LA/GnRH-R unbinding force histograms.

<p>Distribution of unbinding events obtained at a loading rate of 2 µm/s in untreated (A) and LA (10⁻⁶ M)-treated (B) PC-3 cells after 30 days of culture and in HEK293_[SCL60] cells (C). Frequency corresponds to the number of events. In both untreated and LA-treated PC-3 cells a bimodal distribution (blue line) is clearly detected and quantified by means of two Gaussian distribution curves (purple and green dashed lines). The first peak was detected at a lower force (f∼37 pN) while the second one at a higher force (f∼65 pN). In HEK293_[SCL60] cells unbinding events at higher forces are less frequent.</p

LA/GnRH-R unbinding events.

<p>(A) Histograms of LA/GnRH-R unbinding events in LA-treated PC-3 cells. In each measurement, performed every 6 days, the counting is normalized to that obtained with untreated cells (control) set to 1. Columns, mean; bars, SD. *p<0.001 <i>vs</i> control, ^•p<0.001 <i>vs</i> 10⁻⁶ M LA (one-way ANOVA and Tukey's multiple comparison tests). (B) Modelling of the GnRH-R rate expression. The amount of unbinding events detected in PC-3 cells treated with 10⁻⁶ M LA (blue diamonds) or 10⁻¹¹ M LA (red squares) was normalized to the amount of unbinding events in untreated cells (control) set to 1. The Gompertz curve was fitted to both the experimental data (blue or red dashed lines). The GnRH-R increase rates were clearly different [(9.3±0.1) day⁻¹ for 10⁻⁶ M LA and (6.1±0.1) day⁻¹ for 10⁻¹¹ M LA] but tended to the same value (1.9±0.1) at long times (beyond the 30^th day). Data are given as mean ± SD from two independent experiments.</p

GnRH-R map of untreated and LA-treated PC-3 cells.

<p>The homogeneous distribution of the receptor molecules was not influenced by the treatment with the analogue (10⁻⁶ M) and did not vary through the time intervals. In blue are represented unbinding events occurring at force <50 pN while in red those at force in the range of 50–100 pN.</p

Western blot analysis of GnRH-R.

<p>(A) Western blot analysis of GnRH-R in: PC-3 cells, HEK293 cells (not expressing GnRH-R) and HEK293_[SCL60] cells (stably transfected with GnRH-R) after 6 days of culture. (B) Western blots showing the LA-triggered enhancement of GnRH-R in PC-3 cells treated for 6–30 days with the analogue (10⁻¹¹ or 10⁻⁶ M). Representative blots from two separate experiments yielding similar results are shown. (C) Grouped densitometric data of Western blot analysis of GnRH-R. The intensity of the signals was quantified by densitometric scanning and normalized to that of β-actin (used as a loading control). Data are the ratio between values of treated and untreated samples (control, set to 1) and they are shown as mean ± SD of 2 independent experiments. *p<0.001 <i>vs</i> control, ^•p<0.001 <i>vs</i> 10⁻⁶ M LA (one-way ANOVA and Tukey's multiple comparison tests).</p

Force-distance curves.

<p>Two typical force-distance curves when interactions are not detected (black square) and when interactions are detected (red circle). The unbinding event (or rupture point) is highlighted by means of the green arrow.</p

Representative phase contrast microscopy analysis of cultured cells.

<p>(A): top, parental cell lines (LI, U87 and U373 cells) cultured in their standard medium added of 10% serum. Middle, formation of neurospheres on top of the monolayers of U373 cell line. Images were taken after 1, 2 and 3 days in neurosphere medium. Bottom, images of primary NS generated by LI, U87 and U373 cells by day 8. (B) Clones derived from LI (D2 and F11) and U87 (C7 and E8) primary NS. Original magnification 200× or 400×. Scale bar = 100 µm.</p

Immunostaining of brain sections from mice implanted with 500,000 LI cells, 10,000 D2 cells and 10,000 F11 cells.

<p>Original magnification 630×. Scale bar = 50 µm.</p

Doubling time of D2 and F11 clones cultured in NSCM (serum-free; presence of growth factors) or in medium with 10% FBS added.

<p>*hours.</p

Additional file 1: of Characterization of inflammatory cell infiltrate of scleroderma skin: B cells and skin score progression

Table S1. Autoantibody characteristics according to scleroderma skin disease extension. (DOCX 14 kb

Low invasive breast cancer cells and fibroblasts reciprocally influenced their plasma membrane fluidity.

<p>Pseudocoloured GP images of living tumor (MCF-7) and fibroblast (NFs or CAFs) cells (A), and reciprocal effects on membrane fluidity (B) are shown. MCF-7 cells were cultured for 6 days, alone or in presence of NFs or CAFs, in 35 mm glass-bottom Petri dishes and labelled with Laurdan (2 µM). Tumor cell/fibroblast interaction determined an enhancement of cancer cell membrane fluidity. On the other hand, MCF-7 cells promoted an increase in fibroblast membrane packing density. Data are mean±SD (error bars) of three independent experiments. Statistical significance was determined using Student's t-test, *p<0.05 vs respective homotypic culture.</p