Journal: Molecular Cancer

Article Title: Reversal of chemosensitivity and induction of cell malignancy of a non-malignant prostate cancer cell line upon extracellular vesicle exposure

doi: 10.1186/1476-4598-12-118

Figure Lengend Snippet: EV-mediated transfer of proteins via Kinexus phospho-protein microarray analysis in DU145 cells co-cultured with PrEC EVs

Article Snippet: To determine the proteins that are involved in or might be responsible for “phenotypic switching”, we utilized Kinexus phospho-protein microarray analysis (Vancouver, BC) in the DU145 cells co-cultured with PrEC EVs.

Techniques: Microarray

Journal: Bioengineering

Article Title: COVID-19 Diagnostic Strategies Part II: Protein-Based Technologies

doi: 10.3390/bioengineering8050054

Figure Lengend Snippet: The developed protein microarray-based tests for COVID-19 detection.

Article Snippet: PEPperPRINT GmbH [ ] , PEPperCHIP ® Pan-Corona Spike Protein Microarray , Antibodies against S antigen , S proteins derived from seven coronaviruses translated into overlapping peptides , (No info) , (No info) , One array with 4564 peptides in duplicate , RUO , For Serum antibody fingerprint analysis, Immune monitoring and Epitope studies.

Techniques: Microarray, Peptide Microarray, Bioprocessing, Derivative Assay, High Throughput Screening Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Virus, Clinical Proteomics

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Identification of KSR1-regulated phosphoproteome in breast cancer cells. ( A ) Experimental schematic outline of SILAC experiment. ( B ) Scatter plot comparison of phosphosite ratios quantified from control vs KSR1-overexpressed MCF7 cells. ( C ) Gene ontology (GO) Classification of the KSR1-regulated phosphoproteome in MCF7 cells according to molecular functions, biological processes and cellular compartmentalisation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Multiplex sample analysis, Comparison, Phospho-proteomics, Control

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 on p53 transcriptional activity in the presence or absence of etoposide by luciferase assays. ( A ) MCF7 cells were transiently co-transfected with either pCMV6 (vector) or pCMV6-KSR1 plasmids in the presence of four individual p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1, p53-CYCLIN G1 and p53-IGFBP3) following dimethylsulphoxide (DMSO) or etoposide (40 μ M ) treatment for 3 h. ( B ) MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 48 h, followed by transfection of three p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1 and p53-CYCLIN G1) for additional 24 h. DMSO or etoposide (40 μ M ) were subsequently added as described above. Firefly luciferase activity was measured (renilla luciferase activity was used to normalise transfection efficiency). The normalised luciferase activity of empty vector is set as 1. Results shown are the average of at least three independent experiments and error bars represent s.d. Student's t -test was performed using SPSS 16.0 statistical software (SPSS Inc.). (* P <0.05, ** P <0.01).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Construct, Expressing, Control, Software

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 on p53 mRNA, total protein and neddylation levels and on p53 subcellular localisation. ( A ) Effects on p53 mRNA and total protein levels after KSR1 overexpression. MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, relative mRNA levels of TP53 and p53 total protein were measured by RT-qPCR and western blotting, respectively. Gene expression level from cells transfected with pCMV6 was set as 1. Results shown are the average of at least three independent experiments. Similarly, in MCF7 stably overexpressing KSR1 cells, p53 total protein was evaluated by western blot. Blots shown are representatives of at least three independent experiments. ( B ) Immunofluorescence staining of p53 cells after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. p53 was detected with an anti-p53 antibody while the nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI). Representative pictures of three independent experiments are shown. Subcellular fractionation assays were performed after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. Tubulin and histone deacetylase 1 (HDAC1) expression served as positive normalising control for cytoplasmic and nuclear proteins respectively. Blots shown are representatives of at least three independent experiments. ( C ) Neddylation assay on p53 after KSR1 overexpression. MCF7 cells were co-transfected with HA-NEDD8 and pCMV6 or pCMV6-KSR1 plasmids as indicated. p53 was immunoprecipitated using a p53-specific antibody (DO-1) and the neddylated-p53 was detected by immunoblotting using anti-NEDD8 and anti-p53-specific antibodies. Blots shown are representatives of at least three independent experiments. Abbreviations: IgG= immunoglobulin G; IP= immunoprecipitation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Gene Expression, Stable Transfection, Immunofluorescence, Staining, Fractionation, Histone Deacetylase Assay, Expressing, Control, Immunoprecipitation

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Mechanisms of KSR1-regulated p53 transcriptional activity. ( A ) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 overexpression followed by etoposide treatment. MCF7 cells were transiently transfected with pCMV6 (vector) or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with various concentrations of etoposide (20, 40, 80 μ M , 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. ( B ) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 silencing followed by a titration of etoposide treatment. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 72 h followed by etoposide treatment (20, 40, 80 μ M , 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. ( C ) Effect of KSR1 on p53 acetylation is through DBC1. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 in concordance with siCT or siDBC1 for 72 h followed by etoposide treatment (40 μ M , 3 h). Acetylated p53, DBC1 and KSR1 protein levels were assessed by immunoblotting with specific antibodies as indicated. ( D ) Effect of KSR1 on DBC1 phosphorylation is dependent on its intact kinase domain. MCF7 cells were transiently transfected with vector, wild-type KSR1 or mutant KSR1 (R502M) plasmids for 24 h followed by etoposide treatment (40 μ M , 3 h). DBC1 phosphorylation was measured by immunoblotting with specific antibody. ( E ) Interaction of DBC1 and SIRT1 after KSR1 overexpression with etoposide treatment by immunoprecipitation (IP). MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with etoposide (40 μ M , 3 h). The interactions between SIRT1 and DBC1 were detected by IP of SIRT1 or DBC1 followed by immunoblotting with DBC1 and SIRT1 antibodies respectively. Blots shown are representatives of at least three independent experiments. Quantification of blots was analysed by ImageJ software (NIH, Bethesda, MD, USA). ( F ) Schematic model illustrating the role of KSR1 on p53 transcriptional activity in breast cancer cells with (i) basal or (ii) up-regulated levels of KSR1. Abbreviation: IgG= immunoglobulin G.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Phospho-proteomics, Over Expression, Transfection, Plasmid Preparation, Western Blot, Titration, Control, Mutagenesis, Immunoprecipitation, Software

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 silencing on breast cancer cell proliferation in vitro . SRB assays of MCF7, ZR75-1, SKBR3 and MDA231 cells after transfection with 20 n M of either siKSR1 or ‘non-targeting' siRNA (control siRNA) or vehicle (Hiperfect) for 6 days. Error bars represent s.d. of three experiements each in quintuplicates (* P <0.05, compared with control siRNA at day 6; Student's t- test).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: In Vitro, Transfection, Control

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: KSR1 expression is altered in breast cancer tissues. Oncomine analysis was performed to examine KSR1 expression in breast normal and cancer tissues using online TCGA microarray data ( www.oncomine.org ).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Expressing, Microarray

Journal: Nutrition & Diabetes

Article Title: Human mediastinal adipose tissue displays certain characteristics of brown fat

doi: 10.1038/nutd.2013.6

Figure Lengend Snippet: Comparison of gene expression in human subcutaneous and mediastinal adipose tissue. Real-time PCR validation of genes selected from the microarray analysis. Each dot represents one individual ( n =23). Box plots represent median (thick black lines), first and third quartiles (outlined boxes), the lowest data point still within 1.5 times the interquartile range from the first quartile (lower whiskers) and the highest data point still within 1.5 times the interquartile range from the third quartile (upper whiskers) of the expression levels of UCP1 , PPARGC1A , CIDEA , PRDM16 , S HOX2 and HOXC8 in subcutaneous and mediastinal adipose tissue. Gene expression was normalized to reference gene PPIA . P -values were calculated according to Wilcoxon paired-sample test.

Article Snippet: RNA samples from a separate group of 23 patients (see description in ) were used for complementary DNA synthesis with SuperScript III (Invitrogen) and analysed with TaqMan gene expression assays ( PPIA : Hs99999904_m1, TBP : Hs00427620_m1, UCP1 : Hs00222453_m1, PRDM16 : Hs00223161_m1, COBL: Hs00391205_m1, CIDEA : Hs00154455_m1, PPARGC1A : Hs00222453_m1, SHOX2 : Hs00243203_m1 and HOXC8 : Hs00224073_m1; Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Gene Expression, Real-time Polymerase Chain Reaction, Biomarker Discovery, Microarray, Expressing

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) Scatter plot shows an upregulation of the PP2A-A subunit in the tumor samples (p=0.0144). A Mann-Whitney U test was used for comparison between the normal and SCLC samples. (B) IHC for PP2A was conducted on TMA tissue sections and images were captured at 4x or 20x using a 3D-Histech PANNORAMIC SCAN whole slide scanner (3D-Histech, Budapest, Hungary). PP2A subunit A positively immunostained the cytoplasm and nucleus of normal lung and tumor tissue but was highly upregulated in tumor tissue. TMAs were scored in normal (n=24) and tumor (n=79) cores on a scale from 0 (no staining/no protein expression) to 3+ (strong staining/high protein expression). (C) Summary bar graph of the average PP2A subunit staining. IHC staining intensity of normal and tumor cores. There was a statistically significant difference between normal and tumor tissue (***, p<0.001). Student’s t test was used for comparison between the normal and SCLC samples. (D) In order to compare the expression of PP2A subunits A and C, cell lysates from seven SCLC cell lines and HBEC 3KT (non-malignant cell line) were subjected to western blotting (n=3 biological replicates). (E) PP2A activity was determined using a serine/threonine phosphatase activity assay (Millipore) after 24 h exposure to cantharidin (10 µM) and LB100 (5 µM) (n=3 biological replicates). ***, p<0.001, results were analyzed by ANOVA with Tukey post-test. (F) The inset showed reduction of PP2A subunit Aα in H524 cells as well as inhibition of cell proliferation due to PP2A subunit Aα knockdown (n=3 biological replicates). p<0.05, Student’s t test was used for comparison between the groups. LB100 alone or in combination with carboplatin inhibited proliferation and colony formation in SCLC cells. The Cell Counting Kit-8 assay detected cell H524 and H69 cell viability. (n=3 biological replicates). (G, H) Cells were treated with LB100, carboplatin and etoposide, as a single treatment or in combination, at constant ratio. The combination index (CI) was calculated using Chou-Talalay method to find synergism between LB100 with carboplatin and etoposide (CompuSyn software: www.combosyn.com). **, p<0.01, ANOVA with Tukey post-test was used for comparison between LB100, LB100/carboplatin and LB100/etoposide. Colony formation assays were used to count the ability of H524 (I) and H69 (J) cells to form colonies. Drug concentrations are listed for two assays with H524 and H69 respectively: LB100 (2.5 µM; 20 µM), carboplatin (4 µM; 20 µM), etoposide (3 µM; 30 µM), LB100/carboplatin (2.5&4 µM; 20&20 µM) and LB100/etoposide (2.5&3 µM; 20&30 µM). Representative images of colonies at 4x are shown under the graph (n=2). *p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Results were analyzed by ANOVA with Tukey post-test.

Article Snippet: LB100 activity alone and with carboplatin against H69 cells subcutaneous mouse xenograft. ( E ) Chemical structure of LB100 ( https://www.selleckchem.com/products/lb-100.html ). (F) Tumor size was measured.

Techniques: MANN-WHITNEY, Comparison, Staining, Expressing, Immunohistochemistry, Western Blot, Activity Assay, Phosphatase Assay, Inhibition, Knockdown, Cell Counting, Software

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) H524 cells were treated with LB100 (2.5 µM), carboplatin (4 µM), or a combination, and ATP production rate was measured using the Agilent Seahorse XF Real Time ATP rate assay. mitoATP (mitochondrial) and glycoATP (glycolityc) rates were evaluated in H524 cells without and with drug treatments. All drug treatments significantly reduced mitoATP (top, blue) and glycoATP (bottom, red) production rates. (B) Energetic map of H524 cells. After LB100 and drug combination, cells became less glycolytic. (C to E) The Agilent Seahorse XF pH sensor probe measures changes in the concentration of free protons, which corresponds to Extracellular Acidification Rate (ECAR). Real Time ATP rate assay includes an improved metric, Proton Efflux Rate (PER), which detects extracellular acidification from all sources. LB100 drastically reduced PER under basal conditions and after two injections of specific inhibitors of oxidative phosphorylation oligomycin (1.5 µM) and antimycin (0.5 µM)/rotenone (0.5 µM). (F) H69 cells were treated with LB100 (10 µM), carboplatin (10 µM), or a combination with LB100/carboplatin. ATP level in cells was measured using the Agilent Seahorse XF Real Time ATP rate assay. LB100, carboplatin and combination significantly reduced mitoATP. (G) Energetic map of H69 cells. (H to J). H69 cellular Proton Efflux Rate after LB100 treatment from glycolysis of basal and olygomycin and antimycin/rotenone injections. *p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Results were analyzed by ANOVA with Tukey post-test (n=2, six technical replicates).

Article Snippet: LB100 activity alone and with carboplatin against H69 cells subcutaneous mouse xenograft. ( E ) Chemical structure of LB100 ( https://www.selleckchem.com/products/lb-100.html ). (F) Tumor size was measured.

Techniques: Concentration Assay, Phospho-proteomics

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) Morphology of a single spheroid of H446 cells on days one and nine. Spheroids grow continuously and H&E staining is represented. (B) Spheroid’s growth in response to LB100 treatment was recorded with IncuCyte Live-Cell Analysis System. (C) Cytotoxicity effect of LB100 was recorded with IncuCyte Live-Cell Analysis System in the presence of LB100 and IncuCyte Cytotox reagent in green fluorescence. 10 µM of LB 100 significantly affected growth and viability of the cells (n=2, six technical replicates). ***, p<0.001. Student’s t test was used for comparison between control and LB100 treatment. Effect of LB100, carboplatin, etoposide, and drug combination on H446 spheroid morphology and growth (D) Representative images of H&E-stained H446 spheroids with LB100, carboplatin, etoposide, and combination treatment. Scale bar 500 µm. The inserts are enlarged images of spheroids. Scale bar 200 µm. (E, F). Effect of LB100 and carboplatin alone or in combination was monitored using IncuCyte Live Cell system for 70 h. Maximal significant inhibitory effect of LB100, carboplatin or drug combination on spheroid’s size was observed at time point 70 hours. LB100/carboplatin significantly inhibited spheroid’s growth compared to control. **, p<0.01. Results were analyzed with Student’s t test. (G, H) Effect of LB100 and etoposide alone or in combination was monitored using IncuCyte Live Cell system for 72 h. LB100/etoposide significantly inhibited spheroid’s growth compared to control. ***, p<0.001. Results were analyzed with Student’s t test. Maximal significant inhibitory effect of LB100, carboplatin or drug combination on spheroid’s size was observed at time point 70 and 72 h (n=3 biological replicates). * p<0.05; ** p<0.01 results were analyzed by ANOVA with Tukey post-test.

Article Snippet: LB100 activity alone and with carboplatin against H69 cells subcutaneous mouse xenograft. ( E ) Chemical structure of LB100 ( https://www.selleckchem.com/products/lb-100.html ). (F) Tumor size was measured.

Techniques: Staining, Cell Analysis, Fluorescence, Comparison, Control

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A, B) Graphical representation of H524/H69 cell ability to disrupt a confluent HUVEC monolayer using an electrical substrate-impedance sensing system. Arrows indicate time point when cells were added. Inserts show mean values and SD for each group after 20 h of drug treatment. After treatment, cell viability was counted using an Auto T4 Cell Counter (Nexcelom Cellometer). Cell viability was 90–95% for drug-treated groups (n=2). p< 0.001 (***) for control (untreated cells) vs. drug combination (LB100/carboplatin) results were analyzed by ANOVA with Tukey post-test. Whole cell Pt accumulation. Graphical representation of LB100 effect on platinum uptake by SCLC cells. Cells were pretreated with LB100 (H524 – 5µM; H69 – 20µM) overnight, then treated with carboplatin for one or four hours (H524 – 10µM; H69 – 20µM). Whole cell pellet was used for platinum (Pt) measurement. Values are normalized to total protein concentration. (C, D) Panels show mean values and SD of Pt accumulation for each group. Drug combination significantly increased Pt concentrations in H524 and H69 cells. Pt concentrations in control and LB100 samples were below detection limit (n=3, technical replicates). p< 0.001 results were analyzed by ANOVA with Tukey post-test. Effect of LB100 on PP2A expression and apoptosis regulatory proteins in H524 and H69 cells. Cells were treated with indicated concentrations of LB100, carboplatin and combination for 72 h. (E) Representative western blot (WB) panels of the expression of PP2A subunits in H524 and H69 cells. Densitometry analysis shows quantification of PP2A A level in H524 and H69 cells (n=3 biological replicates). (F) Protein phosphorylation of γ-H2AX, caspase 3 and PARP1 cleavage activity was analyzed by WB in H524 and H69 cells after drug treatments. Representative WB panels showed significant increase in γ-H2AX phosphorylation and enhancement of caspase 3 and PARP 1 cleavage activity in H524 and H69 cells after treatment. Pan-actin was used as loading control (n=3 biological replicates).

Article Snippet: LB100 activity alone and with carboplatin against H69 cells subcutaneous mouse xenograft. ( E ) Chemical structure of LB100 ( https://www.selleckchem.com/products/lb-100.html ). (F) Tumor size was measured.

Techniques: Control, Protein Concentration, Expressing, Western Blot, Phospho-proteomics, Activity Assay

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) Significant changes were observed for signal transduction and metabolic pathways. (B) MicroArray analysis showed that overnight treatment with 20 µM treatment with LB100 inhibited utilization of carbon substrate sources. Table includes 10 carbon sources affected by LB100 (n=3). (C) LB100 significantly inhibited two carbon substrates utilization by H69 cells. P < 0.001 (***) for control (untreated cells) vs. LB100. Results were analyzed by ANOVA with Tukey post-test. (D) Amplex Red Glucose/Oxidase assay kit was used to measure glucose level in cell culture media. Glucose level was significantly higher in cell culture medium from cells treated with LB100 (20 µM). Glucose concentration detected in initial medium and counted as 100%. Subtracting final medium level of glucose from initial glucose medium concentration yielded % glucose in the medium with cells. Level of glucose dropped in control with cells and in LB100 treated groups (p < 0.0001 (****). Results were analyzed by ANOVA with Tukey post-test, (n=3 biological replicates). Effect of LB100 on MET phosphorylation. (E) H524 and H69 cells were treated overnight with LB100 (H524 – 5 µM and H69 – 20 µM) following by stimulation with 100 ng/ml HGF in 10 min. Cells were collected and lysed for WB analysis with pMET and total MET antibody. Pan-actin was used as loading control (n=3 biological replicates). (F) H524 cell lysates (control, LB100, carboplatin and combination (LB100/carboplatin) were analyzed by western blots to check phosphorylation status of MET at Ser985 and Tyr1234/1235. Actin was used as a loading control (n=3 biological replicates).

Article Snippet: LB100 activity alone and with carboplatin against H69 cells subcutaneous mouse xenograft. ( E ) Chemical structure of LB100 ( https://www.selleckchem.com/products/lb-100.html ). (F) Tumor size was measured.

Techniques: Transduction, Microarray, Control, Glucose Oxidase Assay, Cell Culture, Concentration Assay, Phospho-proteomics, Western Blot

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) At time point 0, single spheroids in 96 well plate were treated with LB100, atezolizumab and T cells. Column bars present mean values of spheroids at 0h. (B) Measurement of H446 spheroidal cell distribution after 48 h treatment with LB100 and atezolizumab in the presence of T cells. LB100/T cells (**p < 0.01), Atezo/T cells (***p < 0.001) and LB100/Atezo/ T cells (***p < 0.001). Results were analyzed by ANOVA with Tukey post-test (n=2; 6 technical replicates). (C) Sequential images of the same H446 spheroids in control and treated groups. Scale bar 400 µm (D) H&E and immunohistochemical staining (IHC) with CD3 antibody of H446 spheroids after 48h of treatments. Scale bar 50 µm. Before treatment, 5x103 cells were seeded in round bottom 96 well plate and grown for 3 days. LB100 activity alone and with carboplatin against H69 cells subcutaneous mouse xenograft. (E) Chemical structure of LB100 (https://www.selleckchem.com/products/lb-100.html). (F) Tumor size was measured. Inhibition of tumor growth after LB100 (*p < 0.05), carboplatin (***p < 0.001) and their combination (***p < 0.001) were delivered via i.p. injections. P values show significant differences compared with vehicle group. (G) Tumor images from vehicle and drug-treated groups. (H) Columns show total platinum (Pt) concentration in mouse tumors with carboplatin and LB100/carboplatin treatments (n=3 as technical replicates) Pt mass was normalized to tumor total mass. Statistical analysis was performed using an ANOVA with Tukey post-test (*p < 0.05), carboplatin (**p < 0.01).

Article Snippet: LB100 activity alone and with carboplatin against H69 cells subcutaneous mouse xenograft. ( E ) Chemical structure of LB100 ( https://www.selleckchem.com/products/lb-100.html ). (F) Tumor size was measured.

Techniques: Control, Immunohistochemical staining, Staining, Activity Assay, Inhibition, Concentration Assay

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Increases Syk Protein Levels in Hoxa9-Driven Leukemia (A) Kaplan-Meier survival curves of mice transplanted with either H- or H/M-transformed myeloid progenitor cells (n = 11). The p value is from a Mantel-Cox test. (B) Volcano plot relating q values for differential protein expression to average normalized SILAC ratios from six biological replicates. Blue (higher expression in H cells) and orange (higher expression in H/M cells) dots indicate significantly regulated proteins (q < 0.01). (C) Heatmap of SILAC ratios for significantly differentially expressed proteins in H and H/M cells across the six biological replicates. (D) Syk protein expression in H and H/M cells by immunoblotting. Actin was used as loading control for relative protein quantification. (E) Relative Syk mRNA expression as measured by qPCR, normalized to GAPDH expression (mean ± SD, n = 3); ns, not significant (two-sided unpaired t test). (F and G) Immunohistochemical staining of HOXA9, MEIS1, and SYK in bone marrow biopsies from patients with AML. SYK expression levels were analyzed in 21 AML cases with high HOXA9 expression (F) and 28 cases with high HOXA9/MEIS1 expression (G). Proportions of SYK expression levels as determined by two independent pathologists using a three-stage staining score are shown. See also Figure S1 , , and .

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Transformation Assay, Expressing, Multiplex sample analysis, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Syk Is a Direct Target of miR-146a (A) Schematic workflow of the miRNA expression analysis in H- and H/M-transformed myeloid progenitors. (B) Volcano plot relating q values for differential miRNA expression between H and H/M cells to average miRNA expression fold-changes from three biological replicates. Blue (higher expression in H cells) and orange (higher expression in H/M cells) dots indicate significantly regulated miRNAs (q < 0.01). (C and D) Relative mmu-miR-146a expression (C) and pri-miR-146a expression (D) in H/M versus H cells, measured by qPCR and normalized to sno202 and GAPDH expression, respectively (mean ± SD, n = 3). The p values are from a two-sided unpaired t test. (E) Luciferase assay validating binding of miR-146a to the predicted target sites within the 3′ UTR of Syk (mean ± SD, n = 4); WT, predicted miR-146a target sequence; MUT, mutated version thereof. The p values are from a two-sided unpaired t test. ns, not significant. (F) Luciferase assay validating binding of miR-146a to the full-length Syk 3′ UTR (mean ± SD, n = 4). The p value is from a two-sided unpaired t test. (G) Left, secondary structure of mmu-miR-146 as predicted by RNAfold ( Lorenz et al., 2011 ). The CRISPR/Cas9 cleavage site is indicated. Right, relative expression of miR-146a, measured by qPCR and normalized to sno202 expression, in H cells transduced with either a lentiviral non-specific (nsp) control CRISPR or a CRISPR targeting miR-146 (ΔmiR-146) (mean ± SD, n = 3). The p value is from a two-sided unpaired t test. (H) Corresponding Syk protein expression by immunoblotting. Actin was used as loading control for relative protein quantification. (I) Cell-proliferation curves for H cells transduced with either a lentiviral non-specific (nsp) control CRISPR or a CRISPR targeting miR-146 (ΔmiR-146) (mean ± SD, n = 3). (J) Kaplan-Meier survival curves of mice transplanted with H or H/M cells transduced with a lentiviral non-specific (nsp) control CRISPR, or with H cells transduced with a CRISPR targeting miR-146 (ΔmiR-146) (n = 7). The p value is from a Mantel-Cox test. See also Figure S3 .

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Expressing, Transformation Assay, Luciferase, Binding Assay, Sequencing, CRISPR, Transduction, Control, Western Blot

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Downregulates miR-146a through PU.1 (A) Fold enrichment of PU.1 binding over IgG control as measured by ChIP-qPCR in H and H/M cells (mean ± SD, n = 3). The miR-146a −10 kb region spans the transcription start site of the miR-146a host gene; ns, not significant. (B) PU.1 protein expression in H and H/M cells by immunoblotting. Histone H3 was used as loading control for relative protein quantification. (C) Relative PU.1 mRNA expression in H versus H/M cells measured by qPCR and normalized to GAPDH expression (mean ± SD, n = 3). (D and E) Immunohistochemical staining of PU.1 in bone marrow biopsies from patients with AML. PU.1 expression levels were analyzed in 21 AML cases with high HOXA9 expression (D) and 28 cases with high HOXA9/MEIS1 expression (E). Proportions of PU.1 expression levels as determined by two independent pathologists using a three-stage staining score are shown. (F) PU.1 and SYK protein expression by immunoblotting in H cells transfected with either a control shRNA (nsp) or an shRNA targeting PU.1 (KD). Tubulin was used as loading control for relative protein quantification. (G) mmu-miR-146a and pri-miR-146a expression as measured by qPCR after PU.1 knockdown (KD) relative to control shRNA (nsp) (mean ± SD, n = 4). The p values are from a two-sided unpaired t test. See also Figure S4 and .

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Binding Assay, Control, ChIP-qPCR, Expressing, Western Blot, Immunohistochemical staining, Staining, Transfection, shRNA, Knockdown

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Sensitizes Hoxa9-Driven Leukemia to Syk Inhibition (A) Syk protein expression in H/M cells transfected with either a control shRNA (GL2) or two shRNAs targeting Syk. Actin was used as loading control for relative protein quantification. (B) Percentage of BFP-positive shRNA-expressing cells relative to BFP-negative shRNA-negative cells at the times indicated (mean ± SD, normalized to day 0, n = 3). (C) Same as (A), before and after 5 days of doxycycline (dox) treatment in vivo. (D) Kaplan-Meier survival curves of mice transplanted with H/M cells and treated with doxycycline for 43 days to express non-specific control and Syk-specific shRNA (n = 8). The p value is from a Mantel-Cox test. (E) Percentage of YFP-positive cells from peripheral blood of mice transplanted with H (left) or H/M (right) cells after treating for 7 days with R788 or placebo. Measurements were taken at the indicated time points. The black line connects median values. (F) Kaplan-Meier survival curves of mice transplanted with either H or H/M cells and treated for 20 days with R788 or placebo (n = 11). The p value is from a Mantel-Cox test. (G) Relative HOXA9 and MEIS1 mRNA expression in MV4-11 and KG1 cell lines, and in patient-derived AML cells as measured by qPCR, normalized to GAPDH expression (mean ± SD, n = 3). (H) (p)SYK expression in the patient-derived AML cells in (G). Actin was used as loading control for relative protein quantification. avg, average. (I) Half maximal inhibitory concentration (IC 50 ) for R406 (left) and PRT062607 (right) in patient-derived AML cells as determined by an Annexin V/7-AAD apoptosis assay. Cells were treated for 24 hr and DMSO was used as a control (n = 3). Representative dose-response curves for AML no. 1 (HOXA9 high, MEIS1 low) and AML no. 5 (HOXA9 high, MEIS1 high) are shown at the top. Ticks correspond to estimated IC 50 values. (J) Relative viability of CD34 + bone marrow cells from healthy donors. Cells were treated with either R406 or PRT062607. Blue lines indicate the IC 50 for both SYK inhibitors in H cells. (K) Kaplan-Meier survival curves of NSG mice transplanted with patient-derived AML cells indicated in (G) and treated for 14 days with R788 or vehicle (n = 6 for AML no. 1 and 5; n = 5 for AML no. 2 and 6). The p values are from a Mantel-Cox test. See also Figure S6 .

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Inhibition, Expressing, Transfection, Control, shRNA, In Vivo, Derivative Assay, Concentration Assay, Apoptosis Assay

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet:

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Recombinant, Blocking Assay, Lysis, SYBR Green Assay, Reporter Assay, Extraction, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Reverse Transcription, TaqMan microRNA Assay, Mass Spectrometry, Microarray, Gene Expression, Retroviral, Negative Control, Plasmid Preparation, Software, Multiplex sample analysis