Journal: PLoS ONE

Article Title: Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

doi: 10.1371/journal.pone.0003961

Figure Lengend Snippet: Full-length wild-type FGFR3 or its activating mutants N540K, G380R, R248C, Y373C, K650M and K650E were expressed in CHO cells, activated by brief FGF2 treatment and purified by immunoprecipitation as described in . Immunocomplexes were subjected to kinase assay with recombinant STAT1 as a substrate. Cells transfected with GFP vector serve as negative control for immunoprecipitation. Samples utilizing recombinant FGFR3 tyrosine kinase domain (TK) or those with omitted ATP serve as positive or negative control for kinase assay, respectively. Note that only K650M and K650E-FGFR3 mutants cause STAT1 phosphorylation, as evidenced by western blotting with antibody recognizing STAT1 only when phosphorylated at Y701 (P-Y701-STAT1). FGFR3 and STAT1 western blottings serve as controls for kinase or substrate quantity. Note the appearance of both immature and mature (glycosylated) FGFR3 forms expressed by CHO cells.

Article Snippet: Briefly, kinase reactions were performed for 30 minutes at 30°C in 50 μl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 μM Na 3 VO 4 ) supplemented with 2.5 μg polyethylene glycol, 10 μM ATP and 300 ng of recombinant human STAT1 (Active Motif, Carlsbad, CA) as a substrate.

Techniques: Purification, Immunoprecipitation, Kinase Assay, Recombinant, Transfection, Plasmid Preparation, Negative Control, Phospho-proteomics, Western Blot

Journal: PLoS ONE

Article Title: Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

doi: 10.1371/journal.pone.0003961

Figure Lengend Snippet: HeLa, 293T and RCS cells, transfected with vectors expressing wild-type FGFR, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M and K650E), and kinase-inactive mutant K508M were grown for 48 hours, treated with FGF2 for 15 minutes and analyzed for given molecules by western blotting. The left and right panels represent independent experiments. Note that only K650M and K650E-FGFR3 cause strong activatory STAT1(Y701) phosphorylation whereas the other mutants cause weak (HeLa) or no activation (293T and RCS). This contrasts with ERK1/2 activation, which is induced by all six mutants in 293T and RCS cells (samples without FGF2 treatment). The P-Y701-STAT1 signal in HeLa cells was quantified by densitometry and graphed. Also note the differences in FGFR3 maturation, where HeLa and RCS cells but not 293T cells express mostly immature forms (lower arrow) of K650M and K650E-FGFR3. STAT1, ERK1/2 and ACTIN western blottings serve as loading controls. Cells transfected by GFP vector serve as negative control for transfection.

Article Snippet: Briefly, kinase reactions were performed for 30 minutes at 30°C in 50 μl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 μM Na 3 VO 4 ) supplemented with 2.5 μg polyethylene glycol, 10 μM ATP and 300 ng of recombinant human STAT1 (Active Motif, Carlsbad, CA) as a substrate.

Techniques: Transfection, Expressing, Mutagenesis, Western Blot, Phospho-proteomics, Activation Assay, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

doi: 10.1371/journal.pone.0003961

Figure Lengend Snippet: (A) RCS chondrocytes transfected with vectors expressing wild-type FGFR3, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650E and K650M), and kinase-inactive mutant K508M were grown for 48 hours and analyzed for the indicated molecules by western blotting. Note differential STAT1 and ERK activation by the activating FGFR3 mutants. Cells transfected with empty plasmid (pcDNA3) serve as negative control for transfection. (B) RCS chondrocytes were transfected as described in (A), grown for 72 hours and counted. Note the inhibition of RCS growth by wild-type FGFR3 as well as the activating mutants, as compared to cells transfected either by kinase-inactive K508M-FGFR3 or an empty plasmid. The data represent an average from four individually transfected wells with indicated standard deviation. The cell count difference compared between cells transfected with wild-type FGFR3 and empty plasmid, as well as the cell count difference between cells transfected with wild-type FGFR3 and N540K, G380R, R248C, Y373C, K650M and K650E mutants, were statistically significant (Student's t -test, p <0.01). (C) The experiment shown on (B) was repeated five times to eliminate the variance associated with differential transfection efficiency. The differences in percentages of growth compared between cells transfected with wild-type FGFR3 and empty plasmid, and between cells transfected with wild-type FGFR3 and N540K, G380R, R248C, Y373C, K650M and K650E mutants, were statistically significant (Student's t -test, p <0.01).

Article Snippet: Briefly, kinase reactions were performed for 30 minutes at 30°C in 50 μl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 μM Na 3 VO 4 ) supplemented with 2.5 μg polyethylene glycol, 10 μM ATP and 300 ng of recombinant human STAT1 (Active Motif, Carlsbad, CA) as a substrate.

Techniques: Transfection, Expressing, Mutagenesis, Western Blot, Activation Assay, Plasmid Preparation, Negative Control, Inhibition, Standard Deviation, Cell Counting

Journal: PLoS ONE

Article Title: Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

doi: 10.1371/journal.pone.0003961

Figure Lengend Snippet: (A) The N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants used in this study all cause FGFR3-skeletal dysplasias and signal through ERK MAP kinase in contrast to STAT1, that is activated mostly by the K650M and K650E-FGFR3 mutants. (B) According to the study compiling the clinical data of 591 patients suffering from FGFR3-related skeletal dysplasia , the STAT1-activating K650M and K650E account for as little as 4.9% of cases. It is therefore unlikely that activation of STAT1 plays a central role in FGFR3-related skeletal dysplasias as currently believed, but rather represents a signaling feature unique to small subset of patients carrying the K650M and K650E mutations.

Article Snippet: Briefly, kinase reactions were performed for 30 minutes at 30°C in 50 μl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 μM Na 3 VO 4 ) supplemented with 2.5 μg polyethylene glycol, 10 μM ATP and 300 ng of recombinant human STAT1 (Active Motif, Carlsbad, CA) as a substrate.

Techniques: Activation Assay

Journal: British Journal of Cancer

Article Title: Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

doi: 10.1038/bjc.2015.398

Figure Lengend Snippet: Upregulation of IFN -inducible genes and STAT1 phosphorylation in residual tumour foci after AC treatment. ( A ) In vivo response of HBCx-17 to AC (2/100 mg kg −1 ) (mean tumour volume±s.d.) n =11 per group. ( B ) H&E analysis and in situ hybridisation of Alu probes of one residual tumour foci after chemotherapy response (× 25). ( C ) Type I and II interferon distribution by ISG Database. ( D ) qRT–PCR analysis of STAT1 and eight other IFN -inducible genes in untreated samples, residual and regrowing tumours. HBCx-6, HBCx-8 HBCx-10 and HBCx-17 PDX were treated with AC, mean±s.d., n =5 per group. Comparative analysis of all samples from the four experiments in different experimental conditions (Rel: relapse; Rem: remission; Untr: untreated); fold-change refers to mean intensity value ratio between the two experimental condition indicated. P -value was calculated by t -test except for results indicated with *= t -test with Welch's correction or **=Mann–Whitney test. ( E ) Western blotting of P-STAT1 Tyr701/Ser727 , STAT1, IF44, LCN2 and OAS1 in three PDX models responding to AC treatment. Tumours were analysed at the indicated phases: untreated (U), residual (N=nodule) and relapse tumour (R=Relapse).

Article Snippet: Lysates were resolved on 4–12% TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred into nitrocellulose membranes (Bio-Rad) and immunoblotted overnight at 4 °C or 1 h at room temperature with the following antibodies: P-STAT1 Tyr701 , CASP-3 (1/750–1000; Cell Signaling Ozyme), Total STAT1 (1/1000; Santa Cruz antibodies, Nanterre, France), P-STAT1 Ser727 , P- γ H2AX Ser139 (1/1000; Merck Millipore), OAS1, IFI44, LCN2, or Actin, as an internal control loading (1/1000; Sigma-Aldrich, St Louis, MO, USA).

Techniques: In Vivo, In Situ, Hybridization, Quantitative RT-PCR, MANN-WHITNEY, Western Blot

Journal: British Journal of Cancer

Article Title: Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

doi: 10.1038/bjc.2015.398

Figure Lengend Snippet: List of interferon-inducible genes differentially expressed ( P <0.05) between HBCx-6, HBCx-8 and HBCx-17 in residual tumour cells (Nodule) and untreated tumours (CTRL)

Article Snippet: Lysates were resolved on 4–12% TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred into nitrocellulose membranes (Bio-Rad) and immunoblotted overnight at 4 °C or 1 h at room temperature with the following antibodies: P-STAT1 Tyr701 , CASP-3 (1/750–1000; Cell Signaling Ozyme), Total STAT1 (1/1000; Santa Cruz antibodies, Nanterre, France), P-STAT1 Ser727 , P- γ H2AX Ser139 (1/1000; Merck Millipore), OAS1, IFI44, LCN2, or Actin, as an internal control loading (1/1000; Sigma-Aldrich, St Louis, MO, USA).

Techniques: Histone Deacetylase Assay

Journal: British Journal of Cancer

Article Title: Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

doi: 10.1038/bjc.2015.398

Figure Lengend Snippet: Analysis of IFN/STAT1 pathway activity in chemo-responder and chemo-resistant PDX at early stage after chemotherapy. ( A ) mRNA expression of a 21-gene IFN/STAT1 signature analysed by qPCR at days 0, 3 and 7 post-AC treatment in 9 responders and 5 resistant models. Each curve represents the expression of one gene. ( B ) In vivo response to AC and irinotecan in the HER2+ HBCx13 model and analysis of IFN-inducible genes ( BST2 , CLDN1 , DDX60 , IFI44 , IFI44L , IFI6 , IFIT1 , IFIT3 , IFITM1 , IRF9 , MX1 , OAS1 , OAS2 , PARP12 , PARP9 , SAMD9 , STAT1 , STAT2 , UBE2L6 , ZNFX1 ) at D7 and D14.

Article Snippet: Lysates were resolved on 4–12% TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred into nitrocellulose membranes (Bio-Rad) and immunoblotted overnight at 4 °C or 1 h at room temperature with the following antibodies: P-STAT1 Tyr701 , CASP-3 (1/750–1000; Cell Signaling Ozyme), Total STAT1 (1/1000; Santa Cruz antibodies, Nanterre, France), P-STAT1 Ser727 , P- γ H2AX Ser139 (1/1000; Merck Millipore), OAS1, IFI44, LCN2, or Actin, as an internal control loading (1/1000; Sigma-Aldrich, St Louis, MO, USA).

Techniques: Activity Assay, Expressing, In Vivo

Journal: British Journal of Cancer

Article Title: Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

doi: 10.1038/bjc.2015.398

Figure Lengend Snippet: Activation of JAK/STAT1 pathway and expression of IFN- γ in the early response to AC. ( A ) Western blotting analysis of total STAT1 or P-STAT1 Tyr701/Ser727 in four responders and four resistant models at D0, D3 and D7 post-AC treatment. ( B ) Human and murine soluble IFN- γ expression determined by cytokine array in HBCx-10 tumours at D3, D7 and D14 after AC and during residual and regrowing phases. ( C ) Effect of RUX alone on HBCx-10 tumour growth. Mean RTV±s.d., n =10. ( D ) P-STAT1 Tyr701 foci detected by IHC in one untreated xenograft and the number of P-STAT1 Tyr701 -positive tumour cells in untreated and RUX-treated tumour samples. * P ⩽0.05 (Student's t -test). ( E ) Kaplan–Meier survival analysis of mice treated with chemotherapy alone and with chemotherapy and RUX ( P <0.0001, log-rank (Mantel–Cox) test). ( F ) Expression of IFN- inducible genes by qPCR in untreated, AC, RUX alone or RUX/AC groups at 8 days (D8) posttreatment.

Article Snippet: Lysates were resolved on 4–12% TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred into nitrocellulose membranes (Bio-Rad) and immunoblotted overnight at 4 °C or 1 h at room temperature with the following antibodies: P-STAT1 Tyr701 , CASP-3 (1/750–1000; Cell Signaling Ozyme), Total STAT1 (1/1000; Santa Cruz antibodies, Nanterre, France), P-STAT1 Ser727 , P- γ H2AX Ser139 (1/1000; Merck Millipore), OAS1, IFI44, LCN2, or Actin, as an internal control loading (1/1000; Sigma-Aldrich, St Louis, MO, USA).

Techniques: Activation Assay, Expressing, Western Blot