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 downregulation impairs myelofibrosis progression in a TPO high murine model. a In vivo strategy of HSP27 inhibition using OGX-427, or a scrambled oligonucleotide control (CTL) injected intraperitoneally 3 times a week at a dose of 10 mg kg −1 in a TPO high murine model of myelofibrosis (MF). Mice were 2–4 months old. b Expression level of HSP27 proteins in the serum of TPO high mice ( n =9) compared with healthy mice (WT, n =10) measured by ELISA assay. P value was calculated using the Mann–Whitney test. * P < .05. Error bars represent ±s.e.m. c Western blot analysis of HSP27 in splenocytes (whole cell lysate) of MF mice treated with OGX-427 or CTL. Actin served as the loading control. Bar graphs show quantification of mean relative amount of the proteins ( n =2 per group). Uncropped blots presented in Supplementary Fig. d Spleen weight was evaluated in mice ( n =9 per group). Outcomes of the two treatments were compared using the Mann–Whitney test. * P < .05. Error bars represent ±s.e.m. e Representative picture of spleen size of MF mice treated with CTL or OGX-427. f Left panel, haematoxylin and eosin-stained spleen sections revealed hyperplasia of the red pulp (R) in MF mice and partial restoration of the white pulp (W) territories in OGX-427-treated mice. Right panel, assessment of the grade of extramedullary haematopoiesis (EMH), from spleen sections in 18 killed mice. Pie chart: Grade A (light pink): diffuse EMH invasion. Grade B (purple colour): diffuse EMH invasion with atrophy of white pulp. Images were obtained using a Nanozoomer scanner (Hamamatsu, France) at ×23 magnification and Calopics software. g Left panel, histology of bone marrow sections after haematoxylin and eosin staining. Right panel, Pie chart representing percentages of mice according to erythrocyte (right upper panel) and megakaryocyte counts (right lower panel) from bone marrow sections of 18 killed mice: very low cell number (dark blue), decreased cell number (azure blue), normal cell number (light blue) and increased cell number (pink). Arrows show erythrocyte foci and megakaryocytes. Images were obtained using a Nanozoomer scanner (Hamamatsu, France) at ×13 magnification and Calopics software

Article Snippet: Precleared cell lysates were subjected to immunoprecipitation for 3 h using an anti-mouse HSP27 antibody (2 µg, SPA-800, Enzo Life Sciences) or anti-STAT5 mouse (4 µg, clone A-9, sc-74442, Santa-Cruz) or a mouse IgG (I5381, Sigma-Aldrich) as a negative control that had been pre-incubated for 1 h at room temperature (RT) with Protein G UltraLink Resin beads (53132, Pierce).

Techniques: In Vivo, Inhibition, Injection, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Western Blot, Staining, Software

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 downregulation impairs myelofibrosis progression in a JAK2V617F murine model. a In vivo strategy of HSP27 inhibition using OGX-427 or a vehicle, injected intraperitoneally 3 times a week at a dose of 10 mg kg −1 in a JAK2V617F murine model of myelofibrosis (MF). Mice were 2 months old. b Spleen weight was evaluated in mice ( n =5 per group). Outcomes of the two treatments were compared using the Mann–Whitney test. * P < .05. Error bars represent ±s.e.m. c Western blot analysis of HSP27 in bone marrow (whole cell lysate) of MF mice treated with OGX-427 or vehicle. Actin served as the loading control. Bar graphs show quantification of mean relative amount of the proteins ( n =3 per group). Uncropped blots presented in Supplementary Fig. . d Percentage of megakaryocytes progenitors (MkPs) present in the bone marrow or spleen, error bars represent ±s.e.m. ( n =5 per group). P values were calculated using the Mann–Whitney test. * P < .05. e Upper panel, Gordon and Sweet-stained bone marrow sections revealed reduced reticulin fibrosis in MF mice and in OGX-427-treated mice. Lower panel, assessment of the fibrosis grade from bone marrow sections of 5 mice per group. Pie chart: Grade 1 (light pink): few thin reticulin fibres. Grade 2 (light red): networks of thin reticulin fibres. Grade 3 (purple): dense network of thick reticulin fibres. Images were obtained using a Nanozoomer scanner (Hamamatsu, France) at ×23 magnification and Calopics software. f Blood cell parameters assessed in mice before and after OGX-427 ( n =5) or vehicle treatment ( n =5) and on the day of killing. WBC white blood cells. Error bars represent ±s.e.m. P values were calculated using the Mann–Whitney test. * P < .05; ** P < .01. *** P < .001

Article Snippet: Precleared cell lysates were subjected to immunoprecipitation for 3 h using an anti-mouse HSP27 antibody (2 µg, SPA-800, Enzo Life Sciences) or anti-STAT5 mouse (4 µg, clone A-9, sc-74442, Santa-Cruz) or a mouse IgG (I5381, Sigma-Aldrich) as a negative control that had been pre-incubated for 1 h at room temperature (RT) with Protein G UltraLink Resin beads (53132, Pierce).

Techniques: In Vivo, Inhibition, Injection, MANN-WHITNEY, Western Blot, Staining, Software

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 proliferation of JAK2V617F leukaemic cell lines. a HEL92.1.7, SET-2 and K562 cells were transfected with HSP27 siRNA, OGX-427 or an oligonucleotide control (CTL). Bars represent cell proliferation percentages relative to non-transfected cells (NT) from 9 independent experiments. P values were calculated using the Mann–Whitney test. ** P < .01. Error bars represent ±s.e.m. b HEL92.1.7, SET-2 and K562 cells were transfected with HSP27 siRNA, OGX-427 or CTL and lysed 48 h later in Laemmli Buffer. Protein expression was determined by western blot and compared to non-transfected cells (NT). Actin was used as the loading control ( n =3 independent experiments). See quantification of the blot in Supplementary Fig. . Uncropped blots presented in Supplementary Fig.

Article Snippet: Precleared cell lysates were subjected to immunoprecipitation for 3 h using an anti-mouse HSP27 antibody (2 µg, SPA-800, Enzo Life Sciences) or anti-STAT5 mouse (4 µg, clone A-9, sc-74442, Santa-Cruz) or a mouse IgG (I5381, Sigma-Aldrich) as a negative control that had been pre-incubated for 1 h at room temperature (RT) with Protein G UltraLink Resin beads (53132, Pierce).

Techniques: Transfection, MANN-WHITNEY, Expressing, Western Blot

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: Precleared cell lysates were subjected to immunoprecipitation for 3 h using an anti-mouse HSP27 antibody (2 µg, SPA-800, Enzo Life Sciences) or anti-STAT5 mouse (4 µg, clone A-9, sc-74442, Santa-Cruz) or a mouse IgG (I5381, Sigma-Aldrich) as a negative control that had been pre-incubated for 1 h at room temperature (RT) with Protein G UltraLink Resin beads (53132, Pierce).

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

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: Precleared cell lysates were subjected to immunoprecipitation for 3 h using an anti-mouse HSP27 antibody (2 µg, SPA-800, Enzo Life Sciences) or anti-STAT5 mouse (4 µg, clone A-9, sc-74442, Santa-Cruz) or a mouse IgG (I5381, Sigma-Aldrich) as a negative control that had been pre-incubated for 1 h at room temperature (RT) with Protein G UltraLink Resin beads (53132, Pierce).

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

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 is overexpressed in patients with MPN-associated MF. a Flow cytometry analysis of HSP27, HSP70 and HSP90 in circulating hematopoietic progenitor CD34 + cells from patients with myelofibrosis (MF) and healthy control donors (HDs) (number of samples analysed; MF: n =11–18; HD: n =10–12). P values were calculated by using the unpaired t test with Welch’s correction. ** P < .01; n.s. not significant. Expression levels of HSP27, HSP70 and HSP90 from MF patients were plotted as median fluorescence intensities. b Analysis of HSP27, HSP70 and HSP90 levels in the serum of patients with MF ( n =24-27) compared with HDs ( n =15) measured by ELISA. P values were calculated using the Mann–Whitney test. **** P < .0001. c Representative histological sections of bone marrow from MF patients. Images were obtained using Cell Observer (Zeiss, France) at two different magnifications (×23, scale bar 20 µm and ×63, scale bar 10 µm) and Axiovision software. Arrows show megakaryocytes

Article Snippet: Precleared cell lysates were subjected to immunoprecipitation for 3 h using an anti-mouse HSP27 antibody (2 µg, SPA-800, Enzo Life Sciences) or anti-STAT5 mouse (4 µg, clone A-9, sc-74442, Santa-Cruz) or a mouse IgG (I5381, Sigma-Aldrich) as a negative control that had been pre-incubated for 1 h at room temperature (RT) with Protein G UltraLink Resin beads (53132, Pierce).

Techniques: Flow Cytometry, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Software

Journal: Journal of cell science

Article Title: Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors.

doi: 10.1242/jcs.091223

Figure Lengend Snippet: Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) (A,B), Rh1 (C,D), Spam (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).

Article Snippet: The following primary antibodies were used: rat anti-Crb2.8 antibody (1:1000) (Richard et al., 2006), rabbit anti-Sdt-PDZ antibody (1:500) (Berger et al., 2007), mouse anti-Sdt-PDZ antibody (B1-8, 1:200) (Bulgakova et al., 2010), rabbit antiDrosophila-PATJ antibody (1:500) (Richard et al., 2006), rat anti-DE-Cad antibody (DCAD2, 1:50, DSHB), mouse anti-Na+/K+-ATPase antibody (a5, alpha subunit, 1:500 concentrate, DSHB), rabbit anti-Rh1 antibody (1:1000) (Satoh et al., 2005), mouse anti-Rh1 (4C5, 1:100, DSHB), mouse anti-Spam antibody (21A6, 1:100, DSHB) (Zelhof et al., 2006), mouse anti-FLAG M2 antibody (1:1000, Sigma), rabbit anti-FLAG antibody (1:200, Sigma, F7425), rabbit anti-Baz antibody (1:400, kindly provided by Andreas Wodarz) (Wodarz et al., 1999), guinea pig anti-Par-6 antibody (1:1000, kindly provided by Andreas Wodarz) (Kim et al., 2009), mouse anti-Arm antibody (N2 7A1, 1:50, DSHB), rabbit anti-PKC f C20 (1:1000, Santa Cruz Biotechnology), rabbit anti-phosphorylated-Ezrin/Radixin/Moesin (1:100, Cell Signalling Technology), and rabbit anti-bH-spec (1:500, kindly provided by Graham Thomas) (Thomas and Kiehart, 1994).

Techniques: Over Expression, Staining, Expressing

Journal: Journal of Biomedical Optics

Article Title: Detection of novel biomarkers for ovarian cancer with an optical nanotechnology detection system enabling label-free diagnostics

doi: 10.1117/1.JBO.17.8.081412

Figure Lengend Snippet: Assessment of potential biomarkers for differential diagnosis of primary versus metastatic ovarian cancer carcinoma. (a) through (c) Calreticulin protein levels were detected in cell supernatants and media controls using quantitative immunoblotting (a) ...

Article Snippet: The following antibodies were used in the present study: mouse anti-fibronectin (MAB1918; RnD Systems, Minneapolis, MN), mouse anti-apolipoprotein A1 (Cat. # 20-783-73037; GenWay Biotech Inc., San Diego, CA); mouse anti-calreticulin (Cat. # SPA-601F; Assay Designs, Enzo Life Sciences Inc., Farmingdale, NY), rabbit anti-collagen type 1 (Ab34710; AbCam, Cambridge, MA), mouse anti-mitogen-activated protein kinase 13 (Cat. # H00005603-M01; Assay Designs, Enzo Life Sciences Inc., Farmingdale, NY) and mouse anti-tissue inhibitor of metalloproteinases 3 (MAB973; RnD Systems, Minneapolis, MN).

Techniques: Western Blot