rabbit polyclonal antibody against human full length pinx1  (Proteintech)

Journal: OncoTargets and Therapy

Article Title: PinX1 suppresses tumorigenesis by negatively regulating telomerase/telomeres in colorectal carcinoma cells and is a promising molecular marker for patient prognosis

doi: 10.2147/ott.s103141

Figure Lengend Snippet: Figure 1 Expression of PinX1 in human colorectal carcinoma (CRC) tissues. Notes: (A) High expression of PinX1 was observed in a normal colonic mucosa tissue, in which almost all colonic mucosa cells showed positive staining of PinX1 in nuclei. (B) A CRC (case 11) shows negative expression of PinX1. (C) A CRC (case 52) was examined with low expression of PinX1, in which ,50% of carcinoma cells showed positive staining of PinX1 in nuclei. (D) High expression of PinX1 was examined in another CRC sample (case 36) in which .50% carcinoma cells demonstrated positive staining of PinX1. (E) The percentage of the cases with low/negative or high PinX1 expression in tumor and normal tissues (**P,0.01).

Article Snippet: The tissue slides were incubated with the rabbit polyclonal antibody against human full-length PinX1 (ProteinTech Group, Inc., Rosemont, IL, USA; 1:100 dilution) overnight at 4°C.

Techniques: Expressing, Staining

Journal: OncoTargets and Therapy

Article Title: PinX1 suppresses tumorigenesis by negatively regulating telomerase/telomeres in colorectal carcinoma cells and is a promising molecular marker for patient prognosis

doi: 10.2147/ott.s103141

Figure Lengend Snippet: Figure 2 Kaplan–Meier statistical analyses of overall survival (OS) and disease-free survival (DFS) in 86 colorectal carcinoma patients. Notes: Downregulation of PinX1 was significantly associated with poorer OS (A) and poorer DFS (B), according to PinX1 expression levels in the primary tumor (P,0.05, log-rank test).

Article Snippet: The tissue slides were incubated with the rabbit polyclonal antibody against human full-length PinX1 (ProteinTech Group, Inc., Rosemont, IL, USA; 1:100 dilution) overnight at 4°C.

Techniques: Expressing

Journal: OncoTargets and Therapy

Article Title: PinX1 suppresses tumorigenesis by negatively regulating telomerase/telomeres in colorectal carcinoma cells and is a promising molecular marker for patient prognosis

doi: 10.2147/ott.s103141

Figure Lengend Snippet: Figure 3 PinX1 suppresses proliferation and promotes apoptosis in colorectal carcinoma cells in vitro. Notes: (A) Expression of PinX1 was detected by Western blot in stable transfected SW1116 and SW480cells (SW1116-PinX1; SW480-PinX1) relative to empty vector control cells (SW1116-Vector; SW480-Vector) and blank control cells (SW1116-Blank; SW480-Blank). Expression was normalized against endogenous GAPDH. (B) Cell growth rate was suppressed by ectopic overexpression of PinX1 in SW1116 cells detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Results are expressed as mean ± standard deviation (SD) of three independent experiments. (C) PinX1 promoted tumor cell apoptosis in SW1116 cells compared with the vector- transfected control cells and blank control cells under normal conditions. Cell apoptotic death events were monitored by Annexin V/propidium iodide (PI) staining and flow cytometry assays. The percentage of cell apoptosis was shown as the mean ± SD from three independent experiments (**P,0.01, P-value was according to Student’s t-test).

Article Snippet: The tissue slides were incubated with the rabbit polyclonal antibody against human full-length PinX1 (ProteinTech Group, Inc., Rosemont, IL, USA; 1:100 dilution) overnight at 4°C.

Techniques: In Vitro, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Over Expression, Standard Deviation, Staining, Flow Cytometry

Journal: OncoTargets and Therapy

Article Title: PinX1 suppresses tumorigenesis by negatively regulating telomerase/telomeres in colorectal carcinoma cells and is a promising molecular marker for patient prognosis

doi: 10.2147/ott.s103141

Figure Lengend Snippet: Figure 4 Effect of PinX1 on telomerase activity (TA) and telomere length in colorectal carcinoma cells. Notes: TA was measured by TRAP assays and Southern blot analysis of telomeric terminal restriction fragments was used for the determination of the telomere length. Ectopic overexpression of PinX1 in SW1116 and SW480 cells decreased TA (A) and shortened telomere (B).

Article Snippet: The tissue slides were incubated with the rabbit polyclonal antibody against human full-length PinX1 (ProteinTech Group, Inc., Rosemont, IL, USA; 1:100 dilution) overnight at 4°C.

Techniques: Activity Assay, Southern Blot, Over Expression

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Correlation of PinX1 expression in tissue with patients’ clinicopathological variables in 187 cases of UCB

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques: Expressing

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: The expression of PinX1 in UCB and adjacent normal bladder tissues. (A) Down-regulated expression of PinX1 mRNA was examined by qRT-PCR in 8/10 UCB cases, when compared with adjacent normal bladder tissues. Expression levels were normalized for GAPDH. Error bars, SD calculated from three parallel experiments. (B) Down-regulated expression of PinX1 protein was detected by Western blotting in 7/10 UCB cases, when compared with adjacent normal bladder tissues. Expression levels were normalized with GAPDH. (C-F) The expression of PinX1 in UCB and adjacent normal bladder tissues by IHC (100×). An UCB (case 45) tissue showed negative expression of PinX1 (C) , while its adjacent normal bladder urothelial mucosal tissue was positive stained by PinX1, in which more than 90% of tumor cells were positively stained by PinX1 in the nucleus (D) . Negative expression of PinX1 was observed in another UCB tissue (case 73), in which only 10% of tumor cells demonstrated a nuclear staining of PinX1 (E) . An UCB (case 126) was negatively stained by PinX1 (F) .

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Kaplan-Meier survival analysis of PinX1 expression in patients with UCB (log-rank test). (A) Total, probability of overall survival of all patients with UCB: negative expression ( dashed line ), n = 83; positive expression ( solid line ), n = 104. (B) Adjuvant chemotherapy, probability of recurrence-free survival of patients underwent adjuvant chemotherapy with UCB: negative expression ( dashed line ), n = 27; positive expression ( solid line ), n = 20. (C) G1, probability of overall survival of G1 patients with UCB: negative expression ( dashed line ), n = 19; positive expression ( solid line ), n = 27. (D) G2, probability of overall survival of G2 patients with UCB: negative expression ( dashed line ), n = 29; positive expression ( solid line ), n = 37. (E) G3, probability of overall survival of G3 patients with UCB: negative expression ( dashed line ), n = 35; positive expression ( solid line ), n = 40. (F) pT1, probability of overall survival of pT1 patients with UCB: negative expression ( dashed line ), n = 13; positive expression ( solid line ), n = 22. (G) pT2, probability of overall survival of pT2 patients with UCB: negative expression ( dashed line ), n = 38; positive expression ( solid line ), n = 57. (H) pT3, probability of overall survival of pT3 patients with UCB: negative expression ( dashed line ), n = 20; positive expression ( solid line ), n = 17. (I) pT4-, probability of overall survival of pT4 patients with UCB: negative expression ( dashed line ), n = 12; positive expression ( solid line ), n = 8. (J) pN-, probability of overall survival of pN- patients with UCB: negative expression ( dashed line ), n = 64; positive expression ( solid line ), n = 93. (K) pN+, probability of overall survival of pN+ patients with UCB: negative expression ( dashed line ), n = 19; positive expression ( solid line ), n = 11.

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques: Expressing, Adjuvant

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Multivariate analysis of prognostic factors on overall survival (Cox regression model)

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques:

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: PinX1 inhibited growth and proliferation of UCB cells in vitro. (A-B) Ectopic expression of PinX1 in EJ and T24 cell analyzed by western blotting. GAPDH was used as a loading control. (C-D) Ectopic expression of PinX1 inhibited EJ and T24 cell proliferation in MTT assays. Each bar represents the average ± SD of three independent experiments. (E-F) Upregulation of PinX1 inhibited EJ and T24 cell growth in colony formation assays. Representative micrographs (upper) and quantification (lower) of crystal violet-stained cells. (G) RNAi-silencing of PinX1 in shRNA-transduced stable 5637 cell. GAPDH was used as a loading control. (H) Silencing endogenous PinX1 promoted cell growth as determined by MTT assays. Each bar represents the average ± SD of three independent experiments. (I) Downregulation of PinX1 promoted 5637 cell growth in colony formation assays. Representative micrographs (upper) and quantification (lower) of crystal violet-stained cells. * P < 0.05, ** P < 0.01.

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques: In Vitro, Expressing, Western Blot, Control, Staining, shRNA

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: PinX1 inhibited growth and proliferation of UCB cells in vivo. (A-B) Ectopic expression of PinX1 in EJ and T24 cells dramatically inhibited tumor growth and proliferation in vivo as determined by a subcutaneous xenograft mice model. Representative graph of tumor growth (left). Data points are the mean tumor volumes ± SD (middle) and mean tumor weights (right) 48 days after inoculation. (C) Suppression of PinX1 in 5637 cells promoted tumor growth and proliferation in vivo as determined by a subcutaneous xenograft mice model. Representative graph of tumor growth (left). Data points are the mean tumor volumes ± SD (middle) and mean tumor weights (right) 48 days after inoculation. ** P < 0.01.

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques: In Vivo, Expressing

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Effect of PinX1 on telomerase activity and telomere length in UCB cells. Telomerase activity was measured by TRAP assays and Southern blot analysis of telomeric terminal restriction fragments were used for determination of the telomere length. (A) Overexpression of PinX1 in EJ cells decreased telomerase activity and shortened telomere length. (B) Overexpression of PinX1 in T24 cells decreased telomerase activity and shortened telomere length. (C) RNAi-silencing of PinX1 in 5637 cells increased telomerase activity and elongated telomere length.

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques: Activity Assay, Southern Blot, Over Expression

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: PinX1 regulates the G1/S phase transition and cell proliferation through the p16/cyclin D1 pathway in UCB cells. Flow cytometric analysis of the (A) EJ bladder urothelial carcinoma cells infected with vector or PinX1, (B) T24 bladder urothelial carcinoma cells infected with vector or PinX1, and (C) 5637 cells infected with RNAi-vector or PinX1-shRNA. (D) The seven genes, CDKN2A (i.e. p16), CDKN2B (i.e. p15), GADD45A, CCND1 (i.e. cyclin D1), CCND2 (i.e. cyclin D2), ANAPC2 and CDK5R1), were examined >2-fold mRNA differential expression in PinX1-transfected T24 cells compared with that of scramble vector transfected by using a Human Cell Cycle RT 2 Profiler CC PCR Array. (E) Overexpression of PinX1 substantially upregulated p16 expression and downregulated cyclin D1 expression in T24 cells detected by western blotting. * P < 0.05, ** P < 0.01.

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques: Sublimation, Infection, Plasmid Preparation, shRNA, Quantitative Proteomics, Transfection, Over Expression, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Univariate analysis of factors associated with overall survival of 187 patients with UCB

Article Snippet: This was followed by incubation with primary rabbit polyclonal antibody against human PinX1 (Proteintech Group, USA), mouse monoclonal antibodies to p16 (Cell Signaling Technology, USA), cyclin D1 (Cell Signaling Technology, USA), CDKN2B (Cell Signaling Technology, USA), CCND2 (Cell Signaling Technology, USA), rabbit monoclonal antibodies GADD45A (Santa Cruz Biotechnology, USA), ANAPC2 (Santa Cruz Biotechnology, USA), and CDK5R1 (Santa Cruz Biotechnology, USA), respectively.

Techniques: Expressing

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Correlation of PinX1 expression in tissue with patients’ clinicopathological variables in 187 cases of UCB

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques: Expressing

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: The expression of PinX1 in UCB and adjacent normal bladder tissues. (A) Down-regulated expression of PinX1 mRNA was examined by qRT-PCR in 8/10 UCB cases, when compared with adjacent normal bladder tissues. Expression levels were normalized for GAPDH. Error bars, SD calculated from three parallel experiments. (B) Down-regulated expression of PinX1 protein was detected by Western blotting in 7/10 UCB cases, when compared with adjacent normal bladder tissues. Expression levels were normalized with GAPDH. (C-F) The expression of PinX1 in UCB and adjacent normal bladder tissues by IHC (100×). An UCB (case 45) tissue showed negative expression of PinX1 (C) , while its adjacent normal bladder urothelial mucosal tissue was positive stained by PinX1, in which more than 90% of tumor cells were positively stained by PinX1 in the nucleus (D) . Negative expression of PinX1 was observed in another UCB tissue (case 73), in which only 10% of tumor cells demonstrated a nuclear staining of PinX1 (E) . An UCB (case 126) was negatively stained by PinX1 (F) .

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Kaplan-Meier survival analysis of PinX1 expression in patients with UCB (log-rank test). (A) Total, probability of overall survival of all patients with UCB: negative expression ( dashed line ), n = 83; positive expression ( solid line ), n = 104. (B) Adjuvant chemotherapy, probability of recurrence-free survival of patients underwent adjuvant chemotherapy with UCB: negative expression ( dashed line ), n = 27; positive expression ( solid line ), n = 20. (C) G1, probability of overall survival of G1 patients with UCB: negative expression ( dashed line ), n = 19; positive expression ( solid line ), n = 27. (D) G2, probability of overall survival of G2 patients with UCB: negative expression ( dashed line ), n = 29; positive expression ( solid line ), n = 37. (E) G3, probability of overall survival of G3 patients with UCB: negative expression ( dashed line ), n = 35; positive expression ( solid line ), n = 40. (F) pT1, probability of overall survival of pT1 patients with UCB: negative expression ( dashed line ), n = 13; positive expression ( solid line ), n = 22. (G) pT2, probability of overall survival of pT2 patients with UCB: negative expression ( dashed line ), n = 38; positive expression ( solid line ), n = 57. (H) pT3, probability of overall survival of pT3 patients with UCB: negative expression ( dashed line ), n = 20; positive expression ( solid line ), n = 17. (I) pT4-, probability of overall survival of pT4 patients with UCB: negative expression ( dashed line ), n = 12; positive expression ( solid line ), n = 8. (J) pN-, probability of overall survival of pN- patients with UCB: negative expression ( dashed line ), n = 64; positive expression ( solid line ), n = 93. (K) pN+, probability of overall survival of pN+ patients with UCB: negative expression ( dashed line ), n = 19; positive expression ( solid line ), n = 11.

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques: Expressing, Adjuvant

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Multivariate analysis of prognostic factors on overall survival (Cox regression model)

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques:

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: PinX1 inhibited growth and proliferation of UCB cells in vitro. (A-B) Ectopic expression of PinX1 in EJ and T24 cell analyzed by western blotting. GAPDH was used as a loading control. (C-D) Ectopic expression of PinX1 inhibited EJ and T24 cell proliferation in MTT assays. Each bar represents the average ± SD of three independent experiments. (E-F) Upregulation of PinX1 inhibited EJ and T24 cell growth in colony formation assays. Representative micrographs (upper) and quantification (lower) of crystal violet-stained cells. (G) RNAi-silencing of PinX1 in shRNA-transduced stable 5637 cell. GAPDH was used as a loading control. (H) Silencing endogenous PinX1 promoted cell growth as determined by MTT assays. Each bar represents the average ± SD of three independent experiments. (I) Downregulation of PinX1 promoted 5637 cell growth in colony formation assays. Representative micrographs (upper) and quantification (lower) of crystal violet-stained cells. * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques: In Vitro, Expressing, Western Blot, Control, Staining, shRNA

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: PinX1 inhibited growth and proliferation of UCB cells in vivo. (A-B) Ectopic expression of PinX1 in EJ and T24 cells dramatically inhibited tumor growth and proliferation in vivo as determined by a subcutaneous xenograft mice model. Representative graph of tumor growth (left). Data points are the mean tumor volumes ± SD (middle) and mean tumor weights (right) 48 days after inoculation. (C) Suppression of PinX1 in 5637 cells promoted tumor growth and proliferation in vivo as determined by a subcutaneous xenograft mice model. Representative graph of tumor growth (left). Data points are the mean tumor volumes ± SD (middle) and mean tumor weights (right) 48 days after inoculation. ** P < 0.01.

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques: In Vivo, Expressing

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Effect of PinX1 on telomerase activity and telomere length in UCB cells. Telomerase activity was measured by TRAP assays and Southern blot analysis of telomeric terminal restriction fragments were used for determination of the telomere length. (A) Overexpression of PinX1 in EJ cells decreased telomerase activity and shortened telomere length. (B) Overexpression of PinX1 in T24 cells decreased telomerase activity and shortened telomere length. (C) RNAi-silencing of PinX1 in 5637 cells increased telomerase activity and elongated telomere length.

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques: Activity Assay, Southern Blot, Over Expression

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: PinX1 regulates the G1/S phase transition and cell proliferation through the p16/cyclin D1 pathway in UCB cells. Flow cytometric analysis of the (A) EJ bladder urothelial carcinoma cells infected with vector or PinX1, (B) T24 bladder urothelial carcinoma cells infected with vector or PinX1, and (C) 5637 cells infected with RNAi-vector or PinX1-shRNA. (D) The seven genes, CDKN2A (i.e. p16), CDKN2B (i.e. p15), GADD45A, CCND1 (i.e. cyclin D1), CCND2 (i.e. cyclin D2), ANAPC2 and CDK5R1), were examined >2-fold mRNA differential expression in PinX1-transfected T24 cells compared with that of scramble vector transfected by using a Human Cell Cycle RT 2 Profiler CC PCR Array. (E) Overexpression of PinX1 substantially upregulated p16 expression and downregulated cyclin D1 expression in T24 cells detected by western blotting. * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques: Sublimation, Infection, Plasmid Preparation, shRNA, Quantitative Proteomics, Transfection, Over Expression, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: PinX1 suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1 pathway

doi: 10.1186/1476-4598-12-148

Figure Lengend Snippet: Univariate analysis of factors associated with overall survival of 187 patients with UCB

Article Snippet: Subsequently, the TMA slides were incubated overnight at 4°C with rabbit polyclonal antibody against PinX1 (1:200; Proteintech Group, USA), mouse monoclonal anti-Ki-67 (1:100; Sigma-Aldrich, USA), or mouse monoclonal anti-p16 (1:100; Cell Signaling Technology, USA) and anti-cyclin D1 (1:100; Cell Signaling Technology, USA), overnight at 4°C.

Techniques: Expressing

Journal: Oncogene

Article Title: Anthracyclines disrupt telomere maintenance by telomerase through inducing PinX1 ubiquitination and degradation.

doi: 10.1038/onc.2011.214

Figure Lengend Snippet: Figure 3 Anthracyclines block TERT association with telomeres and trigger telomere dysfunction by downregulating PinX1 expression. (a) Flag-TERT-stable HepG2 cells were treated with Et (20 mg/ml), DOX (1 mg/ml) and EPI (1 mg/ml). At the indicated time points, the endogenous PinX1 expression was detected by immunoblotting assay with specific PinX1 antibodies. Tubulin as control to show equal loading amounts for each detected sample. (b) Telomerase-negative BJ normal fibroblasts and U2OS cancer cells were treated with the indicated doses of drugs for 24 h and PinX1 expression was examined by immunoblotting assay. (c) A PinX1 plasmid for PinX1 overexpression and a PinX1 shRNA plasmid to knock down endogenous PinX1 expression were delivered into Flag-TERT HepG2 cells through a lentiviral infection. PinX1 expression was examined by immunoblotting assay before and after the EPI treatment. Tubulin as control to show equal loading samples. Mock, uninfected control cells. (d) IP assays show that the effect of EPI on inhibiting TERT association with the Pot1-containing telomeric protein complex is relieved in PinX1-overexpressing cells. (e) TIFs assays in HepG2 cells after the indicated treatments. Left, representative images of gH2AX (red) and TRF1 (green) and DAPI (blue) staining. Arrows indicate co-localization events. Right, results are mean of triplicate experiments; bars, s.d. (f) BJ cells infected with lentiviruses for PinX1 overexpression and PinX1 knockdown were confirmed by western blotting (left panel). TIFs assays in BJ cells after the indicated treatments (right panel).

Article Snippet: Antibody sources are as follows: Rabbit polyclonal antibodies against PinX1 were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Blocking Assay, Expressing, Western Blot, Control, Plasmid Preparation, Over Expression, shRNA, Knockdown, Infection, Staining

Journal: Oncogene

Article Title: Anthracyclines disrupt telomere maintenance by telomerase through inducing PinX1 ubiquitination and degradation.

doi: 10.1038/onc.2011.214

Figure Lengend Snippet: Figure 4 Anthracyclines downregulate PinX1 expression by inducing this protein degradation through the ubiquitin–proteasome- dependent pathway. (a) HepG2 cells were treated with DOX and EPI for the indicated periods. PinX1 mRNA expression levels were detected by RT–PCR assays. GAPDH as control for total RNA was used in the RT–PCR assays. (b) HepG2 cells were pretreated with ( þ ) or without () the proteasome inhibitor MG132 (10 mM) for 8 h before they were exposed to the indicated dosages of EPI and DOX, respectively. After 16 h of EPI and DOX treatments, nuclear extracts were prepared and subjected to IB assays with PinX1- specific antibodies. Lower panels showed the ratios of PinX1 to tubulin from densitometric analysis. The results were representative of the three experiments. (c) HepG2 cells were transfected with a plasmid encoding Flag-PinX1 or a vector control, and transfected cells were pretreated with MG132 (10 mM) for 4 h before they were exposed to DOX (1 mg/ml) and EPI (1 mg/ml), respectively. Twelve hours after anthracyclines treatments, ectopic-expressed Flag-PinX1 was immunoprecipitated from nuclear extracts by the anti-Flag M2 beads. The immunoprecipitates were subjected to IB assay with an anti-ubiquitin antibody. IB with the anti-Flag antibody shows equal amounts of the precipitated PinX1 proteins for each detecting samples.

Article Snippet: Antibody sources are as follows: Rabbit polyclonal antibodies against PinX1 were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Expressing, Ubiquitin Proteomics, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Oncogene

Article Title: Anthracyclines disrupt telomere maintenance by telomerase through inducing PinX1 ubiquitination and degradation.

doi: 10.1038/onc.2011.214

Figure Lengend Snippet: Figure 5 Modulations of PinX1 expressions affect the cytotoxic effects of EPI in HepG2 cancer cells. (a) Full-length PinX1 for PinX1 overexpression and PinX1 shRNA for PinX1 knockdown were introduced into HepG2 cells through lentiviral infection. Cells were then treated with indicated doses of EPI for 24 h, and EPI-induced cell apoptotic death events were monitored by Alexa Fluor 488 annexin V/PI staining and flow cytometry assays. Viable cells (annexin V and PI negative, D3) are located in the left lower box. Cells undergoing early apoptosis (annexin V positive and PI negative, D4) are located in the right lower box. Cells in the late stage of apoptosis and dead cells (annexin V and PI positive, D2) are located in the right upper box. (b) After treatment, immunoblotting was performed with PARP, PinX1 and tubulin antibody. Lower panels show the ratios of PinX1 to tubulin from densitometric analysis. Mock, uninfected control cells.

Article Snippet: Antibody sources are as follows: Rabbit polyclonal antibodies against PinX1 were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Over Expression, shRNA, Knockdown, Infection, Staining, Cytometry, Western Blot, Control