without (gray bars) doxycycline. (H) Skp2 mRNA levels by RT-PCR in H520 cells treated as in D.

Article Snippet: Proteins were transferred to PVDF membrane (IPVH00010, Millipore) and probed with the following antibodies: Skp2 (sc-7164), p21 (sc-397), and α-tubulin (sc-8035), from Santa Cruz Biotechnology; cleaved caspase-3 (#9661) and p53 (#2524) from Cell Signaling Technology; cullin-1 (ab75812) and p73 (ab40658) from abcam; p27 (#610242, BD Bioscience), and pRb (554136, BD Pharmingen).

Techniques: Transduction, shRNA, Plasmid Preparation, In Vitro, Western Blot, Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Cancer research

Article Title: Targeted inhibition of the E3 ligase SCF Skp2/Cks1 has antitumor activity in RB1 -deficient human and mouse small cell lung cancer (SCLC)

doi: 10.1158/0008-5472.CAN-19-2400

Figure Lengend Snippet: (A) Schematic showing the SCFSkp2 protein complex and its relationship to pRb, p27, and NEDD8. The sites of action of C1, FKA, and pevonedistat are shown. (B) The indicated SCLC (green ●,▲,▼) and NSCLC (red ●,▲) cells were plated in 96 well plates, and treated the next day with the indicated concentrations of C1. Cell numbers were quantified after 72 hours with a CellTiter-Glo assay.

Article Snippet: Proteins were transferred to PVDF membrane (IPVH00010, Millipore) and probed with the following antibodies: Skp2 (sc-7164), p21 (sc-397), and α-tubulin (sc-8035), from Santa Cruz Biotechnology; cleaved caspase-3 (#9661) and p53 (#2524) from Cell Signaling Technology; cullin-1 (ab75812) and p73 (ab40658) from abcam; p27 (#610242, BD Bioscience), and pRb (554136, BD Pharmingen).

Techniques: Glo Assay

Journal: Cancer research

Article Title: Targeted inhibition of the E3 ligase SCF Skp2/Cks1 has antitumor activity in RB1 -deficient human and mouse small cell lung cancer (SCLC)

doi: 10.1158/0008-5472.CAN-19-2400

Figure Lengend Snippet: (A) Western blot showing concentration-dependent increase in p27 in SCLC (RP-Lung, RP-LvMet, H69), but not in NSCLC (H460, H520), cells treated with C1 for 24 hours. (B) Flavokawain A (FKA) and pevonedistat reduce cullin1 neddylation in RP-Lung and H69 SCLC cells. (C) Comparative effect of C1, FKA and pevonedistat on Skp2, p27, p21, and cleaved caspase 3 protein expression in RP-Lung and H69 SCLC cells. (D) Immunoprecipitation of Skp2 from control and Pevonedistat-treated cells showing loss of co-immunoprecipitated p27 in drug-treated cells. (E) Lack of correlation between the IC50s for etoposide with those for C1 (●) and pevonedistat (○) in seven SCLC cell lines. (F) Effect of the combination of C1 and pevonedistat on cell proliferation in SCLC cells. Data are the means ± SD from five cell lines (H69, H146, H196, H446, H720). Each cell line was treated for 72 hours with C1 or pevonedistat at their respective IC50s, with the drugs used alone or in combination. *Indicates significant difference between the drugs used in combination compared to their use individually, p < 0.05.

Article Snippet: Proteins were transferred to PVDF membrane (IPVH00010, Millipore) and probed with the following antibodies: Skp2 (sc-7164), p21 (sc-397), and α-tubulin (sc-8035), from Santa Cruz Biotechnology; cleaved caspase-3 (#9661) and p53 (#2524) from Cell Signaling Technology; cullin-1 (ab75812) and p73 (ab40658) from abcam; p27 (#610242, BD Bioscience), and pRb (554136, BD Pharmingen).

Techniques: Western Blot, Concentration Assay, Expressing, Immunoprecipitation

Journal: Cancer research

Article Title: Targeted inhibition of the E3 ligase SCF Skp2/Cks1 has antitumor activity in RB1 -deficient human and mouse small cell lung cancer (SCLC)

doi: 10.1158/0008-5472.CAN-19-2400

Figure Lengend Snippet: Tumors were initiated by the subcutaneous inoculation of human H69 SCLC cells (A), murine RP-LvMet SCLC cells (B & E), and 1-2 mm fragments of the human SCLC PDXs CTG-0199 (C) and CTG-1252 (D). When tumors reached 100 mm3 in size, mice were treated with C1 (A; 40 mg/kg/day ip), FKA (B,C,D; 600 mg/kg/day by gavage) or pevonedistat (E; 50 or 90 mg/kg/day subcutaneously). Tumor size was measured on the indicated days; data are means ± SEM of at least 5 mice per group. All drug treatments were significantly different from controls (P < 0.0001). (F) Both patient-derived xenografts (PDXs) stain positive for neuroendocrine markers synaptophyosin (Syp) and chromogranin A (ChromA), and had a large population of actively proliferating Ki67-positive cells. (G) Tumors isolated from drug-treated mice had increased apoptosis after staining for cleaved caspase 3 immunohistochemistry (IHC). (H & I) Western blot and quantitation of Skp2, p27 and cleaved caspase 3 in RP-LvMet tumor extracts from control and pevonedistat-treated mice. Each lane in (H) is an individual mouse tumor. (J) Quantitation of cleaved caspase 3 (percent positive-staining cells) in RP-LvMet and PDX tumors isolated from control or drug-treated mice using Image J software. (K) Mouse body weight during daily treatment with C1 or pevonedistat, as indicated. Means ± SD of 5-6 mice per group.

Article Snippet: Proteins were transferred to PVDF membrane (IPVH00010, Millipore) and probed with the following antibodies: Skp2 (sc-7164), p21 (sc-397), and α-tubulin (sc-8035), from Santa Cruz Biotechnology; cleaved caspase-3 (#9661) and p53 (#2524) from Cell Signaling Technology; cullin-1 (ab75812) and p73 (ab40658) from abcam; p27 (#610242, BD Bioscience), and pRb (554136, BD Pharmingen).

Techniques: Derivative Assay, Staining, Isolation, Immunohistochemistry, Western Blot, Quantitation Assay, Software