Journal: Medicina

Article Title: Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27 KIP1 Regulation

doi: 10.3390/medicina60071160

Figure Lengend Snippet: SKP2 is a FASN target in human HCC cell lines. ( A ) The HLF, MHCC97-H, Hep3B, HuH7, and SNU449 cell lines were subjected to FASN knockdown using a specific small interfering siRNA against FASN (si-FASN). Data were collected 48 h after the silencing. The effects of FASN silencing on FASN, SKP2, and p27 KIP1 protein levels were detected by Western blot analysis. β-Actin was used as a loading control. ( B ) The effects of FASN silencing in the same cell lines on FASN , SKP2 , and CDKN1B (encoding p27 KIP1 ) mRNA levels were detected by quantitative real-time PCR. Student’s t -test: p < 0.0001 *** vs. scramble siRNA (Scr). Experiments were conducted three times in triplicate.

Article Snippet: Gene Expression Assays for human FASN (Hs01005622_m1), SKP2 (Hs01021864_m1), CDKN1B (Hs00153277_m1), and β-actin (4333762T), and mouse Fasn (Mm00662319_m1), Skp2 (Mm00449925_m1), Cdkn1b (Mm00438168_m1) and β-actin (mm00607939_S1) were from Applied Biosystems (Foster City, CA, USA).

Techniques: Knockdown, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Medicina

Article Title: Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27 KIP1 Regulation

doi: 10.3390/medicina60071160

Figure Lengend Snippet: Forced overexpression of SKP2 confers resistance to HCC cells against growth restraint by the C75 FASN inhibitor. The Huh7 cell line was subjected to SKP2 overexpression by stable transfection using an HA-tagged SKP2 plasmid (SKP2). Huh7 cells were also transfected with the empty vector (vector) as the control. Cells transfected with either SKP2 or vector were grown untreated or treated with the FASN inhibitor C75 (25 µM) for 48 h. ( A ) Increased levels of SKP2 protein following transfection of the SKP2 plasmid in Huh7 cells, as detected by Western blot analysis. β-Actin was used as a loading control. Transfection of SKP2 increases proliferation ( B ) and diminishes apoptosis ( C ) in the same cells. C75 administration significantly reduces proliferation and augments apoptosis in vector-transfected but not SKP2-transfected cells. ( D – F ) Equivalent results were obtained in the SNU449 cell line. Tukey–Kramer’s test: p < 0.0001; a , versus empty vector (vector); b , versus vector-transfected and C75-treated cells (Vector + C75); c, versus SKP2 overexpression. Experiments were conducted three times in triplicate.

Article Snippet: Gene Expression Assays for human FASN (Hs01005622_m1), SKP2 (Hs01021864_m1), CDKN1B (Hs00153277_m1), and β-actin (4333762T), and mouse Fasn (Mm00662319_m1), Skp2 (Mm00449925_m1), Cdkn1b (Mm00438168_m1) and β-actin (mm00607939_S1) were from Applied Biosystems (Foster City, CA, USA).

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

Journal: Medicina

Article Title: Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27 KIP1 Regulation

doi: 10.3390/medicina60071160

Figure Lengend Snippet: Dot spot graph representative of cell cycle distribution of HCC cells treated with si-FASN and si-SKP2 30 nM for 48 h. For the analysis, cells were recovered, washed with PBS and the pellet solubilized in ethanol 70% and stored at −20 °C overnight. The samples were stained with 7AAD (BD Biosciences, CA, USA) and incubated for 15 min at room temperature before acquisition using the flow cytometer FACS CANTOII (BD Biosciences, CA, USA). A total of 30.000 events for each sample were acquired and data were analyzed with ModFIT LT 6.0 (Verity Software House).

Article Snippet: Gene Expression Assays for human FASN (Hs01005622_m1), SKP2 (Hs01021864_m1), CDKN1B (Hs00153277_m1), and β-actin (4333762T), and mouse Fasn (Mm00662319_m1), Skp2 (Mm00449925_m1), Cdkn1b (Mm00438168_m1) and β-actin (mm00607939_S1) were from Applied Biosystems (Foster City, CA, USA).

Techniques: Staining, Incubation, Flow Cytometry, Software

Journal: Medicina

Article Title: Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27 KIP1 Regulation

doi: 10.3390/medicina60071160

Figure Lengend Snippet: Representative immunohistochemistry patterns of FASN and SKP2 proteins in human hepatocellular carcinoma (HCC; n = 210). ( A ) Example of a liver non-tumorous surrounding tissue exhibiting moderate membranous and cytoplasmic for FASN and weak/absent immunolabeling for SKP2. ( B ) A human HCC (denominated HCC1) displaying robust and diffuse immunoreactivity for FASN and SKP2 proteins. Note that FASN staining is localized in the cytoplasm of HCC cells, whereas SKP2 immunoreactivity is localized in the cytoplasmic and nuclear compartments. ( C ) A second hepatocellular tumor (HCC2) is characterized by intense, homogeneous FASN immunoreactivity and low/absent SKP2 immunolabeling. ( D ) Finally, a third tumor (HCC3) shows faint FASN positivity and absent SKP2 immunoreactivity. Abbreviation: H&E, hematoxylin and eosin staining. Original magnifications: 200× in all panels. Scale bar: 100 µm in all panels.

Article Snippet: Gene Expression Assays for human FASN (Hs01005622_m1), SKP2 (Hs01021864_m1), CDKN1B (Hs00153277_m1), and β-actin (4333762T), and mouse Fasn (Mm00662319_m1), Skp2 (Mm00449925_m1), Cdkn1b (Mm00438168_m1) and β-actin (mm00607939_S1) were from Applied Biosystems (Foster City, CA, USA).

Techniques: Immunohistochemistry, Immunolabeling, Staining

Journal: Medicina

Article Title: Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27 KIP1 Regulation

doi: 10.3390/medicina60071160

Figure Lengend Snippet: FASN and SKP2 are upregulated in human hepatocellular carcinoma (HCC). ( A ) Quantitative real-time RT-PCR values of FASN (first panel) and SKP2 (second panel) are significantly higher in the tumors (HCC) (n = 46) than in corresponding non-tumorous counterparts (ST). ( B ) Quantitative real-time RT-PCR values of FASN (first panel) and SKP2 (second panel) are significantly higher in HCC with poorer prognosis/shorter survival (HCCP; n = 25) than in tumors with better prognosis/longer survival (HCCB; n = 21). HCCP and HCCB are characterized by <3 and ≥3 years’ survival following partial liver resection. Student’s t -test: ***, p < 0.0001; **, p < 0.005. ( C ) Evaluation of the relation between FASN and SKP2 mRNA in HCC samples. p values and correlation r values were calculated using Pearson correlation analysis. ( D , E ) Kaplan–Meyer curves in HCC patients show that FASN ( D ) and SKP2 ( E ) mRNA levels inversely correlate with patients’ survival in this disease.

Article Snippet: Gene Expression Assays for human FASN (Hs01005622_m1), SKP2 (Hs01021864_m1), CDKN1B (Hs00153277_m1), and β-actin (4333762T), and mouse Fasn (Mm00662319_m1), Skp2 (Mm00449925_m1), Cdkn1b (Mm00438168_m1) and β-actin (mm00607939_S1) were from Applied Biosystems (Foster City, CA, USA).

Techniques: Quantitative RT-PCR

Journal: Medicina

Article Title: Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27 KIP1 Regulation

doi: 10.3390/medicina60071160

Figure Lengend Snippet: SKP2 is induced in the liver lesions from AKT mice. ( A , B ) Hydrodynamic gene delivery approach. In brief, FASN fl/fl mice were either injected with the myr-AKT1 construct (AKT mice) ( A ) or co-injected with Myr-AKT1 and Cre recombinase plasmids (AKT/Cre mice) ( B ). Five mice per group were injected and sacrificed 32 weeks post-injection (w.p.i.). ( C ) Immunohistochemical analysis shows that hepatocellular tumor lesions (T) developed in AKT mice display robust immunoreactivity for FASN, HA-AKT, and SKP2 proteins. Note that in the tumor cells, the immunoreactivity for SKP2 is localized in the cytoplasmic and nuclear compartments, as appreciable at the 200× magnification. In contrast, the surrounding non-tumorous liver tissues (ST) show weak/absent staining for the same proteins. Abbreviation: H&E, hematoxylin and eosin staining. Original magnifications: 100× and 200×, as indicated. Scale bar: 100 µm in 100× magnification pictures, 50 µm in the 200× magnification picture. ( D ) Representative Western blot analysis showing the levels of FASN, SKP2, and p27KIP1 in livers from FASN fl/fl mice injected with the empty vector only (Control), Myr-AKT1 (AKT mice), and myr-AKT1/Cre (AKT/Cre mice). Note that AKT mice display upregulation of SKP2 and marked downregulation of p27 KIP1 . Suppression of FASN in AKT/Cre mice, which triggers the inhibition of hepatocarcinogenesis, is accompanied by downregulation of SKP2 and an increase of p27 KIP1 protein levels. β-Actin was used as a loading control. ( E ) Quantitative real-time RT-PCR showing the mRNA levels of Fasn , Skp2 , and Cdkn1b in livers from FASN fl/fl mice injected with the empty vector only (vector), Myr-AKT1 (AKT mice) and myr-AKT1/Cre (AKT/Cre mice). N target = 2 −ΔCt , wherein the ΔCt value of each sample was calculated by subtracting the average Ct value of the target gene from the average Ct value of the β- actin gene. Five mice per group were analyzed. Tukey–Kramer’s test: p < 0.0001; a , versus control livers (vector); b , versus AKT livers.

Article Snippet: Gene Expression Assays for human FASN (Hs01005622_m1), SKP2 (Hs01021864_m1), CDKN1B (Hs00153277_m1), and β-actin (4333762T), and mouse Fasn (Mm00662319_m1), Skp2 (Mm00449925_m1), Cdkn1b (Mm00438168_m1) and β-actin (mm00607939_S1) were from Applied Biosystems (Foster City, CA, USA).

Techniques: Injection, Construct, Immunohistochemical staining, Staining, Western Blot, Plasmid Preparation, Control, Inhibition, Quantitative RT-PCR

Journal: Medicina

Article Title: Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27 KIP1 Regulation

doi: 10.3390/medicina60071160

Figure Lengend Snippet: SKP2 inactivation or non-degradable p27 KIP1 suppresses AKT-dependent hepatocarcinogenesis in mice. In the upper panels, the hydrodynamic gene delivery approach is depicted. In brief, C57BL/6J mice were either co-injected with the HA-tagged myr-AKT1 and empty vector (AKT mice), with Myr-AKT1 and SKP2 dominant negative (AKT/SKP2dn mice), or with Myr-AKT1 and a non-degradable form of V5-tagged p27 KIP1 (p27 KIP1-T187A ; AKT/p27 KIP1 mice). Five mice per group were injected and sacrificed 32 weeks post-injection (w.p.i.). At this time point, as revealed by hematoxylin and eosin staining (H&E), the livers of AKT mice are occupied by several tumor nodules (T). In contrast, the livers of AKT/SKP2dn and AKT/p27 KIP1 mice appear completely normal (better appreciable in the pictures taken at higher magnification). Original magnifications: 40× and 100×. Scale bar: 500 µm in 40× magnification pictures, 200 µm in 100× magnification pictures.

Article Snippet: Gene Expression Assays for human FASN (Hs01005622_m1), SKP2 (Hs01021864_m1), CDKN1B (Hs00153277_m1), and β-actin (4333762T), and mouse Fasn (Mm00662319_m1), Skp2 (Mm00449925_m1), Cdkn1b (Mm00438168_m1) and β-actin (mm00607939_S1) were from Applied Biosystems (Foster City, CA, USA).

Techniques: Injection, Plasmid Preparation, Dominant Negative Mutation, Staining

Journal: Medicina

Article Title: Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27 KIP1 Regulation

doi: 10.3390/medicina60071160

Figure Lengend Snippet: Inactivation of SKP2 or non-degradable p27 KIP1 is detrimental to the growth of HCC cells in vitro. ( A ) Transfection of SKP2dn triggers the upregulation of p27 KIP1 levels in SNU449 cells, as detected by Western blot analysis. As expected, transient transfection of SKP2dn resulted in the expression of a truncated form of SKP2 (transfected) with a lower molecular weight than the endogenous protein. β-Actin was used as a loading control. Transfection of SKP2dn reduces proliferation ( B ) and increases apoptosis ( C ) in the same cells. ( D ) Transfection of p27 KIP1−187A results in the appearance of a second band (transfected) with a higher molecular weight than the endogenous p27 KIP1 protein in the SNU449 cell line. β-Actin was used as a loading control. Similar to that described for SKP2dn, transfection of p27 KIP1−187A decreases proliferation ( E ) and augments apoptosis ( F ) in the same cell line. Student’s t -test: p < 0.0001 *** vs. empty vector (control). Experiments were conducted three times in triplicate.

Article Snippet: Gene Expression Assays for human FASN (Hs01005622_m1), SKP2 (Hs01021864_m1), CDKN1B (Hs00153277_m1), and β-actin (4333762T), and mouse Fasn (Mm00662319_m1), Skp2 (Mm00449925_m1), Cdkn1b (Mm00438168_m1) and β-actin (mm00607939_S1) were from Applied Biosystems (Foster City, CA, USA).

Techniques: In Vitro, Transfection, Western Blot, Expressing, Molecular Weight, Control, 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 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: International journal of molecular medicine

Article Title: eRF3b-37 inhibits the TGF-β1-induced activation of hepatic stellate cells by regulating cell proliferation, G0/G1 arrest, apoptosis and migration.

doi: 10.3892/ijmm.2018.3900

Figure Lengend Snippet: Figure 1. Validation of eRF3b‑37 sequences and determination of the optimum dosage of TGF‑β1 and eRF3b‑37 for LX‑2. (A) Nucleotide sequences and the corresponding amino acid alignments of the eRF3b‑37 in Homo species were obtained from the NCBI BLAST protein database. eRF3b‑37 identified by (B) mass‑spectrogram and (C) liquid chromatogram satisfied the requirements of the experiment. (D) The A490 and (E) proliferation viability of LX‑2 cells were determined by MTT following stimulation with 0, 0.1, 1, 10, 100 or 500 ng/ml TGF‑β1: It reached the plating point at 10 ng/ml at 24 and 48 h. LX‑2 cells were stimulated with 0, 0.1, 1, 10, 100 or 500 ng/ml eRF3b‑37, and the (F) cell proliferation A490 and (G) inhibition rate reached the optimal level at 100 ng/ml at 12, 24, 36 and 48 h, as determined by MTT. Data are presented as the mean ± standard deviation of six independent repeated experiments per group. TGF, trans- forming growth factor; eRF3b, eukaryotic peptide chain releasing factor 3b polypeptide; MTT, 3‑(4,5‑dimethyl‑2‑thiazolyl)‑2,5‑diphenyltetrazolium bromide.

Article Snippet: The anti-eRF3b antibody against human eRF3b was purchased from ProteinTech Group, Inc. (chicago, IL, USA; cat. no. 12989-1-AP).

Techniques: Biomarker Discovery, Inhibition, Standard Deviation

Journal: International journal of molecular medicine

Article Title: eRF3b-37 inhibits the TGF-β1-induced activation of hepatic stellate cells by regulating cell proliferation, G0/G1 arrest, apoptosis and migration.

doi: 10.3892/ijmm.2018.3900

Figure Lengend Snippet: Figure 2. eRF3b‑37 affects the mRNA and protein expression of GSPT2 and TGF‑β1 in LX‑2 cells. eRF3b‑37 (A) promotes GSPT2 and (B) inhibits TGF‑β1 mRNA expression as determined by reverse transcription‑quantitative polymerase chain reaction at 24 h. eRF3b‑37 (C) promotes GSPT2 and (D) inhibits TGF‑β1 protein expression as determined by western blotting at 48 h. Data are presented as the mean ± standard deviation of three independent repeated experiments per group. **P<0.01 vs. Control group; #P<0.05 and ##P<0.01 vs. TGF group. eRF3b, eukaryotic peptide chain releasing factor 3b polypeptide; TGF, transforming growth factor; GSPT2, G1 to S phase transition 2.

Article Snippet: The anti-eRF3b antibody against human eRF3b was purchased from ProteinTech Group, Inc. (chicago, IL, USA; cat. no. 12989-1-AP).

Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Standard Deviation, Control, Sublimation

Journal: International journal of molecular medicine

Article Title: eRF3b-37 inhibits the TGF-β1-induced activation of hepatic stellate cells by regulating cell proliferation, G0/G1 arrest, apoptosis and migration.

doi: 10.3892/ijmm.2018.3900

Figure Lengend Snippet: Figure 3. eRF3b‑37 decreases the mRNA and protein levels of the pro‑fibrotic factors ColI, CTGF and α‑SMA TGF‑β1‑stimulated in LX‑2 cells. eRF3b‑37 inhibited the mRNA expression of (A) ColI, (B) CTGF and (C) α‑SMA, as determined by reverse transcription‑quantitative polymerase chain reaction at 24 h. eRF3b‑37 inhibited the protein expression of (D) Col I, (E) CTGF and (F) α‑SMA as determined by western blotting at 48 h. (G) Doses of 0.1, 1, 10, 100 or 500 ng/ml of eRF3b‑37 increased GSPT2 expression and inhibited the expression of α‑SMA dose‑dependently at 48 h. (H) Statistical analysis indicated that there was a negative correlation between GSPT2 and α‑SMA protein expression. Data are presented as the mean ± standard deviation of three independent repeated experiments per group. **P<0.01 vs. Control group; #P<0.05 and ##P<0.01 vs. TGF group. eRF3b, eukaryotic peptide chain releasing factor 3b polypep- tide; CTGF, connective tissue growth factor; SMA, smooth muscle actin; TGF, transforming growth factor; Col, collagen; GSPT2, G1 to S phase transition 2.

Article Snippet: The anti-eRF3b antibody against human eRF3b was purchased from ProteinTech Group, Inc. (chicago, IL, USA; cat. no. 12989-1-AP).

Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Standard Deviation, Control, Sublimation

Journal: International journal of molecular medicine

Article Title: eRF3b-37 inhibits the TGF-β1-induced activation of hepatic stellate cells by regulating cell proliferation, G0/G1 arrest, apoptosis and migration.

doi: 10.3892/ijmm.2018.3900

Figure Lengend Snippet: Figure 4. eRF3b‑37 inhibits cell proliferation and promotes G0/G1 cell cycle arrest, as well as markedly affecting the expression of proliferative factors in LX‑2 cells stim- ulated with TGF‑β1. (A) eRF3b‑37 inhibited LX‑2 proliferation when stimulated with TGF‑β1, as determined by (4,5‑dimethyl‑2‑thiazolyl)‑2,5‑diphenyltetrazolium bromide assay at 24 and 48 h. (B) eRF3b‑37 increased the number of G0/G1 phase cells and downregulated the number of S phase cells, as determined by FCM. (C) A percentage bar chart and (D) statistical analysis results presenting the effects of eRF3b‑37 on the cell cycle. (E) eRF3b‑37 inhibited the proliferation index (%) of HSCs stimulated with TGF‑β1, as determined by FCM. eRF3b‑37 regulated the mRNA expression of (F) Cyclin D1, (G) CDK4 and (H) P21, as determined by reverse transcription‑quantitative polymerase chain reaction. Data are presented as the mean ± standard deviation of three independent repeated experiments per group. **P<0.01 vs. Control group; #P<0.05 and ##P<0.01 vs. TGF group. eRF3b, eukaryotic peptide chain releasing factor 3b polypeptide; TGF, transforming growth factor; CDK, Cyclin‑dependent kinase; FCM, flow cytometry; HSCs, hepatic stellate cells.

Article Snippet: The anti-eRF3b antibody against human eRF3b was purchased from ProteinTech Group, Inc. (chicago, IL, USA; cat. no. 12989-1-AP).

Techniques: Expressing, 5 Diphenyltetrazolium Bromide Assay, Polymerase Chain Reaction, Standard Deviation, Control, Flow Cytometry

Journal: International journal of molecular medicine

Article Title: eRF3b-37 inhibits the TGF-β1-induced activation of hepatic stellate cells by regulating cell proliferation, G0/G1 arrest, apoptosis and migration.

doi: 10.3892/ijmm.2018.3900

Figure Lengend Snippet: Figure 5. eRF3b‑37 reverses cell apoptosis and reduces cell migration viability by regulating associated factor expression in LX‑2 cells stimulated with TGF‑β1. (A) eRF3b‑37 reverses the cell apoptosis induced by TGF‑β1, as determined by flow cytometry at 48 h. (B) Statistical analysis revealed that the apoptotic rate was higher in the TGF+eRF3b group when compared with the TGF group. eRF3b‑37 regulates the mRNA expression of (C) Bcl‑2, (D) Bax and (E) Fas, as determined by reverse transcription‑quantitative polymerase chain reaction at 24 h. (F) Statistical analysis of the effects of eRF3b‑37 on cell migration. eRF3b‑37 reduced cell migration abilities at (G) 24 and (H) 48 h respectively (fluorescence microscope; magnification, x4.2). Image 1 is day 0; images 2, 3 and 4 are the control, TGF and TGF+eRF3b groups, respectively. Data are presented as the mean ± standard deviation of three independent repeated experiments per group. *P<0.05 and **P<0.01 vs. Control group; ##P<0.01 vs. TGF group. eRF3b, eukaryotic peptide chain releasing factor 3b poly- peptide; TGF, transforming growth factor; Bcl‑2, B‑cell lymphoma 2; Bax, Bcl‑2‑associated X protein.

Article Snippet: The anti-eRF3b antibody against human eRF3b was purchased from ProteinTech Group, Inc. (chicago, IL, USA; cat. no. 12989-1-AP).

Techniques: Migration, Expressing, Flow Cytometry, Polymerase Chain Reaction, Fluorescence, Microscopy, Control, Standard Deviation

Journal:

Article Title: Cell adhesion induces p27 Kip1 -associated cell-cycle arrest through down-regulation of the SCF Skp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

doi: 10.1182/blood-2006-11-060350

Figure Lengend Snippet: Cell adhesion–mediated p27Kip1 protein levels are posttranslationally regulated through down-regulation of the SCFSkp2 ubiquitin ligase Skp2. (A) An RPA for gene expression of p27Kip1, p21, and other cell-cycle–related molecules in Jeko-1 and SUDH-10 cells adhered to HS-5 versus those placed in suspension media. (B) The p27Kip1 and Skp2 expression in Jeko-1 cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) analyzed by Western blot. Cell-cycle distribution (C) and p27Kip1 and Skp2 expression (D) 24 hours after transfection with either Skp2 siRNA (siSkp2) or control siRNA (siCont) in Jeko-1 cells in suspension. (E) Cyclin D1 expression in Jeko-1 and SUDH-10 lymphoma cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) analyzed by Western blot. All data are representative of at least 3 experiments (means ± SD).

Article Snippet: Expressions of Cdh1, Skp2, p27 Kip1 , and GAPDH were analyzed using inventoried primers: TaqMan gene expression assay Hs 00212373_m1, Hs 00261857_m1, Hs 00153277_m1, and Hs 99999905_m1, respectively (Applied Biosystems [ABI], Foster City, CA).

Techniques: Ubiquitin Proteomics, Gene Expression, Suspension, Expressing, Western Blot, Transfection, Control

Journal:

Article Title: Cell adhesion induces p27 Kip1 -associated cell-cycle arrest through down-regulation of the SCF Skp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

doi: 10.1182/blood-2006-11-060350

Figure Lengend Snippet: Cell adhesion–mediated down-regulation of Skp2 requires Cdh1. (A) The Cdh1 expression in Jeko-1 cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) analyzed by Western blot. Cell-cycle distribution (B) and protein levels of p27Kip1, Skp2, and Cdh1 (C) 24 hours after transfection with Skp2 siRNA (siSkp2), Cdh1 siRNA (siCdh1), or control siRNA (siCont) in Jeko-1 lymphoma cells in suspension. All data are representative of at least 3 experiments (means ± SD).

Article Snippet: Expressions of Cdh1, Skp2, p27 Kip1 , and GAPDH were analyzed using inventoried primers: TaqMan gene expression assay Hs 00212373_m1, Hs 00261857_m1, Hs 00153277_m1, and Hs 99999905_m1, respectively (Applied Biosystems [ABI], Foster City, CA).

Techniques: Expressing, Suspension, Western Blot, Transfection, Control

Journal:

Article Title: Cell adhesion induces p27 Kip1 -associated cell-cycle arrest through down-regulation of the SCF Skp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

doi: 10.1182/blood-2006-11-060350

Figure Lengend Snippet: p27Kip1 depletion, Skp2 overexpression, and Cdh1 knock down abolished cell-adhesion–mediated p27Kip1 induction and cell-cycle arrest. (A) The p27Kip1 expression in control siRNA entering RISC-transfected (siCont.RISC[ + ]) or siP27Kip1 RNA-transfected (siP27) Jeko-1 cells in suspension (Sus) versus 12 hours of HS-5 adhesion (HS5-Ad) analyzed by Western blot, and (B) corresponding cell-cycle distribution in suspension (Sus) versus HS-5 adhesion (HS5-Ad) analyzed by flow cytometry. (C) The Skp2 and p27Kip1 expression in nontransfected [pCont(−)] Jeko-1 cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) or stably mock (pCont( + ))- or plasmid Skp2-transfected (pSkp2) Jeko-1 cells in suspension versus to HS-5 adhesion for 12 hours analyzed by Western blot and (D) corresponding cell-cycle distribution in stably mock- or plasmid Skp2-transfected Jeko-1 cells in suspension (pCont( + )-Sus or pSkp2-Sus) versus HS-5 adhesion (pCont( + )-Ad or pSkp2-Ad) for 12 hours analyzed by flow cytometry. (E) Cdh1, Skp2, and p27Kip1 expressions in nonsilencing siControl RNA-transfected (siCont) or siCdh1 RNA-transfected (siCdh1)-Jeko-1 cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) for 12 hours analyzed by Western blot, and (F) corresponding cell-cycle distribution in control siRNA- or Cdh1 siRNA-transfected Jeko-1 cells in suspension (siCont-Sus or siCdh1-Sus) versus HS-5 adhesion (siCont-Ad or siCdh1-Ad) for 12 hours analyzed by flow cytometry. Each blot is representative of 3 experiments, and the bar graphs show means plus or minus SD of 3 experiments.

Article Snippet: Expressions of Cdh1, Skp2, p27 Kip1 , and GAPDH were analyzed using inventoried primers: TaqMan gene expression assay Hs 00212373_m1, Hs 00261857_m1, Hs 00153277_m1, and Hs 99999905_m1, respectively (Applied Biosystems [ABI], Foster City, CA).

Techniques: Over Expression, Knockdown, Expressing, Control, Transfection, Suspension, Western Blot, Flow Cytometry, Stable Transfection, Plasmid Preparation

Journal:

Article Title: Cell adhesion induces p27 Kip1 -associated cell-cycle arrest through down-regulation of the SCF Skp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

doi: 10.1182/blood-2006-11-060350

Figure Lengend Snippet: The molecular mechanisms for cell-adhesion–mediated Skp2 and Cdh1 changes. (A,B) Effect of cell adhesion on Skp2 degradation. Jeko-1 cells were treated with cycloheximide (100 μM) with and without HS-5 cells adhesion for the indicated time periods. At the times indicated, cells were lysed, and Skp2 protein levels were determined by Western blot analysis (A) and quantified (from 3 independent experiments) using densitometry. The data shown (B) represent mean values and standard deviations of the average percentage of Skp2, compared with the amount of Skp2 at time 0 for each treatment group. (C,D) Cell-adhesion–induced changes of Skp2 and CDH1 mRNA expressions measured by real-time qRT-PCR. Fold values were obtained by externally standardizing against identical amplifications in Jeko-1 and SUDH-10 cells in suspension versus HS-5 adhesion and by internally standardizing against GAPDH in each cell line. The mean values and standard deviations from 3 independent experiments are shown. Student t test was used for statistical analysis. *P < .05.

Article Snippet: Expressions of Cdh1, Skp2, p27 Kip1 , and GAPDH were analyzed using inventoried primers: TaqMan gene expression assay Hs 00212373_m1, Hs 00261857_m1, Hs 00153277_m1, and Hs 99999905_m1, respectively (Applied Biosystems [ABI], Foster City, CA).

Techniques: Western Blot, Quantitative RT-PCR, Suspension

Journal:

Article Title: Cell adhesion induces p27 Kip1 -associated cell-cycle arrest through down-regulation of the SCF Skp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

doi: 10.1182/blood-2006-11-060350

Figure Lengend Snippet: Cell-adhesion–induced p27Kip1-associated cell-cycle arrest through down-regulating the SCFSkp2 ubiquitin ligase pathway in primary MCL and other non-Hodgkin B-cell lymphomas. (A,B) The Cdh1, Skp2, and p27Kip1 expressions in primary MCL (A) and DLBCL (B) cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) were analyzed by Western blot. (C) Cell-cycle distribution in DLBCL (DLBL0401) cells in suspension (Sus), or on a confluent HS-5 monolayer (HS5-Ad; 12 hours).

Article Snippet: Expressions of Cdh1, Skp2, p27 Kip1 , and GAPDH were analyzed using inventoried primers: TaqMan gene expression assay Hs 00212373_m1, Hs 00261857_m1, Hs 00153277_m1, and Hs 99999905_m1, respectively (Applied Biosystems [ABI], Foster City, CA).

Techniques: Ubiquitin Proteomics, Suspension, Western Blot

Journal:

Article Title: Cell adhesion induces p27 Kip1 -associated cell-cycle arrest through down-regulation of the SCF Skp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

doi: 10.1182/blood-2006-11-060350

Figure Lengend Snippet: Model for cell-adhesion–mediated cell-cycle arrest. Hypothetically ordered series of signaling events: cell-adhesion–dependent activation of APC/Cdh1 ubiquitin ligase complex, increased ubiquitination and degradation of Skp2, subsequent decreased ubiquitination and degradation of p27Kip1, and ultimately p27Kip1 accumulation, resulting in cell-cycle arrest.

Article Snippet: Expressions of Cdh1, Skp2, p27 Kip1 , and GAPDH were analyzed using inventoried primers: TaqMan gene expression assay Hs 00212373_m1, Hs 00261857_m1, Hs 00153277_m1, and Hs 99999905_m1, respectively (Applied Biosystems [ABI], Foster City, CA).

Techniques: Activation Assay, Ubiquitin Proteomics