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: Cell Death and Differentiation

Article Title: Ablation of galectin-3 induces p27 KIP1 -dependent premature senescence without oncogenic stress

doi: 10.1038/cdd.2014.88

Figure Lengend Snippet: Inhibition of galectin-3 reduces the proliferation of gastric cancer cells in a Skp2- and p27 KIP1 -dependent manner. ( a ) Cell proliferation after silencing of galectin-3 using siRNA transfection with respect to time (1–3 days) and concentration (1–20 nM) in the AGS human gastric cancer cells. The error bars indicate 95% confidence intervals; *, ** P <0.0001 using one-way ANOVA. ( b ) Detection of the levels of galectin-3, Skp2, p27 KIP1 , p14 ARF , p16 INK4A , and p21 WAF1/CIP1 after silencing of galectin-3 using siRNA transfection in the AGS human gastric cancer cells. β -Actin was used as the normalization control. ( c ) Detection of mRNA and protein expression of galectin-3, p27 KIP1 , and Skp2 after transfection with galectin-3 siRNA for 48 h, as analyzed by RT-PCR and immunoblotting, respectively. β -Actin was used as the normalization control. ( d and e ) After transfection of AGS cells with galectin-3 siRNA, p27 KIP1 siRNA, and co-transfection of AGS cells with both siRNAs ( d ) the protein expression of galectin-3, p27 KIP1 , and Skp2 was analyzed by western blotting. β -Actin was used as the normalization control. ( e ) Cell cycle populations were examined by PI staining. A quantitative graph (left panel) and histogram (right panel) are generated. Marking of histogram is as follows: M 1 -SubG 1 , M 2 -G 1 , M 3 -S, and M 4 -G 2

Article Snippet: Galectin-3 depletion reduced Skp2 mRNA but increased p27 KIP1 protein expression without a change in the mRNA expression.

Techniques: Inhibition, Transfection, Concentration Assay, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cotransfection, Staining, Generated

Journal: Cell Death and Differentiation

Article Title: Ablation of galectin-3 induces p27 KIP1 -dependent premature senescence without oncogenic stress

doi: 10.1038/cdd.2014.88

Figure Lengend Snippet: Decreased cell proliferation and increased premature senescence in galectin-3 knockout mouse embryonic fibroblasts (MEFs) and galectin-3-depleted human skin fibroblasts. ( a ) Expression levels of galectin-3 in galectin-3 −/− and galectin-3 +/+ MEFs were analyzed by western blotting (upper panel). Galectin-3 −/− and galectin-3 +/+ MEFs were stained with crystal violet (lower panel), and the graph shows the percentage of cells in the microscopic fields (right panel). The error bars indicate 95% confidence intervals; * P =0.0397 using the two-sided t -test. ( b ) Cell lysates of galectin-3 −/− and galectin-3 +/+ MEFs were prepared and analyzed by RT-PCR using primers specific for p27 KIP1 , Skp2, and galectin-3. GAPDH served as the normalization control. Protein levels were also detected using antibodies against p27 KIP1 , p21 WAF1/CIP1 , Skp2, and galectin-3 by western blotting. β -Actin was used as the normalization control. ( c ) Premature senescence was induced in galectin-3 −/− MEFs. Galectin-3 −/− and galectin-3 +/+ MEFs were stained for β -galactosidase activity. The graph shows the percentage of β -galactosidase-positive cells (right panel). The error bars indicate 95% confidence intervals; * P <0.0001 using the two-sided t -test. The scale bar indicates 100 μ m. ( d ) After 48 h of transfection with control siRNA, galectin-3 siRNA, p27 KIP1 siRNA, and both siRNAs in human skin fibroblasts, the cells were stained with crystal violet. ( e ) Cell lysates were subjected to western blotting using the indicated antibodies. ( f ) Premature senescence was detected by β -galactosidase activity. The graph shows the percentage of β -galactosidase-positive cells (right panel). The error bars indicate 95% confidence intervals; * P <0.0001 and ** P =0.0003 using the two-sided t -test

Article Snippet: Galectin-3 depletion reduced Skp2 mRNA but increased p27 KIP1 protein expression without a change in the mRNA expression.

Techniques: Knock-Out, Expressing, Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction, Control, Activity Assay, Transfection

Journal: Cell Death and Differentiation

Article Title: Ablation of galectin-3 induces p27 KIP1 -dependent premature senescence without oncogenic stress

doi: 10.1038/cdd.2014.88

Figure Lengend Snippet: Increased premature senescence caused by the inhibition of galectin-3 is p27 KIP1 dependent, but p53 independent. ( a ) After 48 h of transfection with control siRNA, galectin-3 siRNA, p27 KIP1 siRNA, and both siRNAs in the ( a and b ) AGS cells and ( c and d ) SNU601 cells, ( a ) cell proliferation was evaluated. The error bars indicates 95% confidence intervals; * P <0.0001 and ** P =0.0047 using a two-sided t -test. ( b ) Premature senescence was detected by β -galactosidase activity. The graph shows the percentage of β -galactosidase-positive cells. The error bars indicate 95% confidence intervals; * P <0.0001 and ** P <0.0001 using the two-sided t -test. ( c ) Cell proliferation was determined and the data were presented as a histogram. The error bars indicate 95% confidence intervals; * P <0.0001 and ** P =0.0139 using the two-sided t -test. ( d ) Premature senescence was analyzed by determining the β -galactosidase activity. The graph shows the percentage of β -galactosidase-positive cells. The error bars indicate 95% confidence intervals; * P =0.0008 and ** P =0.0013 using the two-sided t -test. ( e – g ) After transfection with the galectin-3 plasmids, Skp2 siRNA was additionally transfected into the SNU-638 cells. ( e ) Expression of the indicated proteins was detected by western blotting. ( f ) Cell proliferation and ( g ) cell cycle population analyses were performed by Ez-Cytox assay and PI staining, respectively. The error bars indicate 95% confidence intervals; * P =0.0005 and ** P =0.0067 using the two-sided t -test

Article Snippet: Galectin-3 depletion reduced Skp2 mRNA but increased p27 KIP1 protein expression without a change in the mRNA expression.

Techniques: Inhibition, Transfection, Control, Activity Assay, Expressing, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: Ablation of galectin-3 induces p27 KIP1 -dependent premature senescence without oncogenic stress

doi: 10.1038/cdd.2014.88

Figure Lengend Snippet: Inhibition of Skp2 reduces cell proliferation and induces premature senescence through an increase of p27 KIP1 protein expression. ( a – c ) After transfection with the Skp2 plasmids, galectin-3 siRNA was additionally transfected into the AGS cells. ( a ) Protein expression, ( b ) cell proliferation, and ( c ) cell cycle population analyses were performed by western blotting, Ez-Cytox assay, and PI staining, respectively. The error bars indicate 95% confidence intervals; * P =0.0094 using the two-sided t -test. AGS cells were transfected with control siRNA, Skp2 siRNA, p27 KIP1 siRNA, or both siRNAs for 48 h; ( d ) the protein expression was analyzed by western blotting and ( e ) cell proliferation was evaluated and the data are presented as a histogram. The error bars indicate 95% confidence intervals; * P =0.0005 and ** P =0.0156 using the two-sided t -test. ( f ) Premature senescence was detected by β -galactosidase activity. The graph shows the percentage of β -galactosidase-positive cells. The error bars indicate 95% confidence intervals; * P =0.0013 and ** P =0.0017 using the two-sided t -test. ( g ) The cell cycle populations were analyzed by PI staining

Article Snippet: Galectin-3 depletion reduced Skp2 mRNA but increased p27 KIP1 protein expression without a change in the mRNA expression.

Techniques: Inhibition, Expressing, Transfection, Western Blot, Staining, Control, Activity Assay

Journal: Cell Death and Differentiation

Article Title: Ablation of galectin-3 induces p27 KIP1 -dependent premature senescence without oncogenic stress

doi: 10.1038/cdd.2014.88

Figure Lengend Snippet: Inhibition or overexpression of galectin-3 regulates the phosphorylation of Rb and expression levels of cyclin D1 and CDK4. ( a ) Detection of protein levels of ppRb (at Ser 780 and Ser 807/811), Rb, E2F1, cyclin D1, and CDK4 after the depletion of galectin-3 using siRNA transfection in the AGS human gastric cancer cells. ( b ) Detection of the protein levels of galectin-3, Skp2, Rb, ppRb (at Ser 780 and Ser 807/811), and p27 KIP1 by western blot analysis in SNU-638 cells infected with a lentiviral construct containing galectin-3 or LacZ as the negative control. ( c ) Detection of the levels of galectin-3, Skp2, Rb, ppRb (at Ser 780 and Ser 807/811), and p27 KIP1 by western blot analysis after transfection of cyclin D1 and CDK4 in SNU-638 cells infected with the constructs in ( b ). ( d ) Schematic model of Flag-galectin-3 domain (1–250 aa as the full length; 1–110 aa as the N-terminal tail; and 33–250, 63–250, and 111–250 aa as the CRD). ( e ) Detection of the levels of galectin-3, Skp2, Rb, ppRb (at Ser 780 and Ser 807/811), and p27 KIP1 by western blot analysis after transfection of the galectin-3 domains in SNU-638 cells. β -Actin was used as the normalization control

Article Snippet: Galectin-3 depletion reduced Skp2 mRNA but increased p27 KIP1 protein expression without a change in the mRNA expression.

Techniques: Inhibition, Over Expression, Phospho-proteomics, Expressing, Transfection, Western Blot, Infection, Construct, Negative Control, Control

Journal: Cell Death and Differentiation

Article Title: Ablation of galectin-3 induces p27 KIP1 -dependent premature senescence without oncogenic stress

doi: 10.1038/cdd.2014.88

Figure Lengend Snippet: Galectin-3 directly interacts with Rb and regulates the transcriptional activity of E2F1. ( a ) Immunoprecipitation was performed with antibodies against galectin-3 and Rb to detect the interactions of galectin-3 with E2F1, Rb, cyclin D1, and CDK4 in the AGS cells. Whole cell lysate was used as the positive control. ( b ) Immunoprecipitation with Flag-galectin-3 domains, Cyclin D1, CDK4, and Rb using transfection of the galectin-3 domains in SNU-638 cells. ( c ) Schematic model of the interaction of the Skp2 promoter with the E2F1 binding site. ChIP primers were prepared to detect the E2F1 binding site (−42 to −35) from −95 to +135. The ChIP assay was performed using antibodies against galectin-3, Rb, and E2F1 in scRNA- and galectin-3 siRNA-transfected AGS cells. A PCR primer for the Skp2 promoter was used to detect promoter fragments in the immunoprecipitates. The input lane with total genomic DNA was used as a control for the PCR reaction. ( d–f ) Detection of luciferase activity after transfection of ( d ) SNU638 cells with an E2F1 consensus plasmid and galectin-3 domains for 48 h, ( e ) AGS cells with siRNA targeting galectin-3, Cyclin D1, and CDK4, or LacZ as a negative control for 48 h, and ( f ) galectin-3-overexpressing SNU638 cells with siRNA targeting Cyclin D1 and CDK4 for 48 h. The cells were transfected with 1 μ g of the E2F1 consensus plasmid. The error bars indicate 95% confidence intervals; * P <0.0001 using a two-sided t -test. Three independent experiments were performed

Article Snippet: Galectin-3 depletion reduced Skp2 mRNA but increased p27 KIP1 protein expression without a change in the mRNA expression.

Techniques: Activity Assay, Immunoprecipitation, Positive Control, Transfection, Binding Assay, Control, Luciferase, Plasmid Preparation, Negative Control

Journal: Cell Death and Differentiation

Article Title: Ablation of galectin-3 induces p27 KIP1 -dependent premature senescence without oncogenic stress

doi: 10.1038/cdd.2014.88

Figure Lengend Snippet: Galectin-3 depletion reduces the tumor burden in gastric cancer cell-xenografted mice and these effects are reversed by overexpression of skp2. ( a–d ) Lentiviruses expressing galectin-3 shRNA and overexpressing Skp2 were employed to produce stable AGS cell lines. Lentivirus expressing shRNA targeting LacZ was used as a control. Mice ( n =5 per group) were inoculated subcutaneously into both flanks with 10 6 cells of each of the AGS cell lines. ( a ) Skp2 overexpression and galectin-3 depletion were confirmed by western blot analysis. ( b ) Tumor formation was observed 30 days after inoculation. ( c ) Tumor formation was quantified by measuring the tumor volume 10 days after inoculation ( n =5). The error bars indicate 95% confidence intervals; * P= 0.001 and ** P= 0.0015 using two-sided t -test. All statistical tests were two sided. ( d ) Schematic model of galectin-3-dependent promotion of tumor progression and premature senescence. Galectin-3 interacts with Cyclin D1, CDK4, and Rb and blocks the inhibition of E2F1 transcription. This increases the expression of Skp2 and reduces the stability of p27 KIP1 to promote the proliferation of gastric cancer cells. After galectin-3 depletion, Rb interacts with E2F1 to block the transcriptional activity of E2F1, suppress Skp2 expression, and increase the stability of p27 KIP1 . This results in the suppression of the proliferation of gastric cancer cells and in the induction of premature cellular senescence

Article Snippet: Galectin-3 depletion reduced Skp2 mRNA but increased p27 KIP1 protein expression without a change in the mRNA expression.

Techniques: Over Expression, Expressing, shRNA, Control, Western Blot, Inhibition, Blocking Assay, Activity Assay

Journal: Cell Death and Differentiation

Article Title: Ablation of galectin-3 induces p27 KIP1 -dependent premature senescence without oncogenic stress

doi: 10.1038/cdd.2014.88

Figure Lengend Snippet: Positive association between galectin-3 and Skp2 expression and negative association between galectin-3 and p27 KIP1 expression is found in malignant tissues of gastric cancer patients. ( a ) The correlation between the mRNA expression of galectin-3 and Skp2 in the malignant tissues of 52 gastric cancer patients. The mRNA expression was detected by RT-PCR and quantified by an NIH ImageJ analyzer. GAPDH and β-actin were used as normalization controls. ( b ) The probability of survival of gastric cancer patients whose tumors show high galectin-3 and low p27 KIP1 levels is (red line) compared with that of gastric cancer patients whose tumors show low galectin-3 and high p27 KIP1 levels (blue line) using Kaplan–Meyer analysis. Statistical analysis is described in the Materials and Methods section. ( c ) Protein expression of galectin-3, Skp2, and p27 KIP1 in the malignant tissues of gastric cancer patients, as shown by immunohistochemical staining (brown) with hematoxylin and eosin (H&E). The stained tissue samples were observed using an inverted light microscope. Magnification: × 200 (top) and × 400 (bottom)

Article Snippet: Galectin-3 depletion reduced Skp2 mRNA but increased p27 KIP1 protein expression without a change in the mRNA expression.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Light Microscopy