Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: A. FRAP analysis of cell-to-cell communication. Digital images of fluorescence distribution in a HMEC monolayer at three times during a typical gap-FRAP experiment: prebleach, just after bleaching (0 min) and after fluorescence recovery (8 min). Bars 20 μm. Corresponding fluorescence intensities (% of prebleach value) versus time in tested cells. The fluorescence in one unbleached cell (Ref) was used to correct the artefact loss of fluorescence. Note the fluorescence recovery follows an exponential time course when the bleached cells (circles) are interconnected by open gap-junction channels to unbleached cells (black squares are Ref). The relative permeability of gaps is given by the time constant k . B. Recombinant human HSP27 (rhHSP27) effect on GJIC. Graph represents mean ± SEM of the fluorescence redistribution after photobleaching in coupled HMEC in control (●) or after 60 min (○) with rhHSP27 (5 μg/ml). C. Histogram shows k values measured after the rhHSP27 addition for 0, 30 and 60 min (mean ± SD, n = 8; * P < 0.05 vs control [ t = 0 min]). D. Both rhHSP27 and SW480-conditioned media (-CM; collected after 6 h) increase the GJIC in a TLR3-dependent manner. Cells exposure for 30 min, in the absence or the presence of neutralizing anti-TLR3 antibody (20 μg/ml) (mean ± SD, n = 4; * P < 0.01 vs control). E. LPS (1 μM) blocks GJIC within 60 min. This inhibitory effect was prevented by OxPAPC (30 μg/ml), a TLR4/TLR2 inhibitor (mean ± SD, n = 4; * P < 0.01 vs control).

Article Snippet: Low endotoxin rhHSP27 was purchased from Enzo Life Sciences (Villeurbanne, Fr) and rabbit anti-HSP27 from ABR (AffinityBioReagent, ThermoFisher, Fr).

Techniques: Fluorescence, Permeability, Recombinant

Journal: Oncotarget

Article Title: Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

doi:

Figure Lengend Snippet: A. Immunofluorescence detection of Cx43 in SW480 cells and SW620 cells (Bar 20 μm). The dotted areas are enlarged in the inserts below. Arrows indicated the Cx43 plaques at the plasma membrane in cells. Representative of 5 experiments. B. Western blot of Cx43 in whole cell lysates from SW480 and SW620 cells, exposed or not the HMEC-conditioned media (-CM, collected after 6 h). P0, P1 and P2 denote the three major Cx43 migration bands (Hsc70 as loading control). C. Time-dependent increase in Cx43 phosphorylation induced by the SW480-CM in confluent HMEC. Whole cell lysates in HMEC exposed to SW480-CM (collected after 6 h) for time periods as indicated (Hsc70 as loading control; representative of 3 experiments). D. No change in the phosphorylation state of the endothelial Cx43 was induced by the SW620-CM ( n = 3). E. Serine phosphorylation and Cx43 immuno-precipitate in HMEC exposed to SW480-CM. Note that the Cx43 interaction with the protein 14–3-3 precedes its phosphorylation in serine sites. Data are representative of 3 independent experiments. IP, immunoprecipitation; IB, immunoblot; Input material, total amount of proteins per lane. IgG heavy chain at 55 kDa. F. Neither rhHSP27 nor SW480-CM affect the low amount of CIP75 interacting with Cx43 (representative of 3 experiments).

Article Snippet: Low endotoxin rhHSP27 was purchased from Enzo Life Sciences (Villeurbanne, Fr) and rabbit anti-HSP27 from ABR (AffinityBioReagent, ThermoFisher, Fr).

Techniques: Immunofluorescence, Western Blot, Migration, Immunoprecipitation

Journal: Cancer Research

Article Title: Heat Shock Protein 27 Differentiates Tolerogenic Macrophages That May Support Human Breast Cancer Progression

doi: 10.1158/0008-5472.can-10-1778

Figure Lengend Snippet: Figure 1. Elevated serum Hsp27 levels in breast cancer patients and increased expression and release of Hsp27 in human primary breast tumor cells and cell lines. A, serum samples (n ¼ 32 patients and 26 controls) were tested for Hsp27 by ELISA. B, healthy volunteers’ (normal) primary breast epithelial cells, patients’ breast tumor cells, and normal (HMEC) and breast tumor (MCF-7 and ZR-75-1) cell lines were tested for intracellular expression of Hsp27 by flow cytometry. CD326 (Ep-CAM, an epithelial cell marker) is used for the gating of epithelial cells. Representative data are shown. C, normal primary breast epithelial cells, primary breast tumor cells, and normal and breast cancer cell lines were cultured for 24 hours (5 105 cells/mL), and culture supernates were tested for Hsp27; **, P < 0.01; ***, P < 0.0005; ****, P < 0.0001. D, culture supernates of primary breast tumor cells and cell lines were concentrated and subjected to 4–15% gradient Tris-glycine gel electrophoresis [each containing approximately 10 ng Hsp27 (determined by ELISA) in 30 mL loading volume]. rhHsp27 (20 ng) was loaded as the positive control. Blot was probed with anti-Hsp27 antibody followed by reprobing with anti-phospho-Hsp27 (Ser82) antibody.

Article Snippet: rhHsp27 and phospho-Hsp27 (Ser82) antibody were purchased from Stressgen Laboratories/Enzo Life Science.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Marker, Cell Culture, Nucleic Acid Electrophoresis, Positive Control

Journal: Cancer Research

Article Title: Heat Shock Protein 27 Differentiates Tolerogenic Macrophages That May Support Human Breast Cancer Progression

doi: 10.1158/0008-5472.can-10-1778

Figure Lengend Snippet: Figure 4. Hsp27-differentiated MACs induce anergy in T cells. A, C, and D, monocyte macrophages were cocultured (1:5 ratio) with autologous T cells in the presence of iCD3 for 6 days. T cells were re-isolated, rested for 24 hours, and tested for anergy by restimulation of the T cells (1 105/well) with iCD3þsCD28 in the presence or absence of IL-2 (100 U/mL) for 4 days for assessing proliferation (n ¼ 14; A) and cytokine production (n ¼ 6; B, C; *, P < 0.05; ***, P < 0.0005, compared with adherence alone group; #, P < 0.05; ###, P < 0.0005, compared with M-CSF group; $, P < 0.05, compared with corresponding IL-2–treated group). B, monocytes were cultured in the presence of M-CSF alone or in the presence of ZR-75-1 culture supernate [added to monocyte culture at 20% (v/v) at a final concentration of 100 ng Hsp27/mL, as determined by ELISA]. The tumor culture supernate was also incubated for 1 hour with Hsp27 antibody or control IgG (each 5 mg/mL) before their addition to the monocyte culture. rhHsp27 (250 ng/mL) was also added to the M-CSF group to serve as positive control. Differentiated MACs were then cocultured with autologous T cells, and the proliferation of re-isolated T cells was assessed as described above. Cul Sup, culture supernate.

Article Snippet: rhHsp27 and phospho-Hsp27 (Ser82) antibody were purchased from Stressgen Laboratories/Enzo Life Science.

Techniques: Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control, Positive Control