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. 2021 Mar 17;12(4):286. doi: 10.1038/s41419-021-03555-5

Fig. 2. p97 inhibition reduces breast cancer growth, the CSC population, and mammosphere formation.

Fig. 2

a MDA-MB-231 cells were treated with 1.25, 2.5, 5, and 10 μM Eer I and NMS-873 for 24 h. DMSO was used as a vehicle control. The ubiquitinated proteins in cell lysates were analyzed by immunoblotting. GAPDH was used as a loading control. b Proliferation of MCF-7 and MDA-MB-231 cells treated with Eer I and NMS-873, with IC50 values indicated. c MCF10A, MCF-7, and MDA-MB-231 cells were treated with 1.25, 2.5, 5, and 10 μM Eer I for 24 h and cell viability was measured. d Growth of orthotopic tumors formed by MDA-MB-231 cells and treated with p97 inhibitors or DMSO. e Growth of orthotopic tumors formed by ALDH+ CSCs and ALDH− non-CSC cells and treated with p97 inhibitors or DMSO. f, g qPCR and immunoblotting analysis of p97, OCT4, and SOX2 in adherent and spheroid MDA-MB-231 cells. β-actin serves as a loading control for immunoblotting. h Twenty-four hours after MDA-MB-231 cells were seeded, Eer I and NMS-873 were added to the culture at 0.1, 0.4, 1.6, and 6.4 μM for 7 days to assess their impact on mammosphere formation. DMSO was used as a vehicle control. Left: representative images of the spheres treated with 6.4 μM Eer I or 6.4 μM NMS-873. Bar: 100 μm. Right: the percentage of spheres formed under each condition relative to the control group, with IC50 values indicated. i Eer I and NMS-873 were added to the culture at 0.1, 0.25, 0.5, and 1 μM for 7 days after the secondary spheres were formed to assess their impact on mammosphere maintenance. Left: representative images of the spheres treated with 1 μM Eer I or 1 μM NMS-873. Bar: 100 μm. Right: the percentage of spheres formed under each condition relative to the control group, with IC50 values indicated. Data were shown as mean + or ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.