3451200.pdf

Document containing supplementary information to the manuscrip

Invasion assay.

<p>Number of RM1 cells that invaded the matrigel membrane of the transwell system after 24 h in co-culture with preadipocytes, adipocytes and control media, used to evaluate invasion capacity, which was significantly higher in both co-cultures compared with control media alone (* p<0.05 Unpaired T test) (A). Representative images of the cells invading the matrigel membrane for each tested condition (B).</p

Cell proliferation assay in the presence of conditioned media.

<p>RM1 cell proliferation cultured with different conditioned media, assessed by the percentage of tetrazolium salt reduction. RM1 proliferation cultured with preadipocyte conditioned media (PACM) compared with the control (p>0.05, Mann-whitney U test) (A). RM1 proliferation cultured with 24 and 48h adipocyte conditioned media (ACM) compared with the control (*** p<0.001 one-way ANOVA, Newman-Keuls multiple comparison post-hoc test) (B). RM1 Proliferation with three different conditioned media (* p<0.05 one-way ANOVA, Newman-Keuls multiple comparison post-hoc test) (C).</p

Cell proliferation assay in the presence of insulin e leptin.

<p>RM1 cell proliferation in the presence of different insulin concentrations (0, 200, 800, 1600 nmol/L) evaluated by determination of the % of resazurin reduction at different time points (A) (p<0.01 1600 nmol/L vs 800, 200, 0 nmol/L at 24h, one-way ANOVA, Newman-Keuls multiple comparison post-hoc test). RM1 cell proliferation of in the presence of different leptin concentrations (0, 10, 50, 100 ng/mL) evaluated by determination of the % of resazurin reduction at different time points (B).</p

Cell proliferation assay in co-culture.

<p>Cellular proliferation was measured in RM1 co-cultivated with preadipocyte and adipocytes. Cell number/ well in different conditions tested, co-cultivated in the presence of preadipocytes (A) and adipocytes (B), all compared with the control, and comparing both conditions (C) (** p<0.01; *** p<0.001 Unpaired T test).</p

RM1 cell growth curve.

<p>Growth curve using three different initial cell concentrations (2x10⁴, 5x10⁴, 10x10⁴ cells/mL) evaluated by the determination of the percentage of tetrazolium salt reduction at different time points (12, 24, 36 and 48h).</p

Migration assay.

<p>Width of invaded space by RM1 cells cultured with PACM, ACM and control media in the “wound healing assay” used to measure cell migration capacity (* p<0.05 one-way ANOVA, Newman-Keuls multiple comparison post-hoc test) (A). Photographs representative of the cellular migration in the “wound healing” process at the different experimental conditions measured at baseline (0H) and at 24H (B).</p