Journal: Oncology Letters

Article Title: Siva 1 inhibits proliferation, migration and invasion by phosphorylating Stathmin in ovarian cancer cells

doi: 10.3892/ol.2017.6307

Figure Lengend Snippet: Sequences of real-time PCR primers.

Article Snippet: Subsequent to blocking with 5% skim milk (YILI, Hohhot, Inner Mongolia, China) at room temperature for 1 h, diluted by TBS Tween (TBST), the PVDF membrane was incubated with the following antibodies at 4°C overnight: rabbit anti-human anti-Siva 1 polyclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA, cat. no., sc-48767), rabbit anti-human anti-cleaved caspase-3 polyclonal antibody (dilution, 1:1,000; Abcam, Cambridge, UK, cat. no., ab2302), goat anti-human anti-cleaved caspase-9 polyclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc., cat. no., sc-22182), rabbit anti-human anti-Bcl-2-like protein 4 (Bax) polyclonal antibody (dilution, 1:400; Boster, Hubei, Wuhan, China, cat. no., BA0315), rabbit anti-human anti-Bcl-2 polyclonal antibody (dilution, 1:400; Boster, cat. no., BA0412), rabbit anti-human anti-Stathmin polyclonal antibody (dilution, 1:500; Bioss Antibodies, Beijing, China, cat. no., bs-1902R), rabbit anti-human anti-phosphorylated Stathmin polyclonal antibody (dilution, 1:500; Bioss Antibodies, cat. no., bs-3431R), rabbit anti-human anti-α-tubulin polyclonal antibody (dilution, 1:1,000; Abcam, cat. no., ab178484) or mouse anti-human anti-acetyl-α-tubulin monoclonal antibody (dilution, 1:1,000; Abcam, cat. no., ab24610).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Oncology Letters

Article Title: Siva 1 inhibits proliferation, migration and invasion by phosphorylating Stathmin in ovarian cancer cells

doi: 10.3892/ol.2017.6307

Figure Lengend Snippet: Siva 1 enhances phosphorylation of Stathmin. (A) Siva 1 does not affect Stathmin mRNA level. (B and C) Overexpression of Siva 1 enhances phosphorylation of Stathmin and acetylation of α-tubulin, but did not markedly affect the expression level of Stathmin and α-tubulin detected by western blot. ***P<0.001 vs. pCMV group; ns, no significance.

Article Snippet: Subsequent to blocking with 5% skim milk (YILI, Hohhot, Inner Mongolia, China) at room temperature for 1 h, diluted by TBS Tween (TBST), the PVDF membrane was incubated with the following antibodies at 4°C overnight: rabbit anti-human anti-Siva 1 polyclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA, cat. no., sc-48767), rabbit anti-human anti-cleaved caspase-3 polyclonal antibody (dilution, 1:1,000; Abcam, Cambridge, UK, cat. no., ab2302), goat anti-human anti-cleaved caspase-9 polyclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Inc., cat. no., sc-22182), rabbit anti-human anti-Bcl-2-like protein 4 (Bax) polyclonal antibody (dilution, 1:400; Boster, Hubei, Wuhan, China, cat. no., BA0315), rabbit anti-human anti-Bcl-2 polyclonal antibody (dilution, 1:400; Boster, cat. no., BA0412), rabbit anti-human anti-Stathmin polyclonal antibody (dilution, 1:500; Bioss Antibodies, Beijing, China, cat. no., bs-1902R), rabbit anti-human anti-phosphorylated Stathmin polyclonal antibody (dilution, 1:500; Bioss Antibodies, cat. no., bs-3431R), rabbit anti-human anti-α-tubulin polyclonal antibody (dilution, 1:1,000; Abcam, cat. no., ab178484) or mouse anti-human anti-acetyl-α-tubulin monoclonal antibody (dilution, 1:1,000; Abcam, cat. no., ab24610).

Techniques: Over Expression, Expressing, Western Blot

Journal: Oncotarget

Article Title: Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

doi:

Figure Lengend Snippet: A. Representative IHC staining of high and low expression of STMN1 in the large (400×) and small images (100×). B. Representative IHC staining of high and low expression of multiple phosphor-sites (Ser-16, Ser-25, Ser38, Ser63) in the large (400×) and small images (100×). C. Kaplan-Meier analysis of DFS in the training set. D. Kaplan-Meier analysis of DFS in the validation set.

Article Snippet: For STMN1 and Ser38, TMAs were blocked with 10% normal goat serum for 1 h at room temperature (RT) and incubated in a humid chamber at 4°C overnight with polyclonal rabbit anti-human STMN1 antibody (Proteintech) or polyclonal rabbit anti-human STMN1 Ser38 (Cell Signaling Technologies) antibody diluted to 1:400 or 1:100, respectively.

Techniques: Immunohistochemistry, Expressing, Biomarker Discovery

Journal: Oncotarget

Article Title: Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

doi:

Figure Lengend Snippet: Univariate association of the STMN1-E/P model, clinicopathological characteristics, and single phospho-sites status with disease-free survival

Article Snippet: For STMN1 and Ser38, TMAs were blocked with 10% normal goat serum for 1 h at room temperature (RT) and incubated in a humid chamber at 4°C overnight with polyclonal rabbit anti-human STMN1 antibody (Proteintech) or polyclonal rabbit anti-human STMN1 Ser38 (Cell Signaling Technologies) antibody diluted to 1:400 or 1:100, respectively.

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

doi:

Figure Lengend Snippet: Data are shown as AUC (95% CI) or hazard ratios (95% CI). ROC = receiver operator characteristic. AUC = area under the curve. A. Comparisons of the prognostic accuracy by the STMN1-E/P model and TNM stage in the training set. B. DFS of patients with high- or low-risk scores in the training set. C. Comparisons of the prognostic accuracy by the STMN1-E/P model and TNM stage in the validation set. D. DFS of patients with high- or low-risk scores in the validation set. P values were calculated using the log-rank test.

Article Snippet: For STMN1 and Ser38, TMAs were blocked with 10% normal goat serum for 1 h at room temperature (RT) and incubated in a humid chamber at 4°C overnight with polyclonal rabbit anti-human STMN1 antibody (Proteintech) or polyclonal rabbit anti-human STMN1 Ser38 (Cell Signaling Technologies) antibody diluted to 1:400 or 1:100, respectively.

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

doi:

Figure Lengend Snippet: A. Comparisons of the prognostic accuracy by the STMN1-E/P/C model, STMN1-E/P model and TNM stage in the training set. B. DFS of patients with high- or low-risk scores according to the STMN1-E/P/C model in the training set. C. Comparisons of the prognostic accuracy by the STMN1-E/P/C model, STMN1-E/P model and TNM stage in the validation set. D. DFS of patients with high- or low-risk scores according to the STMN1-E/P/C model in the validation set. P values were calculated using the log-rank test. E. DFS of patients with luminal breast cancer. F. DFS of patients with HER2/neu subtype breast cancer. G. DFS of patients with TNBC subtype breast cancer.

Article Snippet: For STMN1 and Ser38, TMAs were blocked with 10% normal goat serum for 1 h at room temperature (RT) and incubated in a humid chamber at 4°C overnight with polyclonal rabbit anti-human STMN1 antibody (Proteintech) or polyclonal rabbit anti-human STMN1 Ser38 (Cell Signaling Technologies) antibody diluted to 1:400 or 1:100, respectively.

Techniques: Biomarker Discovery

Journal: Cancers

Article Title: The microRNA-210-Stathmin1 Axis Decreases Cell Stiffness to Facilitate the Invasiveness of Colorectal Cancer Stem Cells

doi: 10.3390/cancers13081833

Figure Lengend Snippet: Identification of the miR-210-3p-STMN1 axis in SDCSCs. ( a ) Strategy for identifying miR-210 targets. The putative targets were obtained from the set of differentially expressed genes in SDCSCs versus parental HT29 cells (GSE14773) and subjected to target prediction with SVmicro. Migration-, tumor suppressor- and stemness-related genes were selected. ( b ) A table illustrating the expression levels of five candidate targets of miR-210-3p from microarray analysis. ( c ) RT-qPCR examining the expression of five candidate targets of miR-210-3p in SDCSCs and their parental cells. ( d , e ) RT-qPCR examining the expression of miR-210-3p or LRRC2, RCBTB2 and STMN1 in HCT15 cells ( d ) and HT29 cells transfected with control agomiR (Scramble, 100 nM) or miR-210-3p agomiR (miR-210-AgomiR, 100 nM). ( f ) Western blot showing the expression of STMN1 in HT29 cells transfected with control agomiR (Scramble, Scr, 100 nM) or miR-210-3p agomiR (miR-210-AgomiR, 100nM). ( g ) Analysis of the NCI-60 panel revealed an inverse correlation between STMN1 (200783_s_at) and miR-210-3p expression. The pvalue of the Pearson correlation was assessed, and the correlation coefficient is reported. ( h ) Immunoblots showing STMN1 expression. ( i ) Western blots showing the expression of STMN1 in control SDCSCs (Zip-C) and miR-210-3p-silenced SDCSCs (Zip-210). Data in bar charts are expressed as the mean ± SD values. * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t -test).

Article Snippet: The transfer membrane was blocked and probed with the following antibodies prepared in 5% BSA (Sigma-Aldrich) overnight at 4 °C: anti-STMN1 (1:1000, 13655S, Cell Signaling, Danvers, MA, USA), anti-FLAG-M2-HRP (1:1000, A8592, Sigma-Aldrich), and anti-β-actin (1:5000, A5441, Sigma-Aldrich).

Techniques: Migration, Expressing, Microarray, Quantitative RT-PCR, Transfection, Western Blot

Journal: Cancers

Article Title: The microRNA-210-Stathmin1 Axis Decreases Cell Stiffness to Facilitate the Invasiveness of Colorectal Cancer Stem Cells

doi: 10.3390/cancers13081833

Figure Lengend Snippet: Restoration of STMN1 expression abolishes the migratory and invasive abilities of SDCSCs. ( a ) Western blots showing the expression of STMN1 in control SDCSCs (Vec) and SDCSCs ectopically expressing STMN1. An anti-FLAG antibody was used to detect the expression of exogenous STMN1 with a DKK-Myc tag. ( b ) Relative viability of cells as assessed by an MTT assay. ns, nonsignificant. ( c ) The sphere-forming capacity of control SDCSCs (Vec) and STMN-expressing SDCSCs (STMN1 DKK-Myc). ns, nonsignificant. ( d ) The colony formation of control SDCSCs (Vec) and STMN-expressing SDCSCs (STMN1 DKK-Myc). ns, nonsignificant. ( e ) Representative images showing the colonies generated. ( f ) Histograms showing the relative transwell migration ability of cells. ( g , h ) Representative images of vertical invasion of vector control HT29-SDCSCs (pLenti-C) and STMN-expressing SDCSCs (pLenti-STMN1 Myc-DDK). Data in bar charts are expressed as the mean ± SD values. ** p < 0.01; *** p < 0.001 (Student’s t -test).

Article Snippet: The transfer membrane was blocked and probed with the following antibodies prepared in 5% BSA (Sigma-Aldrich) overnight at 4 °C: anti-STMN1 (1:1000, 13655S, Cell Signaling, Danvers, MA, USA), anti-FLAG-M2-HRP (1:1000, A8592, Sigma-Aldrich), and anti-β-actin (1:5000, A5441, Sigma-Aldrich).

Techniques: Expressing, Western Blot, MTT Assay, Generated, Migration, Plasmid Preparation

Journal: Cancers

Article Title: The microRNA-210-Stathmin1 Axis Decreases Cell Stiffness to Facilitate the Invasiveness of Colorectal Cancer Stem Cells

doi: 10.3390/cancers13081833

Figure Lengend Snippet: Silencing STMN1 expression restored the attenuated migratory and invasive abilities of miR-210-3p-knockdown SDCSCs. ( a ) Western blots showing the expression of STMN1 in miR-210-3p-knockdown HCT15-SDCSCs. ( b ) Relative viability of cells as assessed by an MTT assay. ns, nonsignificant. ( c ) The sphere-forming capacity of miR-210-3p-silenced HCT15-SDCSCs receiving control shRNA (KD-Ctrl) and shRNA against STMN1 (KD-STMN1). ns, nonsignificant. ( d ) The colony formation ability of miR-210-3p-silenced HCT15-SDCSCs receiving control shRNA (KD-Ctrl) and shRNA against STMN1 (KD-STMN1). ns, nonsignificant. ( e ) Representative images showing colonies generated from the indicated cells. ( f ) Left: representative images of migrated cells. Right: histograms showing the relative transwell migration ability of cells. ( g ) Representative images of vertical invasion of the indicated cells. ( h ) RT-qPCR validation of the expression of an epithelial cell marker (E-cadherin, CDH1) and mesenchymal marker (N-cadherin, CDH2) in control HCT15-SDCSCs, miR-210-3p-silenced HCT15-SDCSCs receiving shRNA control (KD-Ctrl) and miR-210-3p-silenced HCT15-SDCSCs receiving shRNA against STMN1 (KD-STMN1). ns, nonsignificant. Data in bar charts are expressed as the mean ± SD values. * p < 0.05 (Student’s t -test).

Article Snippet: The transfer membrane was blocked and probed with the following antibodies prepared in 5% BSA (Sigma-Aldrich) overnight at 4 °C: anti-STMN1 (1:1000, 13655S, Cell Signaling, Danvers, MA, USA), anti-FLAG-M2-HRP (1:1000, A8592, Sigma-Aldrich), and anti-β-actin (1:5000, A5441, Sigma-Aldrich).

Techniques: Expressing, Western Blot, MTT Assay, shRNA, Generated, Migration, Quantitative RT-PCR, Marker

Journal: Cancers

Article Title: The microRNA-210-Stathmin1 Axis Decreases Cell Stiffness to Facilitate the Invasiveness of Colorectal Cancer Stem Cells

doi: 10.3390/cancers13081833

Figure Lengend Snippet: The miR-210-3p-STMN1 axis determines the elasticity of SDCSCs and correlates with liver metastasis in CRC patients. ( a ) A representative image illustrating the biolistic particle delivery system and measurement of the intracellular elastic modulus. ( b ) Representative images showing the distribution of injected fluorescent nanoparticles in HCT15 cells by confocal imaging. The XY and YZ projections are shown. Blue, nuclear staining with Hoechst 33342; red, fluorescent nanoparticles. ( c ) A three-dimensional (3D) constructed image from ( b ). ( d ) Histograms showing the elastic modulus of the indicated HCT15 cells at 100 Hz. Zip-C, scramble control; Zip-210, shRNA against miR-210-3p; KD-Ctrl, scramble control; KD-STMN1, shRNA targeting STMN1. Results are expressed as the mean ± SEM values. ( e ) Upper: trajectory of the indicated cells in the 2.5D assay. Lower: the velocity values in the indicated cells. The box plots show the sample maximum (upper end of the whisker), upper quartile (top edge of the box), median (band in the box), lower quartile (bottom edge of the box), and sample minimum (lower end of the whisker) values. * p < 0.05; *** p < 0.001 (Student’s t -test). ( f ) The bar charts showing the expression of miR-210-3p (upper panel) and STMN1 (lower panel) in GSE54088 and GSE3964 datasets retrieved from the Human Cancer Metastasis Database (HCMDB), respectively. The box plots show the sample maximum (upper end of the whisker), upper quartile (top edge of the box), median (band in the box), lower quartile (bottom edge of the box), and sample minimum (lower end of the whisker) values. ( g ) Kaplan–Meier analysis of the overall survival (upper panel) and disease-specific survival (lower panel) of patients in a TCGA-COAD dataset. The median expression levels of miR-210-3p and STMN1 were used for patient stratification. OS, overall survival; DSS, disease-specific survival. The p -values were estimated by the log-rank test. Hazard ratios are reported. ( h ) A schematic summarizing the observations in this study.

Article Snippet: The transfer membrane was blocked and probed with the following antibodies prepared in 5% BSA (Sigma-Aldrich) overnight at 4 °C: anti-STMN1 (1:1000, 13655S, Cell Signaling, Danvers, MA, USA), anti-FLAG-M2-HRP (1:1000, A8592, Sigma-Aldrich), and anti-β-actin (1:5000, A5441, Sigma-Aldrich).

Techniques: Injection, Imaging, Staining, Construct, shRNA, Whisker Assay, Expressing

Journal: bioRxiv

Article Title: Vector analysis of steerable mechanical tension across nuclear lamina

doi: 10.1101/462275

Figure Lengend Snippet: (A) MCF-7 cells were treated with ADM, H 2 O 2 , and CHX separately. The samples were stained for both β-actin and α-tubulin. The cells were also stained with DAPI to localize the nuclei. The images were captured through confocal laser microscopy after immunofluorescence staining (n = 10). (B) Intracellular nanoparticle size distribution in MCF-7 cells separately treated with ADM, H 2 O 2 , and CHX (n = 10). (C) MCF-7 cell intracellular osmotic pressure was measured via osmometery after the cells were exposed to ADM, H 2 O 2 , and CHX. (**: 0.001 < p < 0.05, ns: p > 0.05, Tukey-b test, n = 10). (D) P-cofilin, cofilin, actin, p-stathmin, stathmin, and tubulin levels in MCF-7 cells that had been treated with ADM, H 2 O 2 , or CHX (n = 6). (E) In the H 2 O 2 group, the ΔpNOP could be divided into two parts by comparing the total intensity of the protein nanoparticles, generated from MF and MT depolymerization, respectively (versus , line 1, row 2). Scale bar, 10 μm. All error bars represent SEM.

Article Snippet: Antibodies were purchased from commercial sources: rabbit anti-lamin B1 antibody (13435S, Cell Signaling Technology, Temecula, USA), rabbit anti-β-actin antibody (4970P, Cell Signaling Technology, Temecula, USA), mouse anti-tubulin-α antibody (T5168, Boster, Wuhan, China), rabbit anti-vimentin antibody (5741S, Cell Signaling Technology, Temecula, USA), rabbit anti-GFP antibody (2956S, Cell Signaling Technology, Temecula, USA), mouse anti-phospho-cofilin (Ser24) (bs-10252R, Bioss, Woburn, USA), mouse anti-cofilin antibody (PB0035, Boster, Wuhan, China), rabbit anti-phospho-stathmin 1 (Ser38) (bs-3432R, Bioss, Woburn, USA), and mouse anti-stathmin 1 antibody (BA1417, Boster, Wuhan, China).

Techniques: Staining, Microscopy, Immunofluorescence, Generated

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Overexpression of stathmin 1 is a poor prognostic biomarker in non-small cell lung cancer.

doi: 10.1038/labinvest.2014.124

Figure Lengend Snippet: Figure 1 High levels of STMN1 are correlated with progression and prognostic significance in NSCLC. (a) STMN1 expression in cancer tissues and adjacent normal tissues and from NSCLC patients. STMN1 expression was significantly higher in tumor tissue compared with adjacent normal tissue (Po0.001). (b) High STMN1 expression levels were observed in NSCLC tissues in 34 (91.9%) cases. (c) A representative western blotting shows the relative STMN1 protein expression level in tumorous tissue samples of two NSCLC patients (d) The Kaplan–Meier disease-specific survival curves by STMN1 levels. Patients with elevated STMN1 expression showed reduced survival times compared with patients with low levels of STMN1 expression (log-rank test; Po0.001).

Article Snippet: The sections were then incubated at 4 1C overnight with a rabbit antihuman STMN1 polyclonal antibody (dilution 1:50; Cell Signaling, USA) or anti-p53 antibody (dilution 1:100; Zhongshan).

Techniques: Expressing, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Overexpression of stathmin 1 is a poor prognostic biomarker in non-small cell lung cancer.

doi: 10.1038/labinvest.2014.124

Figure Lengend Snippet: Figure 2 Negative and positive staining of STMN1 protein expression in human NSCLC and normal adjacent tissues by IHC. Negative and positive staining of STMN1 protein in normal and tumor tissues were classified into four groups ( to þ þ þ ) according to the staining intensity and extension of each tissue. Negative staining ( ) and weak staining ( þ ) were regarded as low STMN1 protein expression (score r3), while moderate staining ( þ þ ) and strong staining ( þ þ þ ) were regarded as high STMN1 protein expression (score 43).

Article Snippet: The sections were then incubated at 4 1C overnight with a rabbit antihuman STMN1 polyclonal antibody (dilution 1:50; Cell Signaling, USA) or anti-p53 antibody (dilution 1:100; Zhongshan).

Techniques: Staining, Expressing, Negative Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Overexpression of stathmin 1 is a poor prognostic biomarker in non-small cell lung cancer.

doi: 10.1038/labinvest.2014.124

Figure Lengend Snippet: Figure 3 Silencing STMN1 inhibits the proliferation of NSCLC cells. Silencing STMN1 in two siRNA-transduced stable NSCLC cell lines. Relative gene expression determinations were made using the qRT-PCR (a) and western blotting (b) analysis. The A549 (c) or H1299 (d) cells were transfected with scrambled siRNA or two individual siRNAs against STMN1 as indicated. Cell viability was detected at the indicated time points after transfection using the CCK-8 assays. (*Po0.05).

Article Snippet: The sections were then incubated at 4 1C overnight with a rabbit antihuman STMN1 polyclonal antibody (dilution 1:50; Cell Signaling, USA) or anti-p53 antibody (dilution 1:100; Zhongshan).

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Overexpression of stathmin 1 is a poor prognostic biomarker in non-small cell lung cancer.

doi: 10.1038/labinvest.2014.124

Figure Lengend Snippet: Figure 4 Silencing STMN1 inhibits the metastasis of NSCLC cells. The representative pictures (top panel) and quantification (bottom panel) of invaded cells were analyzed using the Transwell matrix penetration assay. Significant reduction in the invasive ability was shown on STMN1 knockdown (*Po0.05). The cell number was counted in five random view fields, and the error bars represent s.d.s.

Article Snippet: The sections were then incubated at 4 1C overnight with a rabbit antihuman STMN1 polyclonal antibody (dilution 1:50; Cell Signaling, USA) or anti-p53 antibody (dilution 1:100; Zhongshan).

Techniques: Knockdown

Journal: Oncotarget

Article Title: Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

doi:

Figure Lengend Snippet: A. Representative IHC staining of high and low expression of STMN1 in the large (400×) and small images (100×). B. Representative IHC staining of high and low expression of multiple phosphor-sites (Ser-16, Ser-25, Ser38, Ser63) in the large (400×) and small images (100×). C. Kaplan-Meier analysis of DFS in the training set. D. Kaplan-Meier analysis of DFS in the validation set.

Article Snippet: For STMN1 and Ser38, TMAs were blocked with 10% normal goat serum for 1 h at room temperature (RT) and incubated in a humid chamber at 4°C overnight with polyclonal rabbit anti-human STMN1 antibody (Proteintech) or polyclonal rabbit anti-human STMN1 Ser38 (Cell Signaling Technologies) antibody diluted to 1:400 or 1:100, respectively.

Techniques: Immunohistochemistry, Expressing, Biomarker Discovery

Journal: Oncotarget

Article Title: Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

doi:

Figure Lengend Snippet: Univariate association of the STMN1-E/P model, clinicopathological characteristics, and single phospho-sites status with disease-free survival

Article Snippet: For STMN1 and Ser38, TMAs were blocked with 10% normal goat serum for 1 h at room temperature (RT) and incubated in a humid chamber at 4°C overnight with polyclonal rabbit anti-human STMN1 antibody (Proteintech) or polyclonal rabbit anti-human STMN1 Ser38 (Cell Signaling Technologies) antibody diluted to 1:400 or 1:100, respectively.

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

doi:

Figure Lengend Snippet: Data are shown as AUC (95% CI) or hazard ratios (95% CI). ROC = receiver operator characteristic. AUC = area under the curve. A. Comparisons of the prognostic accuracy by the STMN1-E/P model and TNM stage in the training set. B. DFS of patients with high- or low-risk scores in the training set. C. Comparisons of the prognostic accuracy by the STMN1-E/P model and TNM stage in the validation set. D. DFS of patients with high- or low-risk scores in the validation set. P values were calculated using the log-rank test.

Article Snippet: For STMN1 and Ser38, TMAs were blocked with 10% normal goat serum for 1 h at room temperature (RT) and incubated in a humid chamber at 4°C overnight with polyclonal rabbit anti-human STMN1 antibody (Proteintech) or polyclonal rabbit anti-human STMN1 Ser38 (Cell Signaling Technologies) antibody diluted to 1:400 or 1:100, respectively.

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Stathmin and phospho-stathmin protein signature is associated with survival outcomes of breast cancer patients

doi:

Figure Lengend Snippet: A. Comparisons of the prognostic accuracy by the STMN1-E/P/C model, STMN1-E/P model and TNM stage in the training set. B. DFS of patients with high- or low-risk scores according to the STMN1-E/P/C model in the training set. C. Comparisons of the prognostic accuracy by the STMN1-E/P/C model, STMN1-E/P model and TNM stage in the validation set. D. DFS of patients with high- or low-risk scores according to the STMN1-E/P/C model in the validation set. P values were calculated using the log-rank test. E. DFS of patients with luminal breast cancer. F. DFS of patients with HER2/neu subtype breast cancer. G. DFS of patients with TNBC subtype breast cancer.

Article Snippet: For STMN1 and Ser38, TMAs were blocked with 10% normal goat serum for 1 h at room temperature (RT) and incubated in a humid chamber at 4°C overnight with polyclonal rabbit anti-human STMN1 antibody (Proteintech) or polyclonal rabbit anti-human STMN1 Ser38 (Cell Signaling Technologies) antibody diluted to 1:400 or 1:100, respectively.

Techniques: Biomarker Discovery

Journal: Gland Surgery

Article Title: The prognostic role of a phospho-Stathmin 1 signature in breast cancer treated with neoadjuvant chemotherapy

doi: 10.21037/gs-22-628

Figure Lengend Snippet: Relationship between STMN1 and its phosphorylation-related proteins and clinical outcomes for breast cancer patients. (A) Relationship between Kaplan-Meier analysis of DFS and STMN1 level by IHC (400×). (B) Relationship between Kaplan-Meier analysis of DFS and STMN1 phosphorylation at Ser16 levelby IHC (400×). (C) Relationship between Kaplan-Meier analysis of DFS and STMN1 phosphorylation at Ser25 levelby IHC (400×). (D) Relationship between Kaplan-Meier analysis of DFS and STMN1 phosphorylation at Ser38 levelby IHC (400×). (E) Relationship between Kaplan-Meier analysis of DFS and STMN1 phosphorylation at Ser63 levelby IHC (400×). (F) Relationship between Kaplan-Meier analysis of DFS and GRP78 level by IHC (400×). DFS, disease-free survival.

Article Snippet: Polyclonal rabbit anti-human STMN1 Ser38 antibody (Cell Signaling Technologies) was diluted to 1:100 and incubated with samples in a humid chamber at 4 °C overnight. respectively.

Techniques: Phospho-proteomics

Journal: Gland Surgery

Article Title: The prognostic role of a phospho-Stathmin 1 signature in breast cancer treated with neoadjuvant chemotherapy

doi: 10.21037/gs-22-628

Figure Lengend Snippet: Relationship between STMN1 phosphorylation and pathologic response. (A) Correlation between pathologic response and STMN1 expression. (B) Correlation between pathologic response and STMN1 phosphorylation at Ser-16. (C) Correlation between pathologic response and STMN1 phosphorylation at Ser-25. (D) Correlation between pathologic response and STMN1 phosphorylation at Ser-38. (E) Correlation between pathologic response and STMN1 phosphorylation at Ser-63. (F) Correlation between pathologic response and GRP78. pCR, pathological complete response.

Article Snippet: Polyclonal rabbit anti-human STMN1 Ser38 antibody (Cell Signaling Technologies) was diluted to 1:100 and incubated with samples in a humid chamber at 4 °C overnight. respectively.

Techniques: Phospho-proteomics, Expressing

Journal: Gland Surgery

Article Title: The prognostic role of a phospho-Stathmin 1 signature in breast cancer treated with neoadjuvant chemotherapy

doi: 10.21037/gs-22-628

Figure Lengend Snippet: p-STMN1/GRP78 model powerfully predicts DFS for patients treated with NACT. (A) Kaplan-Meier analysis of DFS in breast cancer patients from high and low-risk groups classified by the p-STMN1/GRP78 model. (B) ROC curves to test the prognostic accuracy of the p-STMN1/GRP78 model. DFS, disease-free survival; ROC, receiver operating characteristic; AUC, area under the receiver operating characteristic curve.

Article Snippet: Polyclonal rabbit anti-human STMN1 Ser38 antibody (Cell Signaling Technologies) was diluted to 1:100 and incubated with samples in a humid chamber at 4 °C overnight. respectively.

Techniques:

Journal: PLoS ONE

Article Title: The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

doi: 10.1371/journal.pone.0140612

Figure Lengend Snippet: (A) Yeast two-hybrid assays were performed with STMN homologues STMN1 and STMN2 and FANCC constructs. Yeast strain AH109 was co-transformed with full-length STMN2, full-length STNM1 or truncated STMN1 constructs (STMN1 1–41 and STMN1 42–149 ) along with FANCC constructs or empty vectors as indicated. Yeast transforments were assayed for interaction by plating on selective media. Shown are STMN regulatory region (purple) containing the phosphorylation sites (black lines), the interaction domain (green) and subtype-specific N-terminal region of varying size (orange or grey). A positive interaction is indicated as + and a negative interaction with a −. Negative controls included STMN constructs co-transformed with pGADT7 or pGBKT7 empty vectors. Experiments were performed at least 3 times in triplicates. (B) Pull-down experiments using GST-STMN1 (STMN1) or empty-GST (GST) constructs and protein extracts from 293T cells transfected with empty EGFP plasmids or EGFP-FANCC, EGFP-FANCC L554P or EGFP-FANCC 55–558 expression plasmids. Ponceau stained membrane showing GST or GST-STMN1 fusion proteins (upper panels) and immunoblot (IB) of FANCC constructs using anti-GFP antibodies (lower panels). WCE, whole cell extracts.

Article Snippet: The following antibodies were used: anti-FANCC, (8F3, NovusBiologicals, Littleton, CO), anti-STMN1 (EP1573Y, NovusBiologicals), anti-P16-STMN1 (3353, Cell Signaling Technology, Whitby, ON), anti-P38-STMN1 (ab47399, Abcam, Cambridge, MA or D19H10, Cell Signaling Technology), anti-GFP (clone B2; Santa Cruz Biotechnology, Dallas, TX), anti-α-tubulin (mouse T6199; Sigma-Aldrich), anti-γ-tubulin (mouse T5326, Sigma-Aldrich, Oakville, ON), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Centrin 2 (N-17-R; Santa Cruz Biotechnology), anti-CDK1 (SC-34, Santa Cruz Biotechnology) anti-Aurora-B (NB500-185; NovusBiologicals) and donkey or goat-raised anti-mouse or anti-rabbit Alexa Fluor 488 or 555 (Invitrogen, Burlington, ON).

Techniques: Construct, Transformation Assay, Transfection, Expressing, Staining, Western Blot

Journal: PLoS ONE

Article Title: The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

doi: 10.1371/journal.pone.0140612

Figure Lengend Snippet: (A) HeLa cells double-stained with anti-FANCC (red) and anti-STMN1 (green) antibodies were visualized by confocal microscopy using a Nikon E800 microscope equipped with a C1 confocal system at 100x magnification. Data shown are representative of 3 experiments in which at least 20 cells were analyzed. The white squares indicate the zoomed area shown on the right. (B) Immunofluorescence microscopy of mitotic HeLa cells labeled with anti-FANCC and anti-phospho-serine 38 STMN1. Arrows indicate centrosomes. (C) Immunofluorescence microscopy of HeLa cells during the different phases of mitosis as indicated. Cells were labeled with anti-FANCC (green) and anti-γ-tubulin (red) antibodies. (D) Confocal microscopic visualization and 3D reconstruction of 293T cells labeled with anti-FANCC and anti-γ-tubulin (upper panel) or anti-Centrin-2 (lower panel) antibodies using a Olympus IX81-ZDC fluorescent microscope equipped with a Spinning Disc confocal system and the Metamorph NX software. (E) Wild type (WT) and FancC -/- fibroblasts labeled with anti-FANCC (green) and anti-γ-tubulin (red) antibodies (upper panel). HeLa cells labeled with control IgG followed by secondary antibodies as indicated (Lower panel). (F) FANCC fluorescence intensity relative to γ-tubulin at centrosomes scored in at least 20 mitotic cells from each genotype. P value is indicated in the graph. (G) Immunoprecipitation of EGFP-FANCC with endogenous γ-tubulin from EGFP-FANCC transfected 293T cells and immunoprecipitated with anti-GFP antibodies or control IgG (mouse). Immunoprecipitates were immunoblotted with anti-γ-tubulin or anti-FANCC antibodies. Shown is a representative experiment out of 3. (H) 293T cells were transfected with EGFP-FANCC constructs as indicated in the figure prior to immunofluorescence detection. Cells were labeled with anti-γ-tubulin (red) antibodies. Cells were visualized with a Carl Zeiss Axio Imager M2 at 100X magnification. Nuclear DNA was stained with DAPI. White line indicates scale of 1 μm.

Article Snippet: The following antibodies were used: anti-FANCC, (8F3, NovusBiologicals, Littleton, CO), anti-STMN1 (EP1573Y, NovusBiologicals), anti-P16-STMN1 (3353, Cell Signaling Technology, Whitby, ON), anti-P38-STMN1 (ab47399, Abcam, Cambridge, MA or D19H10, Cell Signaling Technology), anti-GFP (clone B2; Santa Cruz Biotechnology, Dallas, TX), anti-α-tubulin (mouse T6199; Sigma-Aldrich), anti-γ-tubulin (mouse T5326, Sigma-Aldrich, Oakville, ON), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Centrin 2 (N-17-R; Santa Cruz Biotechnology), anti-CDK1 (SC-34, Santa Cruz Biotechnology) anti-Aurora-B (NB500-185; NovusBiologicals) and donkey or goat-raised anti-mouse or anti-rabbit Alexa Fluor 488 or 555 (Invitrogen, Burlington, ON).

Techniques: Staining, Confocal Microscopy, Microscopy, Immunofluorescence, Labeling, Software, Fluorescence, Immunoprecipitation, Transfection, Construct

Journal: PLoS ONE

Article Title: The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

doi: 10.1371/journal.pone.0140612

Figure Lengend Snippet: (A-C) FancC -/- and wild-type cells were immunostained with antibodies against total STMN1 (in A), STMN1-phospho-Serine 16 (in B), or STMN1-phospho-serine 38 (in C) along with γ-tubulin during mitosis and visualized using a Carl Zeiss Axio Imager M2 at 100X magnification. White line indicates scale of 1 μm. Ratio of pS16-STMN1, pS38-STMN1 or STMN1 fluorescence intensity relative to γ-tubulin at centrosomes were scored in at least 20 mitotic cells each from 3 separate experiments. P values are indicated in each graph.

Article Snippet: The following antibodies were used: anti-FANCC, (8F3, NovusBiologicals, Littleton, CO), anti-STMN1 (EP1573Y, NovusBiologicals), anti-P16-STMN1 (3353, Cell Signaling Technology, Whitby, ON), anti-P38-STMN1 (ab47399, Abcam, Cambridge, MA or D19H10, Cell Signaling Technology), anti-GFP (clone B2; Santa Cruz Biotechnology, Dallas, TX), anti-α-tubulin (mouse T6199; Sigma-Aldrich), anti-γ-tubulin (mouse T5326, Sigma-Aldrich, Oakville, ON), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Centrin 2 (N-17-R; Santa Cruz Biotechnology), anti-CDK1 (SC-34, Santa Cruz Biotechnology) anti-Aurora-B (NB500-185; NovusBiologicals) and donkey or goat-raised anti-mouse or anti-rabbit Alexa Fluor 488 or 555 (Invitrogen, Burlington, ON).

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

doi: 10.1371/journal.pone.0140612

Figure Lengend Snippet: FANCC-mutant cells PD331 and FANCC-corrected cells (PD331/C) were immunostained with antibodies against total STMN1 (A), STMN1-phospho-Serine 16 (in B), or STMN1-phospho-serine 38 (in C) along with γ-tubulin during mitosis and visualized using a Carl Zeiss Axio Imager M2 at 100X magnification. White line indicates scale of 1 μm. Ratio of total STMN1, pS16-STMN1 or pS38-STMN1 fluorescence intensity relative to γ-tubulin at centrosomes were scored in at least 20 mitotic cells each from 3 separate experiments. P values are indicated in each graph.

Article Snippet: The following antibodies were used: anti-FANCC, (8F3, NovusBiologicals, Littleton, CO), anti-STMN1 (EP1573Y, NovusBiologicals), anti-P16-STMN1 (3353, Cell Signaling Technology, Whitby, ON), anti-P38-STMN1 (ab47399, Abcam, Cambridge, MA or D19H10, Cell Signaling Technology), anti-GFP (clone B2; Santa Cruz Biotechnology, Dallas, TX), anti-α-tubulin (mouse T6199; Sigma-Aldrich), anti-γ-tubulin (mouse T5326, Sigma-Aldrich, Oakville, ON), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Centrin 2 (N-17-R; Santa Cruz Biotechnology), anti-CDK1 (SC-34, Santa Cruz Biotechnology) anti-Aurora-B (NB500-185; NovusBiologicals) and donkey or goat-raised anti-mouse or anti-rabbit Alexa Fluor 488 or 555 (Invitrogen, Burlington, ON).

Techniques: Mutagenesis, Fluorescence

Journal: PLoS ONE

Article Title: The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

doi: 10.1371/journal.pone.0140612

Figure Lengend Snippet: (A) Western blot analysis of protein extracts from FANCC mutant (PD331) and FANCC-corrected (PD331/C) cells. Cells were unsynchronized (UN) or synchronized (S) using nocodazol (10 μM for 16 hours). Total protein extracts were immunoblotted (IB) with the indicated antibodies. Arrows indicate total STMN1 and STMN1 phosphorylated species. (B) Graph displaying the mean relative ratio of STMN1/γ-tubulin protein expression from 3 separate experiments. (C) Western blot analysis of protein extracts from cells overexpressing serine-to-alanine Myc-tagged STMN1 mutants. Total protein extracts were immunoblotted (IB) with the indicated antibodies

Article Snippet: The following antibodies were used: anti-FANCC, (8F3, NovusBiologicals, Littleton, CO), anti-STMN1 (EP1573Y, NovusBiologicals), anti-P16-STMN1 (3353, Cell Signaling Technology, Whitby, ON), anti-P38-STMN1 (ab47399, Abcam, Cambridge, MA or D19H10, Cell Signaling Technology), anti-GFP (clone B2; Santa Cruz Biotechnology, Dallas, TX), anti-α-tubulin (mouse T6199; Sigma-Aldrich), anti-γ-tubulin (mouse T5326, Sigma-Aldrich, Oakville, ON), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Centrin 2 (N-17-R; Santa Cruz Biotechnology), anti-CDK1 (SC-34, Santa Cruz Biotechnology) anti-Aurora-B (NB500-185; NovusBiologicals) and donkey or goat-raised anti-mouse or anti-rabbit Alexa Fluor 488 or 555 (Invitrogen, Burlington, ON).

Techniques: Western Blot, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

doi: 10.1371/journal.pone.0140612

Figure Lengend Snippet: Graphs displaying the ratio of total STMN1, pS16-STMN1 or pS38-STMN1 fluorescence intensity relative to γ-tubulin at centrosomes in FA patient-derived PD220 and PD20 cells ant their corrected counterparts PD220/A and PD20/D2 cells. Intensity of at least 20 mitotic cells were scored for each cell line from 3 separate experiments. P values are indicated in each graph.

Article Snippet: The following antibodies were used: anti-FANCC, (8F3, NovusBiologicals, Littleton, CO), anti-STMN1 (EP1573Y, NovusBiologicals), anti-P16-STMN1 (3353, Cell Signaling Technology, Whitby, ON), anti-P38-STMN1 (ab47399, Abcam, Cambridge, MA or D19H10, Cell Signaling Technology), anti-GFP (clone B2; Santa Cruz Biotechnology, Dallas, TX), anti-α-tubulin (mouse T6199; Sigma-Aldrich), anti-γ-tubulin (mouse T5326, Sigma-Aldrich, Oakville, ON), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Centrin 2 (N-17-R; Santa Cruz Biotechnology), anti-CDK1 (SC-34, Santa Cruz Biotechnology) anti-Aurora-B (NB500-185; NovusBiologicals) and donkey or goat-raised anti-mouse or anti-rabbit Alexa Fluor 488 or 555 (Invitrogen, Burlington, ON).

Techniques: Fluorescence, Derivative Assay

Journal: PLoS ONE

Article Title: The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

doi: 10.1371/journal.pone.0140612

Figure Lengend Snippet: In normal cells (left panel), FANCC interacts with STMN1 and facilitates the action of CDK1 for STMN1 phosphorylation on serine 38. Subsequent phosphorylation of STMN1 requires a functional FA signaling pathway to ensure proper centrosome number and mitotic spindle size. In FA-deficient cells (right panel), lack of interaction between FANCC and STMN1 and CDK1 would impinge on S38-mediated phosphorylation of STMN1 and reduced mitotic kinases would affect subsequent STMN1 phosphorylation resulting in abnormal spindle sizes and centrosome numbers.

Article Snippet: The following antibodies were used: anti-FANCC, (8F3, NovusBiologicals, Littleton, CO), anti-STMN1 (EP1573Y, NovusBiologicals), anti-P16-STMN1 (3353, Cell Signaling Technology, Whitby, ON), anti-P38-STMN1 (ab47399, Abcam, Cambridge, MA or D19H10, Cell Signaling Technology), anti-GFP (clone B2; Santa Cruz Biotechnology, Dallas, TX), anti-α-tubulin (mouse T6199; Sigma-Aldrich), anti-γ-tubulin (mouse T5326, Sigma-Aldrich, Oakville, ON), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Centrin 2 (N-17-R; Santa Cruz Biotechnology), anti-CDK1 (SC-34, Santa Cruz Biotechnology) anti-Aurora-B (NB500-185; NovusBiologicals) and donkey or goat-raised anti-mouse or anti-rabbit Alexa Fluor 488 or 555 (Invitrogen, Burlington, ON).

Techniques: Functional Assay

Journal: Gland Surgery

Article Title: The prognostic role of a phospho-Stathmin 1 signature in breast cancer treated with neoadjuvant chemotherapy

doi: 10.21037/gs-22-628

Figure Lengend Snippet: Relationship between STMN1 and its phosphorylation-related proteins and clinical outcomes for breast cancer patients. (A) Relationship between Kaplan-Meier analysis of DFS and STMN1 level by IHC (400×). (B) Relationship between Kaplan-Meier analysis of DFS and STMN1 phosphorylation at Ser16 levelby IHC (400×). (C) Relationship between Kaplan-Meier analysis of DFS and STMN1 phosphorylation at Ser25 levelby IHC (400×). (D) Relationship between Kaplan-Meier analysis of DFS and STMN1 phosphorylation at Ser38 levelby IHC (400×). (E) Relationship between Kaplan-Meier analysis of DFS and STMN1 phosphorylation at Ser63 levelby IHC (400×). (F) Relationship between Kaplan-Meier analysis of DFS and GRP78 level by IHC (400×). DFS, disease-free survival.

Article Snippet: Polyclonal rabbit anti-human STMN1 antibody (Proteintech) was diluted to 1:400 and incubated with samples in a humid chamber at 4 °C overnight.

Techniques: Phospho-proteomics

Journal: Gland Surgery

Article Title: The prognostic role of a phospho-Stathmin 1 signature in breast cancer treated with neoadjuvant chemotherapy

doi: 10.21037/gs-22-628

Figure Lengend Snippet: Relationship between STMN1 phosphorylation and pathologic response. (A) Correlation between pathologic response and STMN1 expression. (B) Correlation between pathologic response and STMN1 phosphorylation at Ser-16. (C) Correlation between pathologic response and STMN1 phosphorylation at Ser-25. (D) Correlation between pathologic response and STMN1 phosphorylation at Ser-38. (E) Correlation between pathologic response and STMN1 phosphorylation at Ser-63. (F) Correlation between pathologic response and GRP78. pCR, pathological complete response.

Article Snippet: Polyclonal rabbit anti-human STMN1 antibody (Proteintech) was diluted to 1:400 and incubated with samples in a humid chamber at 4 °C overnight.

Techniques: Phospho-proteomics, Expressing

Journal: Gland Surgery

Article Title: The prognostic role of a phospho-Stathmin 1 signature in breast cancer treated with neoadjuvant chemotherapy

doi: 10.21037/gs-22-628

Figure Lengend Snippet: p-STMN1/GRP78 model powerfully predicts DFS for patients treated with NACT. (A) Kaplan-Meier analysis of DFS in breast cancer patients from high and low-risk groups classified by the p-STMN1/GRP78 model. (B) ROC curves to test the prognostic accuracy of the p-STMN1/GRP78 model. DFS, disease-free survival; ROC, receiver operating characteristic; AUC, area under the receiver operating characteristic curve.

Article Snippet: Polyclonal rabbit anti-human STMN1 antibody (Proteintech) was diluted to 1:400 and incubated with samples in a humid chamber at 4 °C overnight.

Techniques: