Figure 3.

Quantification of cell proliferation of compressed (MAG+) or free (MAG−) spheroids. (A) Percentage of proliferating cells for MAG+ and MAG− spheroids (respectively red and grey bars) at day 3 and day 4 of spheroid maturation. MAG+ spheroids experience an increase in the number of proliferating cells for the two days. Proliferation is evaluated as the ratio between the number of Ki67-positive nuclei and the total number of nuclei (at day 3, n = 3 for both conditions, at day 4, n = 3 for MAG−, n = 5 for MAG +, * p < 0.05). (B) Average of the normalized distance <R/R0> for the two conditions at the two maturation times (days 3 and 4). This distance represents the localization from the spheroid center of a Ki67 positive nucleus. This decreases for the MAG+ spheroids, demonstrating an increase in the proliferative nuclei toward the MAG+ spheroid center (n = 3 spheroids for both conditions at day 3; n = 3 and n = 5 spheroids at day 4 for MAG+ and MAG− conditions, respectively; p < 0.05). (C) Cumulative distributions of the normalized distance <R/R0>. To obtain these distributions, the Ki67 localizations were pooled for every image of a single condition. It demonstrates a shift of the proliferation toward the center of the MAG+ spheroids, whereas the MAG− distribution is grouped around 1, which represents nuclei localized at the edge of the spheroid (at day 3, n = 1915 Ki67-positive nuclei for the MAG+ spheroids, n = 2653 Ki67-positive nuclei for the MAG− spheroids, p < 0.005; at day 4, n = 937 Ki67-positive nuclei for the MAG+ spheroids, n = 2359 Ki67-positive nuclei for the MAG− spheroids, p < 0.005). (D) Metabolic Alamar Blue assay. The relative fluorescence measured is shown for MAG+ and MAG− spheroids (red and striped bars, respectively) for day 3 and day 4. For both times, the proliferation of MAG+ spheroids is increased compared to MAG− spheroids (at day 3, n = 15 for the MAG− spheroids and n = 26 for the MAG+ spheroids, *** p < 0.005, at day 4, n = 15 for MAG+ and n = 14 for MAG−, *** p < 0.005).