Journal: Cancers

Article Title: Growth Hormone Upregulates Mediators of Melanoma Drug Efflux and Epithelial-to-Mesenchymal Transition In Vitro and In Vivo

doi: 10.3390/cancers12123640

Figure Lengend Snippet: Subcutaneous B16-F10 mouse melanoma growth in bGH and GHRKO syngeneic mice. ( A ) Subcutaneous B16-F10 tumor growth in male bGH (n = 5) vs. WT mice (n = 6). ( B ) Subcutaneous B16-F10 tumor growth in female bGH (n = 7) and WT mice (n = 6). ( C ) Subcutaneous B16-F10 tumor growth in male GHRKO (n = 6) vs. WT (n = 8) mice. ( D ) Subcutaneous B16-F10 tumor growth in female GHRKO (n = 4) and WT mice (n = 5). Tumor sizes were analyzed by repeated measures (SPSS). ( E ) Weight of subcutaneous B16-F10 tumors from bGH vs. WT mice at dissection. ( F ) Weight of subcutaneous B16-F10 tumors from GHRKO vs. WT mice at dissection. ( G ) GH levels were measured in protein lysates isolated from tumors of bGH and WT mice using ELISA and normalized to total protein concentrations (n = 4). ( H ) Similar GH measurements were performed in protein lysates isolated from tumors of GHRKO and WT mice (males n = 3, females n = 4). ( I ) IGF-1 levels were measured in protein lysates isolated from tumors of bGH and WT mice using ELISA and normalized to total protein concentrations (n = 4). ( J ) Similar IGF-1 measurements were performed in protein lysates isolated from tumors of GHRKO and WT mice (males n = 3, females n = 4). ( K ) Representative images of western blot analysis of phosphorylation (p) and total (t) levels of STAT1, STAT3 and STAT5 in protein lysates isolated from tumors of bGH and WT mice. Densitometry analysis was performed and normalized against β-Actin. The relative expression levels (fold change relative to WT) are labeled under each band. The ratio of phosphorylated vs. total protein levels in tumors from bGH and WT mice are presented in bar graphs (n = 4). ( L ) Representative images of western blot analysis of phosphorylation and total levels of STAT1, STAT3, and STAT5 in protein lysates isolated from tumors of GHRKO and WT mice. (males n = 3, females n = 4). Data are presented as mean ± standard errors (*, p < 0.05, unpaired student’s t -test).

Article Snippet: Efficiency of GHR RNA knockdown was verified by real-time RT-qPCR and confirmed via western blotting for GH-induced tyrosine phosphorylated STAT5 (CST #9351).

Techniques: Dissection, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Labeling

Journal: Journal of Cancer

Article Title: CSF2 Overexpression Is Associated with STAT5 Phosphorylation and Poor Prognosis in Patients with Urothelial Carcinoma

doi: 10.7150/jca.14281

Figure Lengend Snippet: Summary of two significantly and differentially expressed genes related to positive regulation of tyrosine phosphorylation of pSTAT5 in the published transcriptome of UBUC (GSE32894).

Article Snippet: The endogenous peroxidase was quenched by saline for 15 minutes and then incubated with primary monoclonal antibodies against CSF2 (1:100, Cat. No. ab77768, rabbit polyclonal, abcam, Cambridge, MA) and phosphor-STAT5 (pSTAT5, Tyr 694/Tyr 699) (1:50, Cat. No. sc-11761, goat polyclonal, Santa Cruz, CA) for an hour.

Techniques: Phospho-proteomics, Cell Differentiation, Expressing, Activity Assay, Binding Assay, Transduction, Migration, Protein Binding

Journal: Nature Communications

Article Title: HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis

doi: 10.1038/s41467-018-03627-9

Figure Lengend Snippet: HSP27 affects de-phosphorylation of STAT5. a The phosphorylation of STAT5 by JAK2 is HSP27 independent. An in vitro kinase assay was performed using the recombinant protein JAK2 and STAT5 (produced in reticulocyte lysate) in the presence or absence of HSP27 (produced in reticulocyte lysate). The level of STAT5 phosphorylation, and the amount of JAK2 and STAT5 proteins were determined by western blot ( n =3 independent experiments). Uncropped blots presented in Supplementary Fig. . b HSP27 impairs the in vitro de-phosphorylation of STAT5 induced by SHP2. Following an in vitro kinase assay using the recombinant protein JAK2 and STAT5 (same batch produced in reticulocyte lysate), samples were incubated or not with the recombinant phosphatase, SHP2, in the presence or absence of HSP27 (produced in reticulocyte lysate). The level of phosphorylated STAT5 was detected by western blot using an antibody specific for Ph-STAT5. The expressions of JAK2, SHP2 and HSP27 are also shown. Bar graphs show normalized ratios of Ph-STAT5_SHP2/Ph-STAT5 bands quantified from the western blots ( n =4 independent experiments), error bars represent the ±s.d. P values were calculated using the Student’s t test. ** P < .01. Uncropped blots presented in Supplementary Fig. . c HEL92.1.7 cells were transfected with HSP27 siRNA or CTL for 48 h. Then, cells were treated or not with the JAK2 inhibitor, AG490 (100 μM), at indicated times and lysed in Laemmli Buffer. Level of STAT5 phosphorylation was determined by western blot. Actin was used as the loading control ( n =3 independent experiments). Uncropped blots presented in Supplementary Fig.

Article Snippet: After boiling, the immunoprecipitates were resolved in 10% SDS-PAGE and immunoblots were performed using an anti-rabbit STAT5, JAK2 (clone D2O6Y, #9363 and clone D2E12, #3230, Cell Signalling technologies) or HSP27 antibody (polyclonal, SPA-803, Enzo Life Sciences).

Techniques: De-Phosphorylation Assay, Phospho-proteomics, In Vitro, Kinase Assay, Recombinant, Produced, Western Blot, Incubation, Transfection, Control

Journal: Nature Communications

Article Title: HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis

doi: 10.1038/s41467-018-03627-9

Figure Lengend Snippet: HSP27 interacts with JAK2/STAT5. a The binding of recombinant JAK2 and STAT5a/b at 125 nM to immobilized biotinylated HSP27 was determined by biolayer interferometry Crystallin alpha B and the CBP RING domain serves as a positive control and negative control, respectively (Supplementary Fig. ) ( n =3 independent experiments). b Immunoprecipitation from HEL92.1.7 cell extracts of endogenous HSP27 was followed by immunodetection of endogenous STAT5 and JAK2. CTL: unstimulated cells starved for 16 h; +SVF: Cells starved for 16 h and then stimulated with SVF (10%) for 30 min. Inputs: proteins in total cell lysates. IP IgG: immunoprecipitation with a non-relevant antibody (IgG mouse) ( n =2 independent experiments). Uncropped blots presented in Supplementary Fig. . c Immunoprecipitation of endogenous STAT5 was followed by immunodetection of endogenous HSP27 and JAK2. Inputs: proteins in total cell lysates. IP IgG: immunoprecipitation with a non-relevant antibody (IgG mouse) ( n =2 independent experiments). Uncropped blots presented in Supplementary Fig. . d Immunofluorescence analysis of the endogenous interaction (red foci) of JAK2 (upper panel) or STAT5 (middle panel) with HSP27 visualized in situ by PLA in HEL92.1.7 cells transfected or not (NT) with a HSP27 siRNA. Nuclei are stained with DAPI. Images were taken randomly and obtained using an Axio Imager 2 at ×40 magnification and analysed using ICY software. Cells were segmented manually, and the number of interaction foci in each cell was counted using the spot detector plugin. Right panel, graphs represent quantification of the interaction of JAK2 or STAT5 with HSP27 visualized in situ by PLA. Each data point corresponds to an analysed cell, placed according to the number of detected interaction foci (lower panel). Immunofluorescence analysis of the endogenous interaction (red foci) of JAK2 with STAT5 visualized in situ by PLA in HEL92.1.7 cells transfected with a scramble (CTL) or a HSP27 siRNA (siHSP27). Nuclei are stained with DAPI. Images were obtained using an Axio Imager 2 at ×63 magnification and analysed using ICY software as in upper panel. Right panel, graphs represent quantification of the interaction of endogenous JAK2 with STAT5 visualized by PLA. P values were calculated using the Mann–Whitney test. **** P < .0001. Scale bar 10 µm

Article Snippet: After boiling, the immunoprecipitates were resolved in 10% SDS-PAGE and immunoblots were performed using an anti-rabbit STAT5, JAK2 (clone D2O6Y, #9363 and clone D2E12, #3230, Cell Signalling technologies) or HSP27 antibody (polyclonal, SPA-803, Enzo Life Sciences).

Techniques: Binding Assay, Recombinant, Positive Control, Negative Control, Immunoprecipitation, Immunodetection, Immunofluorescence, In Situ, Transfection, Staining, Software, MANN-WHITNEY