Journal: Scientific reports

Article Title: AEG-1 silencing attenuates M2-polarization of glioma-associated microglia/macrophages and sensitizes glioma cells to temozolomide.

doi: 10.1038/s41598-021-96647-3

Figure Lengend Snippet: Figure 6. AEG-1 activates Wnt/β-catenin signaling via targeting GSK-3β in glioma cells. (A) Enriched top 10 KEGG pathways for Affymetrix microarray of AEG-1 NC (N = 3) and KD (N = 3) glioma cells. 22 DEGs were enriched in KEGG_WNT_SIGNALING PATHWAY. P-value = 1.02E−06. [drawn by R 3.6.0 (https://cran.r- project.org/doc/FAQ/R-FAQ.html#Citing-R)]. (B) Enrichment plot of the Wnt signaling pathway from GSEA; ‘h’ and ‘l’ represented AEG-1 high and low expression, respectively. NES = 1.523, NOM P-value = 0.008, FDR q-value = 0.216 [drawn by GSEA tool (version 4.1.0)]. (C) Western blot bands of AEG-1, β-catenin, GSK-3β, cyclin D1, and CD44 in NC and shAEG-1 glioma cell lines. (D) Relative protein abundance was calculated by ImageJ and GraphPad Prism 8.2.1 software. (E, F) In U251 and U87 cells, Co-IP assays showed the direct interaction of AEG-1 and GSK-3β. (G) Immunofluorescence assays were used to detect the localization of AEG-1 (red) and GSK-3β (green) in U251 and U87 cells. All data were presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Cells were incubated with rabbit anti-AEG-1 antibody (Proteintech, 1:1000), mouse anti-GSK-3β antibody (Cell Signaling Technology, 1:1000), and rabbit anti-γH2AX (Cell Signaling Technology, 1:400), diluted using 1% FBS, at 4 °C overnight.

Techniques: Microarray, Expressing, Western Blot, Quantitative Proteomics, Software, Co-Immunoprecipitation Assay, Immunofluorescence

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

Article Title: Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway.

doi: 10.1038/s41374-021-00652-z

Figure Lengend Snippet: Fig. 2 Vitamin E downregulates EGFR and inactivates MAPK signaling pathway. A Venn diagram of the intersection of drug and disease targets. B Network diagram of gene-drug-disease interaction relationship. C KEGG enrichment analysis results of 30 candidate targets. D Box diagram of EGFR expression in COPD attained through microarray dataset GSE3320, wherein blue box on the left indicates normal tissue samples and red box on the right indicates COPD samples. E EGFR expression in lung tissues of rats after CS or vitamin E treatment was detected by immunohistochemistry. F EGFR, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 protein expression in lung tissues of rats after CS or vitamin E treatment was detected by Western blot. G Statistical diagram of Panel F. The data in the figure are measurement data, which are expressed by the mean ± standard deviation. The data of each group are analyzed by unpaired t-test, and the data among multiple groups are compared by one-way ANOVA and Tukey’s post hoc test. *p < 0.05 vs. the control group, #p < 0.05 vs. the CS group, n = 6.

Article Snippet: Then the membrane was probed with primary antibodies against COX2 (1: 1000, ab179800, Abcam), EGFR (1:2000, ab52894, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab76315, Abcam), p38 (1:1000, 9212, Cell Signaling Technologies [CST], Beverly, MA,USA), p-p38 (1:1000, 9211, CST), JNK (1:2000, 9252, CST), p-JNK (1:2000, 9255, CST), ERK1/2 (1:1000, 4695, CST), p-ERK1/2 (1:1000, 4370, CST), and GAPDH (1:5000, ab8254, Abcam) overnight at 4 °C.

Techniques: Expressing, Microarray, Immunohistochemistry, Western Blot, Standard Deviation, Control

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

Article Title: Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway.

doi: 10.1038/s41374-021-00652-z

Figure Lengend Snippet: Fig. 3 Vitamin E relieves CS-induced HBE cell damage. A RT-qPCR was used to assess the expression of EGFR in HBE cells of each group. B Western blot was used to assess the expression of EGFR, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2 in HBE cells of each group. C Statistical map of Panel B. D CCK8 assay was used to detect the viability of HBE cells in each group. E Annexin V/PI staining was used to detect the apoptosis of HBE cells in each group. F ELISA was used to detect the expression of inflammatory factors in the supernatant of HBE cells of each group. G ROS level in HBE cells of each group. H The SOD activity and MDA content in HBE cells of each group. The data in the figure were all measurement data, expressed as the mean ± standard deviation. The data of each group were analyzed by unpaired t-test, and the comparison of data among multiple groups was performed by one-way ANOVA and Tukey’s post hoc test. *p < 0.05 vs. the Vector group, #p < 0.05 vs. the vector + CS group, and $p < 0.05 vs. the vector + CS + vitamin E group.

Article Snippet: Then the membrane was probed with primary antibodies against COX2 (1: 1000, ab179800, Abcam), EGFR (1:2000, ab52894, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab76315, Abcam), p38 (1:1000, 9212, Cell Signaling Technologies [CST], Beverly, MA,USA), p-p38 (1:1000, 9211, CST), JNK (1:2000, 9252, CST), p-JNK (1:2000, 9255, CST), ERK1/2 (1:1000, 4695, CST), p-ERK1/2 (1:1000, 4370, CST), and GAPDH (1:5000, ab8254, Abcam) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Comparison, Plasmid Preparation

Journal: Cancer science

Article Title: Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

doi: 10.1111/j.1349-7006.2011.01847.x

Figure Lengend Snippet: Fig. 1. Gene expression levels of epidermal growth factor receptor (EGFR) ligands, EGFR family members (a) and a disintegrin and metalloproteinases (ADAMs) (b) in the stomach of the respective models (mean ± SD) calculated from microarray results. Asterisks indicate P < 0.05 versus the wild-type level. (c) Fluorescence immunostaining for phosphorylated EGFR at Tyr845 (green) in the gastric mucosa of the indicated genotype mice. DAPI staining for nuclei is visualized in red. Bars indicate 100 lm.

Article Snippet: Antibody for phosphorylated EGFR (Tyr845) (cell signaling), Ki-67 (DakoCytomation, Carpenteria, CA, USA), or active b-catenin (Millipore, Billerica, MA, USA) was used as the primary antibody.

Techniques: Gene Expression, Microarray, Fluorescence, Immunostaining, Staining

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a , c ) Lung sections evaluated by H&E staining of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice at the age of ( a ) 3 and ( c ) 6 months. ( b ) Immunofluorescence for Mac-2 and p-S6 in lung sections of 3 month-old mice. Boxed region shows p-S6 staining only. ( d ) Image of a Tsc2 fl/fl Lyz2 -Cre mouse at the age of 6 months (left) and immunohistochemistry of Mac-2 of a paw (right). ( e ) Immunofluorescence for Mac-2 and F4/80 in paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice. ( f ) Flow cytometric scatterplot of the G1 population of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre peritoneal macrophages. ( g ) Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre bone marrow was injected into irradiated wild-type recipient mice and evaluated after 3 months by histology. Left panel: H&E staining of lung sections. Right panel: IHC of Mac-2 in liver. ( h ) Flow cytometry analysis of hypertrophic macrophages in the lungs of Tsc2 fl/fl (n=5) and Tsc2 fl/fl Lyz2 -Cre (n= 4) mice. Shown are means. ( i ) Expression of the indicated mRNAs in the lungs of 6 month-old mice. Shown are means ± SE (n= 5). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant (Student's t test). Representative histological images or plots are from one mouse; each analysis was performed on three ( b , d , e , g ) or five ( a , c,f ) mice per genotype. Numerical data are representative of two independent ( h ) or cumulative of two independent ( i ) experiments. Scale bars, 100 μm ( a , c , e , g ), 10 μm ( b ), 1 mm ( d ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining, Immunofluorescence, Immunohistochemistry, Injection, Irradiation, Flow Cytometry, Expressing

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Immunoblots of CSF1-deprived Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre bone marrow-derived macrophages (BMDM), treated for 18 hours with 100 nM rapamycin or solvent control, hybridized with the indicated antibodies. ( b ) Surface expression of the indicated proteins on Tsc2 fl/fl (n=6) and Tsc2 fl/fl Lyz2 -Cre (n=4) BMDM after differentiation. ( c ) Left: Images of IL-4 stimulated (10 ng/ml) Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM treated with solvent or 100 nM rapamycin for four days. Right: Quantification of cluster formation. Clusters in three pictures per condition (4x magnification) were counted (n= 3). ( d ) Flow cytometry scatterplot of CSF1-deprived BMDM on day 7. ( e,f ) Cell cycle analysis of ( e ) BMDM by 7-AAD and EdU staining and of ( f ) peritoneal macrophages by PI staining from Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice (n= 5). ( g ) IHC of Ki-67 in lung sections of 3 month-old Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre mice. ( h ) Proliferation analysis of BMDM stimulated with 40 ng/ml CSF1 and treated with solvent or 100 nM rapamycin (n=4). Shown are means ± SE ( b , f,h ) or SD ( c ). *p < 0.001 (Student's t test) ( c , f ). Data are representative of three ( a , d , g,c ) or two ( e ) or cumulative from two independent experiments ( b,f,h ) Scale bar, 100 µm ( c ), 50 μm ( g ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Western Blot, Derivative Assay, Solvent, Control, Expressing, Flow Cytometry, Cell Cycle Assay, Staining

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Scatterplot of global gene-expression profiles of CSF1-deprived Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM. Blue dots, 426 genes significantly higher expressed in Tsc2 fl/fl BMDM; red dots, 401 genes significantly higher expressed in Tsc2 fl/fl Lyz2 -Cre BMDM. ( b ) Gene-set enrichment analysis (GSEA) of hallmark gene sets (H.all) from the Molecular Signatures Database of the Broad Institute, showing the most significantly enriched gene sets in Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM and their normalized enrichment scores (NES) as well as their false discovery rates (FDR). ( c,d ) GSEA plot of the E2F targets (c) and Tnfa signaling via NFKB (d) gene signature in Tsc2 fl/fl Lyz2 -Cre BMDM relative to Tsc2 fl/fl BMDM from the analysis in (b) with members of the gene set presented in the ranked list of genes ('bar code' below) and the signal-to-noise ranking metric (bar at bottom). ( e ) GSEA of the 'KEGG_apoptosis' gene signature in Tsc2 fl/fl Lyz2 -Cre BMDM relative to Tsc2 fl/fl BMDM. ( f ) The most significantly enriched transcription factor target gene sets (C3.TFT) from the Molecular Signatures Database in unstimulated Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM derived by GSEA. NES, normalized enrichment score; FDR, false discovery rate. Data are from one experiment with four biological replicates per genotype obtained from two independent experiments.

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Gene Expression, Derivative Assay

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a,b,c ) BMDM were treated with 100 nM rapamycin, solvent control and 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. ( d ) IHC of CDK4 in lung sections of 3 month-old mice. ( e ) Left, analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of the CKD4 inhibitor PD-0332991 (n= 4). Right, cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 µM PD-0332991 for 18 h (n= 3). ( f,g ) Tsc2 fl/fl Lyz2 -Cre bone marrow was injected into irradiated wild-type recipient mice. After ten days mice were treated daily with PD-0332991 (n=10) or solvent control (n=10) daily for two month. ( f ) Lung (left panel) and liver (right panel) sections were evaluated by IHC of Mac-2. ( g ) Area of Mac-2 positive cells compared to total lung area of the treated mice. Two random images per animal were evaluated. ( h ) BMDM were treated with 1 µM PD-0332991 or solvent control and stimulated with 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SE ( e ) or SD ( g ). *p < 0.01, **p < 0.001 (Student's t test). Data are representative of three ( a , b , c , d ), one ( f,g ) or two ( h ) independent, or cumulative of two ( e ) experiments. Scale bar, 40 μm ( d ), 100 μm ( f ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Solvent, Control, Western Blot, Cell Cycle Assay, Injection, Irradiation

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMDM reported in units of mpH/min and pmol/min, respectively (n=8). ( b ) Uptake of 2-NBDG in BMDM treated with solvent control or 100 nM rapamycin for 18 h; analyzed by flow cytometry (n=4). ( c ) Uptake of 2-NBDG in peritoneal macrophages from Tsc2 fl/fl (n=4) and Tsc2 fl/fl Lyz2 -Cre (n=5) mice. ( d ) Total amount of glucose in lung tissue of mice (n=10). ( e ) Mitotracker Green staining analyzed by flow cytometry of BMDM treated with solvent control or 100 nM rapamycin for 18 h (n=4). ( f ) Immunofluorescence for F-ATPase β of BMDM deprived of CSF1. ( g ) ECAR of Tsc2 fl/fl Lyz2 -Cre BMDM that were treated for 30 min with solvent or 1 µM PD-0332991 (n=8). ( h ) ECAR of Tsc2 fl/fl that were treated overnight with solvent or 10 ng/ml CSF1 and at the indicated time point with 1 µM PD-0332991 (n=8). ( i ) Images of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity on frozen lung sections of mice in situ . Sections were additionally stained for Mac-2, p-S6, and DAPI. ( j ) Fraction (in %) of p-S6-positive Mac-2 macrophages in lungs of mice with high activities of GAPDH, lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and succinate dehydrogenase (SDH) (n=3-4). ( k ) Analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of 2-DG (n= 4). ( l ) Cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 mM 2-DG for 18 h (n= 3). ( m ) BMDM were treated with 1 mM 2-DG or solvent control and then stimulated with 10 ng/ml CSF1 for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SD ( a , g , h , j ) or SE ( b , c , e , k , l ) or boxplots with means, 25 th to 75 th percentile and minimum to maximum bars ( d ). *p<0.05, **p<0.001, ***p<0.001 (Student's t test). Data are representative of one ( d ) or two ( a,f-j,m ) independent experiments or cumulative of 2 experiments ( b,c,e,k,l) . Scale bar, 10 μm ( f ), 100 μm ( i ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Solvent, Control, Flow Cytometry, Staining, Immunofluorescence, Activity Assay, In Situ, Cell Cycle Assay, Western Blot

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) Sections of three sarcoidosis patients stained with p-S6 by IHC. ( b ) Immunofluorescence for Mac-2 and p-S6 in a sarcoidosis granuloma. ( c ) GSEA analysis of the ‘mTORC1 signaling’, ‘E2F targets’, and ´Glycolysis´ gene signatures in progressive relative to self-limiting sarcoidosis from the data of Lockstone et al . ( d ) An unsupervised cluster analysis of the microarray data of Tsc2 fl/fl and Tsc2 fl/fl Lyz2 -Cre BMDM performed with the genes that were differentially expressed in progressive relative to self-limiting sarcoidosis patients. ( e ) IHC for p-S6 and Ki-67 of granulomas from sarcoidosis patients. ( f ) Immunofluorescence for Mac-2 and Ki-67 in a sarcoidosis granuloma. ( g ) Relationship between p-S6 and Ki-67 expression in granulomas of 27 human sarcoidosis biopsies. Ki-67 high, > 5 % Ki-67 positive cells in the granuloma; Ki-67 low, < 5 % Ki-67 positive cells in the granulomas. The relationship was investigated using fisher’s exact test. Data are representative of 27 human sarcoidosis patients ( a , b , e , f ) or from 8 self-limiting and 7 progressive sarcoidosis patients ( c , d ). Scale bar, 200 μm ( a ), 20 μm ( b ), 100 μm ( e,f ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining, Immunofluorescence, Microarray, Expressing

Journal: Nature immunology

Article Title: Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

doi: 10.1038/ni.3655

Figure Lengend Snippet: ( a ) 2 month-old and ( b ) 6 month-old Tsc2 fl/fl Lyz2 -Cre mice were treated with placebo or everolimus for three weeks and lung sections were evaluated by H&E staining. ( c ) Paw and tail images of a 6 month-old Tsc2 fl/fl Lyz2 -Cre mouse at day 0 and 14 of everolimus treatment. ( d ) IHC for Mac-2 of paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice treated with placebo or everolimus. ( e ) IHC for p-S6 and cleaved caspase 3 of paw sections of 6 month-old Tsc2 fl/fl Lyz2 -Cre mice treated with placebo or everolimus for two days. Representative histological images are from one mouse; each analysis was performed on four ( a , b , c ) or three ( d , e ) mice per genotype. Scale bar, 40 μm ( a ), 100 μm ( b ), 1 mm ( d ), 50 μm ( e ).

Article Snippet: Primary antibodies were TSC2 (D93F12) 1:1000, p-Akt S473 (D9E) 1:1000, 4E-BP1 (53H11) 1:1000, p-S6 S240/244 (rabbit polyclonal) 1:3000, S6 (54D2) 1:1000, p-Akt T308 (D25E6) 1:1000, pan-Akt (40D4) 1:1000, β-tubulin (9F3) 1:1000 , p-RB Ser807/811 (D20B12) 1:1000, PDCD4 (D29C6) 1:1000, cleaved caspase 3 Asp175 (5A1E or rabbit polyclonal) 1:500, survivin (71G4B7) 1:1000 and p-S6K1 T389 (108D2) 1:1000, p-TSC2 S939 (rabbit polyclonal) 1:1000 obtained from Cell Signaling Technology.

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

doi: 10.1083/jcb.201702058

Figure Lengend Snippet: ATF4 is activated through the ISR. (A) Western blot analysis showing the increased phosphorylation of eIF2α (Ser51) upon 6 h of treatment with the different mitochondrial stressors. Bottom, ratio between P-eIF2α and eIF2α total levels. (B and C) mRNA expression analysis of ATF4 and its target genes, CHOP (DDIT3) , ASNS , CHAC1 , PCK2 , and the ER stress marker BIP , upon 6 h of treatment with the different mitochondrial stressors and the ER stressor tunicamycin (Tn at 2.5 µg/ml) in HeLa cells, together with the inhibitor of the integrated stress response (ISRIB at 500 nM). (D) Boxplots showing an increase in basal and ATP-dependent respiration of HeLa cells treated with 500 nM of ISRIB for 24 h. OCR: oxygen consumption rate. (E) mRNA expression analysis of eIF2α kinases upon knock down with specific shRNAs. Data are presented as mean ± SEM of two independent shRNAs for each gene. Statistical differences were calculated compared with pLKO1. (F) mRNA expression analysis of ATF4 and some of its target genes upon knock down of the eIF2α kinases and 6 h of treatment with FCCP. Data are presented as mean ± SEM of two independent shRNAs for each gene. No statistical differences were found between the FCCP treated conditions. All experiments were independently performed at least two times, using triplicates for each condition; data are presented as mean ± SEM of a representative experiment; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6.

Article Snippet: The following primary antibodies were used: mouse anti–β-actin (sc-47778; Santa Cruz Biotechnology, Inc.); mouse anti-HSP90 (BD Biosciences, 610418); OXPHOS antibody cocktail (mouse mAbs, ab110413; Abcam); mouse anti-HSP60 (ADI-SPA-806; Enzo Life Sciences); mouse anti-CLPP (WH0008192M1-100; Sigma-Aldrich); mouse anti-HSPA9 (ABIN361739; Antibodies online); rabbit anti-LONP1 (HPA002192; Sigma-Aldrich); rabbit anti-OTC (sc-102051; Santa Cruz Biotechnology, Inc.); mouse anti-OPA1 (BD, 612606); rabbit anti-CREB-2 (ATF4, sc-200; Santa Cruz Biotechnology, Inc.); phospho-eIF2α (Ser51) rabbit mAb (9721; Cell Signaling Technology); eIF2α rabbit mAb (9722; Cell Signaling Technology); and α tubulin (T5168; Sigma-Aldrich).

Techniques: Western Blot, Phospho-proteomics, Expressing, Marker, Knockdown

Journal: The Journal of Cell Biology

Article Title: Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

doi: 10.1083/jcb.201702058

Figure Lengend Snippet: Genetic link of ATF4 and mitochondrial stress in human and mouse populations. (A and B) Multitissue correlation network analysis of transcript levels across (A) 49 human tissues in GTEx and (B) 16 mouse tissues in the BXD genetic reference population. Nodes represent the transcripts and the width of the ties among nodes indicate the probability to show a significant positive correlation in all tissues analyzed. The human network shows a tighter clustering likely caused by the higher number of tissues and samples per tissues. (C) Heatmap representing the KEGG pathway analysis of the top negative-correlated genes across 49 tissues using GTEx data for ATF4 , CEBPB , DDIT3/CHOP , and NCOR1 (used as positive control). Color key represents the negative logarithm base 10 of the p-value of each pathway obtained in the analysis. (D) Expression quantitative trait locus (eQTL) analysis of Atf4 transcript level in the prefrontal cortex. The yellow mark represents the Atf4 gene locus on chromosome 15, whereas the red mark represent the position of a trans-eQTL for Atf4 expression levels on chromosome 1 (Chr1). (E) eQTL mapping of Atf4 transcript levels across several tissues identifies a common and strong trans-eQTL on chromosome 1 (170–180 Mb). (F) Representation of the chromosome 1 locus (170–180 Mb) containing 142 genes, 6 of which have nonsynonymous substitutions, and only one gene, Fh1 , encodes a mitochondrial protein. Fh1 contains a nonsynonymous sequence variant (A296T; rs32536342 ) that segregates in the BXDs. This sequence variant regulates the expression levels of Fh1 , which in turn regulates Atf4 expression. (G) mRNA expression analysis of HeLa cells after knockdown of fumarate hydratase (shFH) and treatment with monomethyl fumarate at 2.5 mM for 24 h. Data are presented as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Enrichment score plot from GSEA using microarray data from renal cysts of mice with renal tubule specific inactivation of Fh1 ( Fh1 −/− ; GSE29988 ). The list of mitochondrial stress genes (mt-stress genes; Table S5) was used as the gene set of interest. FDR q-val: false discovery rate adjusted p-value; NES, normalized enrichment score; NOM p-val, nominal p-value. (I) Scheme summarizing our working hypothesis. Mitochondrial stress stimulates the phosphorylation of the eIF2α, which inhibits cytosolic translation and activates the ATF4 pathway. At the same time, mitochondrial stress also reduces the expression of MRPs to inhibit mitochondrial translation and protect mitochondrial function.

Article Snippet: The following primary antibodies were used: mouse anti–β-actin (sc-47778; Santa Cruz Biotechnology, Inc.); mouse anti-HSP90 (BD Biosciences, 610418); OXPHOS antibody cocktail (mouse mAbs, ab110413; Abcam); mouse anti-HSP60 (ADI-SPA-806; Enzo Life Sciences); mouse anti-CLPP (WH0008192M1-100; Sigma-Aldrich); mouse anti-HSPA9 (ABIN361739; Antibodies online); rabbit anti-LONP1 (HPA002192; Sigma-Aldrich); rabbit anti-OTC (sc-102051; Santa Cruz Biotechnology, Inc.); mouse anti-OPA1 (BD, 612606); rabbit anti-CREB-2 (ATF4, sc-200; Santa Cruz Biotechnology, Inc.); phospho-eIF2α (Ser51) rabbit mAb (9721; Cell Signaling Technology); eIF2α rabbit mAb (9722; Cell Signaling Technology); and α tubulin (T5168; Sigma-Aldrich).

Techniques: Positive Control, Expressing, Sequencing, Variant Assay, Knockdown, Microarray, Phospho-proteomics

Journal: Stem cells (Dayton, Ohio)

Article Title: LGR5 Promotes Breast Cancer Progression and Maintains Stem-Like Cells Through Activation of Wnt/β-Catenin Signaling.

doi: 10.1002/stem.2083

Figure Lengend Snippet: Figure 3. LGR5 promotes cell migration, tumor metastasis, and activation of the Wnt/b-catenin pathway to induce epithelial- mesenchymal transition. (A): Transwell invasion assay demonstrating that LGR5 promotes cell invasion. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. The results are expressed as the mean 6 SD of three independent experiments (**, p < .01, independent Student’s t test). (B): Metastatic nodules (arrows) on the lung surface. The number of nodules was quantified on lungs of nude mice (n 5 6 per group) 8 weeks after tail vein injection of LGR5- and empty vector- transfected MCF-7 cells and sh-LGR5- and sh-ctr-transfected MDAMB231 cells (**, p < .01, independent Student’s t test). (C): Represen- tative images of hematoxylin and eosin stained sections derived from metastatic nodules at the lung surface. Original magnification, 3100. (D): Representative IF images showing expression of E-cadherin, vimentin, and b-catenin in LGR5-MCF7 cells compared with Vec- MCF7 cells (upper). sh-LGR5- and sh-ctr-transfected MDAMB231 cells (bottom). Nuclei were counterstained with DAPI. Original magnifi- cation, 3400. (E): Relative expressions of E-cadherin, b-catenin, vimentin, fibronectin, snail, and slug were compared between Vec- MCF7 and LGR5-MCF7 or shc-MDAMB231 and shLGR5-MDAMB231 cells by Western blotting (left). The Western blot image in the right panel illustrates that the Wnt inhibitor Dkk1 could effectively decrease b-catenin, snail, C-myc, and cyclinD1 expression induced by LGR5 in MCF-7 cells, whereas Wnt3a could reactivate expression of these genes in shLGR5-MDAMB231 cells.

Article Snippet: Cells were incubated overnight at 48C with the following primary antibodies: LGR5 (Abcam, 1:100); E-cadherin, vimentin, and b-catenin (Cell Signaling Technology, Danvers, MA, 1:100); cytokeratin14 and cytokeratin18 (Bioss People’s Republic of China; 1:100).

Techniques: Migration, Activation Assay, Transwell Invasion Assay, Injection, Plasmid Preparation, Transfection, Staining, Derivative Assay, Expressing, Western Blot

Journal: Stem cells (Dayton, Ohio)

Article Title: LGR5 Promotes Breast Cancer Progression and Maintains Stem-Like Cells Through Activation of Wnt/β-Catenin Signaling.

doi: 10.1002/stem.2083

Figure Lengend Snippet: Figure 5. LGR5 enhances Wnt/b-catenin pathway activity to induce stemness. (A): Cellular morphology is shown at the second day after passaging the LGR5high cells (0 hour, upper) and culturing in the absence of other factors (control) or the presence of either Wnt3a-conditioned medium (Wnt3a) or Dickkopf1 (Dkk1) (96 hours, lower). (B): Cells treated with R-spondin1 and Wnt3a generated sig- nificantly more spheres, and treatment with DKK1 significantly reduced the number of spheres. The data represent the mean 6 SD of three independent experiments, *, p < .05; **, p < .01. (C): The spheroid formed from LGR5high cells expressed different levels of b-catenin in the control medium or when treated with Wnt3a or DKK1. Nuclei were stained with DAPI. Original magnification: 3400. (D): Breast cancer specimens were analyzed by immunohistochemical staining, and the representative LGR5, b-catenin, c-myc, and cyclinD1 expression are shown. Scale bar 5 50 mm. (E): Analysis of immunohistochemistry tissue microarray data showing linear regres- sions and significant Pearson correlations of LGR5 with b-catenin (n 5 129), LGR5 with C-myc (n 5 123), and LGR5 with cyclinD1 (n 5 109) in breast cancer.

Article Snippet: Cells were incubated overnight at 48C with the following primary antibodies: LGR5 (Abcam, 1:100); E-cadherin, vimentin, and b-catenin (Cell Signaling Technology, Danvers, MA, 1:100); cytokeratin14 and cytokeratin18 (Bioss People’s Republic of China; 1:100).

Techniques: Activity Assay, Passaging, Control, Generated, Staining, Immunohistochemical staining, Expressing, Immunohistochemistry, Microarray

Journal: Stem cell research & therapy

Article Title: Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

doi: 10.1186/s13287-017-0507-4

Figure Lengend Snippet: Fig. 2 Expression and promoter methylation of DNA methyltransferases in intestinal epithelium. a, b Decreased Dnmt1 expression is detected in db/db crypts and intestinal epithelium stem cells (IESCs) by qRT-PCR using the FACS cell population (a) and Western blot using the MACS cell population (b left) and quantification of immunoblot bands from Western blot (b right). mRNA and protein levels are expressed relative to β-actin. Mean ± SE; n ≥6; *P < 0.05, **P < 0.01 by t test. C represents the control db/+ group; D represents the diabetic db/db group. c Promoter methylation microarray of Dnmt3b indicates promoter hypermethylation not only in db/db mice (orange bars) but also in db/+ mice (light blue bars). Values in brackets indicate the peak score that reflects the probability of positive enrichment and average P value scores. d, e Validation of Dnmt3b microarray results by bisulfite sequence. The table indicates the results of bisulfite sequencing analyses of 10 bacterial colonies per group (d). The CpG sites validated are the sites that exhibit a peak signal in the microarray. Black circles represent methylated CpG sites; white hollow circles represent unmethylated CpG sites; crosses represents methylation status not determined . Bar graph showing the ratio of methylated cyto- sine in CpG within the regions analyzed (e). f, g Immunohistochemical staining of Dnmt1 (f) and Dnmt3a (g). Scale bars = 50 μm. Diff differenti- ated cells, N.S. not significant

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot, Control, Microarray, Biomarker Discovery, Sequencing, Methylation Sequencing, Immunohistochemical staining, Staining

Journal: Stem cell research & therapy

Article Title: Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

doi: 10.1186/s13287-017-0507-4

Figure Lengend Snippet: Fig. 3 Promoter methylation levels and gene expression of Wnt signaling-related genes. a (left) Hierarchical clustering analysis on the basis of the DMEP-related genes related with Wnt signaling. Each row indicates a DMEP-related gene, and the corresponding gene name is indicated on the right. Each column indicates a sample analyzed. Methylation levels range from unmethylated (green) to fully methylated (red), as indicated by the color legend at the top of the graph. a (right) The degree of methylation of Wnt signaling pathway-related genes among DMEP-related genes. The bar height reflects the degree of methylation. b, c The mRNA levels of Wnt signaling-related genes in crypts and intestinal epithelium stem cells (IESCs) using the FACS cell population. d (upper) Western blot analysis of Sox9 using the MACS cell population, and d (lower) quantification of immunoblot bands from three repetitions of western blot experiments. e Immunohistochemical staining of Sox9 in the small intestinal epithelium. The arrows indicate cells expressing low levels of Sox9, which were considered IESCs. Scale bars = 50 μm. mRNA and protein levels are expressed relative to β-actin. Mean ± SE; n ≥6 (mRNA); n ≥3 (protein); *P < 0.05, **P < 0.01, ***P < 0.001 by t test. N.S. not significant

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Methylation, Gene Expression, Western Blot, Immunohistochemical staining, Staining, Expressing

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Overexpression of P‐SIRT1 Ser27 in CRC tissues and its implications for SIRT1 stability and proliferation and migration of colon cancer cells. (A) SW480 cells (5 × 10 6 ) stably expressing mock or SIRT1 vector were injected subcutaneously into the flank of BALB/c nude mice. Photographs show the xenograft tumors resected from mice at the time of harvest (day 50). The tumor volume and the tumor weight of the mice at the time of harvest were measured. Comparison of the means between mock and SIRT1 vector group was determined by Student's t ‐test. The results are shown as the mean ± S.D. of four xenografts for each group: * P < 0.05. Scale bar represents 1 cm. (B) Histological structures in sections from excised xenograft tumors were analyzed by H&E staining (first column). The yellow arrow indicates neovascularization. The expression of SIRT1, IL‐6, and 8 in excised xenograft tumors were examined by immunohistochemical analysis (second to last columns). Each scale bar represents 100 µm. The data are presented as the mean ± S.D. of three independent samples for each group in Fig. S1H: * P < 0.05; ** P < 0.01. (C) Kaplan–Meier survival curve of overall survival in colon cancer patients with high ( n = 497) and low ( n = 506) levels of SIRT1 based on the data collected from the GENT2 database ( http://gent2.appex.kr ). (D) Kaplan–Meier survival curve of disease‐specific survival in colon cancer patients with high ( n = 238) and low ( n = 238) levels of SIRT1 based on the data obtained from the GENT2 database ( http://gent2.appex.kr ) (E) Immunofluorescence staining of tissue microarray containing thirty‐five pairs of colon adenocarcinoma and matched adjacent normal colon tissues was conducted using anti‐SIRT1 and P‐SIRT1 Ser27 antibodies. The adjacent normal area is 1.5 cm distant from the tumor, which was taken by a histologist. Images of H&E‐stained tissue sections were offered by US Biomax Inc. Each scale bar represents 200 µm. (F) The fluorescence intensity was measured by image analysis software ImageJ, and results are shown as a violin plot. Statistics were calculated using both paired t ‐test and Wilcoxon matched‐pairs test. (G) The protein levels of SIRT1 and P‐SIRT1 Ser27 in colon cancer and corresponding normal tissues were measured by Western blot analysis. The data are presented as the mean ± S.D. of eleven pairs of human tissue specimens in Fig. D: * P < 0.05.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Over Expression, Migration, Stable Transfection, Expressing, Plasmid Preparation, Injection, Comparison, Staining, Immunohistochemical staining, Immunofluorescence, Microarray, Fluorescence, Software, Western Blot

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Comparison of the effects of SIRT1‐WT and SIRT1‐S27A on the tumorigenicity of HCT‐116 cells. (A) HCT‐116 cells (5 × 10 6 ) transfected with mock, SIRT1‐WT, or SIRT1‐S27A vector were inoculated subcutaneously into the flank of BALB/c nude mice. Photographs show the xenograft tumors collected from mice at the end of the experiment (day 20). The tumor volume and the tumor weight of the mice at the time of harvest were measured, and the one‐way ANOVA with Tukey's multiple comparisons test was performed for the statistical comparison. The results are shown as the mean ± S.D. of six xenografts for each group: * P < 0.05; ** P < 0.01. Scale bar represents 1 cm. (B) H&E staining and immunohistochemical analysis of SIRT1, P‐SIRT1 Ser27 , IL‐6, and IL‐8 were performed on sections from resected xenograft tumor. Each scale bar represents 100 µm. For quantification of each target, the DAB intensity was measured by image analysis software FIJI (the enhanced version of imagej2 ). Statistics were calculated using the one‐way ANOVA with Tukey's multiple comparisons test, and results are presented as the mean ± S.D. ( n = 4 per group): ** P < 0.01; *** P < 0.001. (C) Identification of high‐confidence kinase candidates contributing phosphorylation of SIRT1 at serine 27 by gps 5.0 ( http://gps.biocuckoo.cn/ ). (D) Multiple sequence alignment using clustalw software ( https://www.genome.jp/tools‐bin/clustalw ) showing an evolutionarily conserved phosphorylation site of SIRT1 at serine 27 (arrow) between different species. The positions which have a fully conserved residue are indicated by asterisks. (E) HCT‐116 cells were treated with two different concentrations (10 and 20 µ m ) of a JNK inhibitor (SP600125), an ERK inhibitor (U0126) or a p38 inhibitor (SB203580) for 3 h. The protein levels of P‐SIRT1 Ser27 and SIRT1 were measured by Western blot analysis. One‐way ANOVA with Tukey's multiple comparisons test was used to determine the significance. The results are shown as the mean ± S.D. ( n = 3): ** P < 0.01; *** P < 0.001. (F) HCT‐116 cells were treated with CHX (20 μg·mL −1 ) for indicated periods in the absence or presence of SP600125 (20 μ m ). Comparison of the means between two groups was determined by Student's t ‐test. The values are presented as the mean ± S.D. of three independent experiments: *** P < 0.001.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Comparison, Transfection, Plasmid Preparation, Staining, Immunohistochemical staining, Software, Phospho-proteomics, Sequencing, Residue, Western Blot

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Strong inactivation of Snail in HCT‐116 cells expressing SIRT1‐S27A. (A) Nuclear extracts prepared from HCT‐116 cells transfected with SIRT1‐WT or SIRT1‐S27A were subjected to the transcription factor activation profiling array to analyze the activity of ninety‐six different transcription factors. The top 10 hits are listed in the Table in an ascending order of the SIRT1‐S27A to SIRT1‐WT ratio. The red arrow denotes the top hit whose activity was most reduced in HCT‐116 cells harboring the SIRT1‐S27A mutation. (B) Following transfection of HCT‐116 cells with mock, SIRT1‐WT, or SIRT1‐S27A vector for 48 h, the mRNA (upper panels) and protein (lower panels) levels of Snail were measured by RT‐PCR and immunoblotting analyses, respectively. Differences in the means among the groups were assessed by one‐way ANOVA with Tukey's multiple comparisons test. Data are presented as the mean ± S.D. ( n = 3 per group): ** P < 0.01; *** P < 0.001. NS, not significant. (C) Immunohistochemical (IHC) analysis of Snail was carried out on sections from resected xenograft tumor corresponding to the Fig. . Each scale bar represents 100 µm. The DAB intensity was assessed by image analysis software fiji (the enhanced version of imagej2 ). Statistics were calculated using one‐way ANOVA with Tukey's multiple comparisons test, and the data are presented as the mean ± S.D. ( n = 4 per group): *** P < 0.001. (D) IHC analysis of Snail and P‐SIRT1 Ser27 was performed on sections from resected xenograft tumor corresponding to the Fig. . Each scale bar represents 100 µm. The results are shown as the mean ± S.D. of three independent samples for each group in Fig. B: * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Expressing, Transfection, Activation Assay, Activity Assay, Mutagenesis, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Software

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: A proposed mechanism underlying contribution of aberrantly stabilized SIRT1 to oncogenicity of human colon cancer cells. JNK‐dependent phosphorylation of SIRT1 at Ser27 contributes to its stabilization and deacetylation of Snail to enhance the production of IL‐6 and IL‐8, thereby promoting proliferation and migration of human colon cancer cells.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Phospho-proteomics, Migration

Journal: Cell Reports

Article Title: Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis

doi: 10.1016/j.celrep.2021.110056

Figure Lengend Snippet: Statin-induced morphodynamic changes are mediated by Rho-GTPases and Integrin signaling (A) Sphere formation of YAPC cells with 48 h pretreatment as indicated; yellow boxes: area chosen for magnification below. Scale bar: 75 μm (top panel), 20 μm (bottom panel). (B and C) Western blot for pERK (Thr202/Tyr204) and total ERK after treatment as indicated for 48 h. (D) GSEA for TGF-β signaling of microarray data from ASPC1 cells treated with 5 μM fluvastatin or control for 48 h. (E) Western blot for pSMAD2 (Ser465/467) and total SMAD2 treated as indicated for 48 h. (F) Top panel: YAPC cells in 2D cell culture after 48 h of treatment as indicated. Middle panel: Sphere formation of YAPC cells with pretreatment for 48 h as indicated; yellow boxes: area chosen for magnification below (bottom panel). Scale bar: 50 μm (top and middle panel) and 20 μm (bottom panel). (G) GSEA for Integrin-mediated signaling of microarray data from ASPC1 cells treated for 48 h with 5 μM fluvastatin or control. (H) Western blot for pSRC (Tyr416), total SRC, and Integrin alpha 2 after treatment as indicated for 48 h. (I) Number of cells in the lungs of mice intravenously injected with ASPC1-GFP cells pretreated as depicted in ( Figure 4 O); quantified by FACS as [%] of total lung cells; each dot represents a mouse, and the black line is the mean; n = 5 mice per group. Significance test: Mann-Whitney test. (J) Tumor weight of subcutaneous tumors in mice injected with ASPC1-GFP cells pretreated as depicted in ( Figure 4 V); each dot represents a mouse, and the black line is the mean; n = 5 mice per group. Significance test: Mann-Whitney test. (K–M) Western blot for pSRC (Tyr416), total SRC, Integrin alpha 2, phospho-ERK (Thr202/Tyr204), total ERK, E-cadherin, and RhoB in Panc89 cells treated for 48 h as indicated. (N) Western blot for NSDHL in Panc89 shcontrol, sh NSDHL #1, and sh NSDHL #2 cells. (O) qRT-PCR of NSDHL in Panc89 shcontrol, sh NSDHL #1, and sh NSDHL #2 cells; bar graphs indicate the mean, and error bars indicate ± SEM of one representative out of three independent experiments. Significance test: one-way ANOVA. (P) Anoikis survival assay of Panc89 shcontrol, sh NSDHL #1, and sh NSDHL #2 cells after 24 h from one representative out of three independent experiments; significance test: ANOVA. (Q) Western blot for Integrin alpha 2, pERK (Thr202/Tyr204), total ERK, E-cadherin, and RhoB in Panc89 cells treated for 48 h as indicated and Panc89 shcontrol, sh NSDHL #1, and sh NSDHL #2 cells. (R) Western blot for Integrin alpha 2, pERK (Thr202/Tyr204), total ERK, E-cadherin, and RhoB in ASPC1 cells treated for 48 h as indicated. (S) Schematic of morphological and signaling effects of statin treatment on PDAC cells. Dashed arrows indicate hypothesized; continuous arrows indicate data-based interactions. See also and .

Article Snippet: Rabbit monoclonal anti Smad2 (D43B4) , Cell Signaling Technology , Cat# 5339; RRID: AB_10626777.

Techniques: Western Blot, Microarray, Control, Cell Culture, Injection, MANN-WHITNEY, Quantitative RT-PCR, Clonogenic Cell Survival Assay

Journal: Cell Reports

Article Title: Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis

doi: 10.1016/j.celrep.2021.110056

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti Smad2 (D43B4) , Cell Signaling Technology , Cat# 5339; RRID: AB_10626777.

Techniques: Recombinant, Mass Spectrometry, Microarray, Western Blot, shRNA, Software

Journal: Oncogene

Article Title: Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer

doi: 10.1038/s41388-022-02314-w

Figure Lengend Snippet: A Transcriptional upregulation of I d2 in suspended mouse ILC (mILC) cells. Id2 mRNA levels were quantified in three independent anchorage-independent mILC cell lines cultured in adherent (Adh) or suspension (Sus/24 h) conditions using mRNA sequencing. Expression is depicted as Reads per kilo base per million mapped (RPKM) values. B Western blot showing Id2 protein expression in cells from A . Akt was used as a loading control. Western blots were quantified by comparing pixel intensity of control compared to the condition. C Western blot analysis showing E-cadherin levels in isogenic MCF7 control (WT) versus two MCF7 knock-out clones (MCF7::Δ CDH1) (left panel), and two SUM44PE ILC cells transduced with a DOX-inducible full length and wild type E-cadherin (SUM44PE::i CDH1 )(right panel). Akt was used as a loading control. D Anoikis resistance assay performed in cell lines from C . MCF7 control (WT) and MCF7::Δ CDH1 isogenic clones 1 and 2 were kept in anchorage independent conditions for 96 h before harvesting, whereas SUM44PE were kept in anchorage independent conditions for 168 h. Note the approx. 2-fold increase in both MCF7::Δ CDH1 clones versus MCF7 control, and approx. 1.5-fold reduction in anoikis resistance for SUM44PE::i CDH1 cell line and 2. The Student’s t test was used to determine statistical significance. *** p < 0.001, ** p < 0.01. Error bars show the standard deviation (SD). E E-cadherin loss induces Id2 expression in suspended cells versus adherent cells. Shown are the RT-qPCR mRNA expression fold increases for Id2 in isogenic MCF7 control and two E-cadherin knock-out (∆ CDH1 ) cell lines and the ILC cell line SUM44PE:i CDH1 . Note the approximate 3 to 5-fold induction of Id2 mRNA expression in the E-cadherin mutant cells in anchorage-independent conditions (Sus). Also note that E-cadherin reconstitution in SUM44PE::i CDH1 upon DOX administration reduces the Id2 mRNA expression in control SUM44PE::i CDH1 cells by an average approximate of 70%. Relative Id2 mRNA expression levels were normalized to either GAPDH or ACTB. MCF7 control (WT), MCF7:: Δ CDH1 were kept in anchorage independent conditions for 96 h, and SUM44PE for 168 h/7 days, before harvesting and analysis. The Student’s t test was used to determine statistical significance. **** p < 0.0001, *** p < 0.001, ** p < 0.01. Error bars show the standard deviation (SD). (All) Representative results are shown from at least three independent experiments.

Article Snippet: The following primary antibodies were used for western blot analysis: monoclonal rabbit Id2 D39E8 (1:1,000; #3431; Cell Signaling Technology), polyclonal rabbit Id2 C-20 (1:1,000; sc-489; Santa Cruz Biotechnology), polyclonal rabbit Rb M-153 (1:1,000; sc-7905; Santa Cruz Biotechnology), rabbit monoclonal phospho-Rb Ser 780 (1:2000; #9307; Cell Signaling Technology), monoclonal rabbit phospho-Rb Ser 807/811 (1:2,000; #9308; Cell Signaling Technology), monoclonal mouse E-cadherin (ECH6; 1:1000; #MSK033; Zytomed); purified mouse p120-Catenin (Clone98; 1;1000; #610134; BD Biosciences), polyclonal rabbit GFP (1:1000; sc-8334; Santa Cruz Biotechnology), and polyclonal goat Akt1 C-20 (1:1,000; sc-1618; Santa Cruz Biotechnology).

Techniques: Cell Culture, Suspension, Sequencing, Expressing, Western Blot, Control, Knock-Out, Clone Assay, Transduction, Standard Deviation, Quantitative RT-PCR, Mutagenesis

Journal: Oncogene

Article Title: Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer

doi: 10.1038/s41388-022-02314-w

Figure Lengend Snippet: Id2 controls anchorage-independent survival. Shown are the levels of anoikis resistance upon knockdown of Id2 in mouse mILC ( A ) or E-cadherin mutant human SKBR3 ( B ) after 24 or 96 h in suspension respectively. Scrambled shRNA (shScr) and Doxycycline (DOX) controls are depicted in white bars. The Student’s t test was used to determine statistical significance. *** p < 0.001, ** p < 0.01, * p < 0.05. Results are expressed as mean and error bars represent standard deviation (SD). All experiments were repeated at least 3 times. C , D Id2 expression is necessary for lung colonization. Shown are bioluminescence signals (relative counts) from luciferase-tagged mILC cells injected intra-venously (iv) in the tail vain of recipient Nude mice ( D ). The effect of Id2 knockdown (shId2) was assessed and compared to controls (shScr). The number of mice showing lung colonization 4 weeks post injection are depicted below, together with the total number of mice injected in parenthesis. A two-tailed Fisher’s exact test was used to determine statistical significance. *** p < 0.001. Panel D shows a representative H&E stained lung sections (magnification 100X) of the mice depicted in C . Arrows point to metastatic outgrowth. For all mouse experiments, animals were randomized, and observers blinded for the experimental conditions.

Article Snippet: The following primary antibodies were used for western blot analysis: monoclonal rabbit Id2 D39E8 (1:1,000; #3431; Cell Signaling Technology), polyclonal rabbit Id2 C-20 (1:1,000; sc-489; Santa Cruz Biotechnology), polyclonal rabbit Rb M-153 (1:1,000; sc-7905; Santa Cruz Biotechnology), rabbit monoclonal phospho-Rb Ser 780 (1:2000; #9307; Cell Signaling Technology), monoclonal rabbit phospho-Rb Ser 807/811 (1:2,000; #9308; Cell Signaling Technology), monoclonal mouse E-cadherin (ECH6; 1:1000; #MSK033; Zytomed); purified mouse p120-Catenin (Clone98; 1;1000; #610134; BD Biosciences), polyclonal rabbit GFP (1:1000; sc-8334; Santa Cruz Biotechnology), and polyclonal goat Akt1 C-20 (1:1,000; sc-1618; Santa Cruz Biotechnology).

Techniques: Knockdown, Mutagenesis, Suspension, shRNA, Standard Deviation, Expressing, Luciferase, Injection, Two Tailed Test, Staining

Journal: Oncogene

Article Title: Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer

doi: 10.1038/s41388-022-02314-w

Figure Lengend Snippet: A Schematic overview of the promoter regions of mouse and human Id2 depicting the position of the canonical cKBS (CTGCNA) relative to the transcription start site (TSS). Id2 -specific primers used in (B) are indicated by arrows. B Id2 is a Kaiso target gene. Chromatin immunoprecipitated (ChIP) samples from mILC or human E-cadherin deficient MDA-MB-231 cells were amplified using an Id2 -specific PCR using primary flanking the cKBS consensus sites (top panels). Negative controls consisted of Id2 -specific non-cKBS primers (bottom panels), no input/Kaiso antibodies (control), IgG mouse antibodies (IgG) and a positive control was based on an anti-H3 antibody ChIP. C , D E-cadherin mutant cells upregulate Id2 under anchorage-independent conditions. Mouse syngeneic breast cancer and isogenic MCF7 control (WT) versus MCF7::Δ CDH1 were transduced with an Id2 -mCherry reporter system and fluorescence was quantified using FACS analysis in adherent (Ad, gray bars) and suspension (Sus, blue bars). Timing of suspension conditions: mouse cells 24 h and human cells 96 h. Note the increase in mCherry fluorescence in the E-cadherin mutant mouse (mILC-1) and human MCF7::Δ CDH1 cells compared to the E-cadherin expressing cells (Trp53 ∆ and MCF7 WT) (D). **** p < 0.0001. E , F Id2 is a Kaiso target gene regulated by p120. Quantification of Id2 reporter activity based on transcriptional luciferase activity. HEK293T cells were co-transfected with either an empty pCDNA vector (control), Kaiso, or p120-1A and cultured in adherent conditions. E Note the 2-fold decrease in Kaiso co-transfected cells and the 3-fold increase in reporter activity in cells co-transfected with p120. The western blot depicts the effect of p120 depletion using Doxycycline (DOX)-inducible knockdown (iKD) of both p120 isoforms on Id2 protein expression. Note the approximate 3-fold reduction in Id2 protein expression upon DOX addition. For F Western blots were quantified by comparing the pixel intensity of controls versus experimental conditions. ( All ) Representative experiments are shown from at least three independent biological replicates. For C – E , the mean and standard deviation are shown. The Student’s t test was used to determine statistical significance. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The following primary antibodies were used for western blot analysis: monoclonal rabbit Id2 D39E8 (1:1,000; #3431; Cell Signaling Technology), polyclonal rabbit Id2 C-20 (1:1,000; sc-489; Santa Cruz Biotechnology), polyclonal rabbit Rb M-153 (1:1,000; sc-7905; Santa Cruz Biotechnology), rabbit monoclonal phospho-Rb Ser 780 (1:2000; #9307; Cell Signaling Technology), monoclonal rabbit phospho-Rb Ser 807/811 (1:2,000; #9308; Cell Signaling Technology), monoclonal mouse E-cadherin (ECH6; 1:1000; #MSK033; Zytomed); purified mouse p120-Catenin (Clone98; 1;1000; #610134; BD Biosciences), polyclonal rabbit GFP (1:1000; sc-8334; Santa Cruz Biotechnology), and polyclonal goat Akt1 C-20 (1:1,000; sc-1618; Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Amplification, Control, Positive Control, Mutagenesis, Transduction, Fluorescence, Suspension, Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Knockdown, Standard Deviation

Journal: Oncogene

Article Title: Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer

doi: 10.1038/s41388-022-02314-w

Figure Lengend Snippet: A – C Id2 controls viability through inhibition of cell cycle progression under anchorage-independent culture conditions. FUCCI-2-tagged mILC-1 cells were cultured in suspension for 24 h and stained for apoptotic cells using cleaved Caspase-3 (blue; A ). Cell cycle stages of mILC-1 cells in which either Id2 or Rb expression were inhibited, were quantified in B and viability was assessed in parallel using FACS analysis for Annexin-V and propidium iodine incorporation C . C The Student’s t test was used to determine statistical significance. ** p < 0.01, * p < 0.05. Error bars depict the standard deviation. Size bar = 5 µm. D , E Id2 depletion causes an increase in cycling cells in a Rb and CDK4/6-dependent manner. Phosphorylated Rb (pRb Ser807/811) IF staining of Adh and Sus (24 h) mILC-1 cells ( D ). Note the absence of p-Rb signals (blue signals) and the reduction of G2/M cells in the Id2-depleted condition in the presence of the CDK4/6 inhibitor ribociclib (CDK4/6i; 3 µM). The number of cells expressing p-Rb from D are quantified in E . Outlines indicate cells with high pRb staining in G2/M phase. E HPF High Power Fields, showing one microscope image in high resolution. Five HPFs containing at least 120 cells per cell cycle state were quantified. E The Student’s t test with Welch’s correction was used to determine statistical significance. ** p < 0.01, * p < 0.05. Error bars depict the standard deviation. (All) Representative experiments are shown from at least three biological independent replicates.

Article Snippet: The following primary antibodies were used for western blot analysis: monoclonal rabbit Id2 D39E8 (1:1,000; #3431; Cell Signaling Technology), polyclonal rabbit Id2 C-20 (1:1,000; sc-489; Santa Cruz Biotechnology), polyclonal rabbit Rb M-153 (1:1,000; sc-7905; Santa Cruz Biotechnology), rabbit monoclonal phospho-Rb Ser 780 (1:2000; #9307; Cell Signaling Technology), monoclonal rabbit phospho-Rb Ser 807/811 (1:2,000; #9308; Cell Signaling Technology), monoclonal mouse E-cadherin (ECH6; 1:1000; #MSK033; Zytomed); purified mouse p120-Catenin (Clone98; 1;1000; #610134; BD Biosciences), polyclonal rabbit GFP (1:1000; sc-8334; Santa Cruz Biotechnology), and polyclonal goat Akt1 C-20 (1:1,000; sc-1618; Santa Cruz Biotechnology).

Techniques: Inhibition, Cell Culture, Suspension, Staining, Expressing, Standard Deviation, Microscopy

Journal: Oncogene

Article Title: Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer

doi: 10.1038/s41388-022-02314-w

Figure Lengend Snippet: A Western blot analysis showing E-cadherin levels in the classical ILC KCL320 and in the E-cadherin expressing non-classical ILC PDO model P008 (top panel). Akt was used as loading control (bottom panel). B Schematic representation of the CDH1 mutation S180Y and the consequences for cell-cell contacts. Note that although E-cadherin S180Y is unable to establish homotypic in trans interactions with adjacent cells, it retains p120-catenin at the cell membrane. C Immunofluorescence showing expression and localization of E-cadherin (red; top panels), p120-catenin (p120; green, middle panels). Merged images are shown in the bottom panels. Note the cytosolic p120 expression in E-cadherin deficient cells versus the co-localization of E-cadherin and p120 in E-cadherin-expressing P008 ILC cells. Isogenic MCF7 and MCF::Δ CDH1 cell lines were used as a controls. Size bar = 5 µm. D RT-qPCR analysis of Id2 mRNA upregulation in anchorage-independent cells (Sus/168 h). Note the 4-fold upregulation of Id2 in KCL320 suspended cells compared to adherent conditions (Ad). Relative Id2 mRNA expression levels were normalized to either GAPDH or ACTB. The Student’s t test was used to determine statistical significance. **** p < 0.0001, *** p < 0.001. Error bars show the standard deviation. E Western blot analysis showing E-cadherin levels in the isogenic P008 and P008::Δ CDH1 PDO models (top panel). Akt was used as a loading control (bottom panel). F Representative differential interference contrast microscopy images of P008 and P008::Δ CDH1 cultured in 3D basement membrane extract-derived matrix. Note the noncoherent “grape-like” phenotypic appearance of both PDO models. Size bar = 50 µm. G RT-qPCR analysis of Id2 mRNA upregulation in anchorage-independent cells. Note the approximate 6-fold upregulation in P008::Δ CDH1 cells compared to P008 suspended cells. Both organotypic cell lines were kept in suspension for 7 days before harvesting and analysis. Relative Id2 mRNA expression levels were normalized to either GAPDH or ACTB. The Student’s t test was used to determine statistical significance. **** p < 0.0001, *** p < 0.001. Error bars show the standard deviation. (All) Representative results are shown from at least three independent experiments.

Article Snippet: The following primary antibodies were used for western blot analysis: monoclonal rabbit Id2 D39E8 (1:1,000; #3431; Cell Signaling Technology), polyclonal rabbit Id2 C-20 (1:1,000; sc-489; Santa Cruz Biotechnology), polyclonal rabbit Rb M-153 (1:1,000; sc-7905; Santa Cruz Biotechnology), rabbit monoclonal phospho-Rb Ser 780 (1:2000; #9307; Cell Signaling Technology), monoclonal rabbit phospho-Rb Ser 807/811 (1:2,000; #9308; Cell Signaling Technology), monoclonal mouse E-cadherin (ECH6; 1:1000; #MSK033; Zytomed); purified mouse p120-Catenin (Clone98; 1;1000; #610134; BD Biosciences), polyclonal rabbit GFP (1:1000; sc-8334; Santa Cruz Biotechnology), and polyclonal goat Akt1 C-20 (1:1,000; sc-1618; Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Control, Mutagenesis, Membrane, Immunofluorescence, Quantitative RT-PCR, Standard Deviation, Microscopy, Cell Culture, Derivative Assay, Suspension

Journal: Oncogene

Article Title: Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer

doi: 10.1038/s41388-022-02314-w

Figure Lengend Snippet: A – C Cytosolic Id2 expression is prevalent in ILC. Shown are representative tissue micro array (TMA) cores (top panels) from IDC-NST and ILC stained for Id2 expression ( A ) (brown). A total of 148 invasive breast cancer samples were scored as binary output and quantified in B . Note the overall increase in Id2 expression when comparing ILC versus IDC-NST in B , and the increase in the number of cases that express cytosolic Id2 when comparing ILC versus IDC-NST ( C ). Pearson Chi-square tests were used to determine statistical significance. P -values are stated in the table. D Shown are Id2 mRNA expression levels in the TCGA dataset. Wilcoxon rank tests were used to assess statistical significance. In total 533 breast cancer samples were analyzed (388 IDC-NST versus 145 ILC). E Id2 controls sustained indolent proliferation in ILC. Cartoon depicting a proposed model for E-cadherin dependent regulation of cell cycle progression. Show are E-cadherin expressing (left panel) and E-cadherin negative breast cancer (right panel). Note the consequences of E-cadherin loss (right panel), inducing cytosolic and nuclear translocation of p120 and a subsequent de-repression of Id2 expression by Kaiso. Id2 will accumulate in the cytosol, where it binds hypo-phosphorylated Rb and inhibits phosphorylation of Rb by CDK4/6. Cell cycle progression will be dampened, underpinning the indolent and low-grade features of E-cadherin mutant ILC. Under anchorage independent conditions (dissemination), Id2 expression is further increased due to a nuclear influx of p120 , leading to a full prevention of Rb phosphorylation, clustering E2F, and inhibition of cell cycle progression in G0/G1.

Article Snippet: The following primary antibodies were used for western blot analysis: monoclonal rabbit Id2 D39E8 (1:1,000; #3431; Cell Signaling Technology), polyclonal rabbit Id2 C-20 (1:1,000; sc-489; Santa Cruz Biotechnology), polyclonal rabbit Rb M-153 (1:1,000; sc-7905; Santa Cruz Biotechnology), rabbit monoclonal phospho-Rb Ser 780 (1:2000; #9307; Cell Signaling Technology), monoclonal rabbit phospho-Rb Ser 807/811 (1:2,000; #9308; Cell Signaling Technology), monoclonal mouse E-cadherin (ECH6; 1:1000; #MSK033; Zytomed); purified mouse p120-Catenin (Clone98; 1;1000; #610134; BD Biosciences), polyclonal rabbit GFP (1:1000; sc-8334; Santa Cruz Biotechnology), and polyclonal goat Akt1 C-20 (1:1,000; sc-1618; Santa Cruz Biotechnology).

Techniques: Expressing, Microarray, Staining, Translocation Assay, Phospho-proteomics, Mutagenesis, Inhibition