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. 2021 Apr 2;218(5):e20191360. doi: 10.1084/jem.20191360

Figure 2.

Figure 2.

Stiff ECM enhances apoptosis in response to treatment. (A) Bar graph showing mean ± SEM with individual values of loss of metabolic activity by MTT assay after paclitaxel treatment in MCF10A cells cultured on soft or stiff substrates. (B) Bar graph showing mean ± SEM with individual values of the percentage of cleaved caspase 3–positive cells from MCF10A cultures on soft and stiff substrates with increasing doses of paclitaxel. At least 300 cells on soft and 900 cells on stiff substrates were counted in each experiment. (C) Bar graph showing mean ± SEM with individual values of the percentage of cleaved caspase 3–positive cells from MCF10A cultures on soft and stiff substrates with increasing doses of IR. At least 300 cells on soft and 500 cells on stiff substrates were counted in each experiment (n = 4 or 5). (D) Bar graph showing mean ± SEM with individual values of the percentage of MCF10A cells cultured on a soft or stiff substrate that are capable of reforming colonies after IR. (E) Representative maximum-intensity projection of T4-2 cells cultured on soft and stiff substrates and treated with 10 nM paclitaxel or DMSO vehicle stained for cleaved caspase 3 (green) and with DAPI (gray). (F) Bar graph showing mean ± SEM with individual values of the percentage of cleaved caspase 3–positive cells from T4-2 cultures on soft and stiff substrates with increasing doses of paclitaxel. At least 900 cells on soft and stiff substrates were counted in each experiment. (G) Bar graph showing mean ± SEM with individual values of the percentage of cleaved caspase 3–positive cells from T4-2 cultures on soft and stiff substrates with increasing doses of IR. At least 800 T4-2 cells on soft substrate and 1,000 T4-2 cells on stiff substrate were counted in each experiment (n = 4). (H) Bar graph showing mean ± SEM with individual values of the percentage of cleaved caspase 3–positive cells from HCC70 cultures on soft and stiff substrates with increasing doses of paclitaxel. At least 750 cells on soft and stiff substrates were counted in each experiment (n = 3 or 4). (I) Bar graph showing mean ± SEM of the change in tumor volume after 14 d of paclitaxel treatment of animals with orthotopically injected T4-2 cells in soft (n = 7) or stiff (n = 8) collagen gels. (J) Representative images of FFPE tumor tissue sections stained for cleaved caspase 3 from orthotopically injected T4-2 tumors in soft or stiff collagen after 14 d of paclitaxel treatment. (K) Bar graph showing mean ± SEM with individual values of the change in tumor volume after 21 d of paclitaxel treatment of animals with orthotopically injected HCC70 cells expressing empty vector control (control; n = 6) or V737N β1 integrin mutant (n = 6). Statistical analyses were performed using two-tailed t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (L) Bar graph showing mean ± SEM with individual values of the time to paclitaxel resistance of animals with orthotopically injected HCC70 cells expressing empty vector control (control; n = 9) or V737N β1 integrin mutant (n = 7). (M) Bar graph showing mean ± SEM with individual values of the time to paclitaxel resistance of animals with orthotopically implanted BCM2665 triple-negative PDX pieces in soft (n = 7) or stiff (n = 7) collagen gels. Statistical analyses of time to resistance were performed using two-tailed Mann-Whitney test (*, P < 0.05). Unless otherwise noted, n = 3 for all experiments. All scale bars are 100 µm.