IGFBP-1, -3, -5 and -6, but not IGFBP-2 and -4, associate with C2 cell surface.

<p>A. C2 cells were incubated for 1 hour at 4°C with or without IGFBP-1 to -6 (5 µg.mL^-1), fixed and incubated with specific primary antibodies and fluorescent secondary antibodies as indicated in Materials and Methods. Staining profiles were analyzed by FACS and mean fluorescence intensities (MFI) quantified. Data presented are representative of three independent experiments. B. C2 cells growing on coverslips were incubated with IGFBP-1 to -6 (5 µg.mL^-1) and then fixed without detergent. Cells were incubated with specific antibodies directed against IGFBP-1 to -6 processed with secondary antibodies stained either with Alexa 488 (IGFBP-1, -3, -4, -5) or Alexa 633 (IGFBP-2, -6). For each incubation condition, upper panel shows confocal fluorescent images and lower panel shows differential interference contrast images. Results are representative of four independent experiments.</p

IGFBPs increase intracellular calcium concentration via pertussis toxin-sensitive and -insensitive signaling pathways in MCF-7 and C2 proliferative cells.

<p>S: sensitive, NS: non sensitive.</p

IGFBP-1 to -6 associate with MCF-7 cell surface.

<p>A. MCF-7 cells were incubated for 1 hour at 4°C with or without IGFBP-1 to -6 (5 µg.mL^-1), fixed and incubated with specific primary antibodies and fluorescent secondary antibodies as indicated in Materials and Methods. Staining profiles were analyzed by FACS and mean fluorescence intensities (MFI) quantified. Data presented are representative of four independent experiments. B. MCF-7 cells growing on coverslips were incubated with IGFBP-1 to -6 (5 µg.mL^-1) and then fixed without detergent. Cells were incubated with specific antibodies directed against IGFBP-1 to -6 processed with secondary antibodies stained either with Alexa 488 (IGFBP-1, -3, -4, -5) or Alexa 633 (IGFBP-2, -6). For each incubation condition, upper panel shows confocal fluorescent images and lower panel shows differential interference contrast images. Results are representative of three independent experiments.</p

IGFBP-2, -3, -4 and -6 increase intracellular calcium concentrations in MCF-7 cells.

<p>MCF-7 cells cultured on glass coverslips were incubated with Fura-2/AM. A. After background recording for 40 seconds to determine basal intracellular calcium concentrations as described in Methods, cells were incubated with IGFBP-6 (20 nM), then with IGFBP-3 (20 nM) and then with thapsigargin (Tp) (1 µM). The results of a typical experiment are shown in which the whole field (red line) was analysed and intracellular calcium quantified. The slides from left to right show representative views of the cells (a) before addition of IGFBP-6, (b) after addition of IGFBP-6, (c) after addition of IGFBP-3, and (d) after addition of thapsigargin. The results presented are representative of 5 independent experiments. B and C. Same experiments as in panel A excepted that cells were incubated with either 20 nM IGFBP-2 (panel B) or 20 nM IGFBP-4 (panel C). Slides show representative views of the cells after addition of IGFBP-2 or -4, respectively. Results presented are representative of 4 independent experiments. D. Quantitative analysis of the calcium response (maximal calcium response - basal calcium level) obtained for IGFBP-2, -4 and -6 in calcium free (empty bars) or calcium containing (hatched bars) medium. Results are the means ± SEM for three to five independent experiments. E. Dose-response curves for intracellular calcium concentrations were established as described in Materials and Methods. Values are expressed as percentages of the maximal response measured with 50 nM IGFBP-2 and are the mean for two independent experiments. F. Graphs present the average values of the intracellular calcium concentration modulated by addition of 20 nM of IGFBP-2, -4 or -6 following thapsigargin (1 µM) treatment in calcium free or containing medium as indicated.</p

IGFBP-5, but not IGFBP-3 and -6, increases intracellular calcium concentrations via a pertussis toxin-sensitive signaling pathway in C2 cells.

<p>C2 cells cultured on glass coverslips were treated with or without 200 ng.mL^-1 pertussis toxin (PTX) for 16 hours at 37°C and incubated with Fura-2/AM. After background recording for 40 seconds to determine basal intracellular calcium concentrations as described in Methods, cells were incubated (A) with IGFBP-3 (20 nM), then IGFBP-6 (20 nM) and then with thapsigargin (1 µM) or incubated (B) with IGFBP-5 (20 nM), then IGFBP-3 (20 nM) and then with thapsigargin (1 µM). The results of a typical experiment are shown in which the whole field (red line) was analysed and intracellular calcium quantified. The slides from left to right show representative views of the cells: panel A (a) after addition of IGFBP-3, (b) after addition of IGFBP-6, panel B: (c) after addition of IGFBP-5, and (d) after addition of IGFBP-3. Results presented are representative of 4 independent experiments.</p