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. 2019 Jun 27;13(8):1651–1668. doi: 10.1002/1878-0261.12503

Figure 7.

Figure 7

HDAC7 influences the transformation properties of RAS‐transformed cells. (A) Immunoblot analysis of MCF10/RAS‐transformed cells expressing or not HDAC7. Cellular lysates were generated and after blotting incubated with the indicated antibodies. Actin was used as loading control. (B) S‐phase determination by BrdU incorporation in RAS‐transformed MCF10A/HDAC7 +/+ and MCF10A/HDAC7 −/− cells. Data are presented as mean ± SD (n = 3). We marked with *P(Kruskal–Wallis) < 0.05, ***P < 0.005. (C) Representative phase‐contrast microscope images of typical spheroid and stellate‐shaped acini obtained after 8 days of 3D culture. (D) Representative confocal images showing the comparison between typical stellate and spheroid acini generated by MCF10A/RAS cells grown under 3D conditions. AF546‐phalloidin was used to stain F‐actin (red), and nuclei were stained with anti‐HMGA2 (green). In the lower part, the Z‐section is shown as indicated. Bar 50 μm. (E) Percentage of spheroid and stellate acini in MCF10A/RAS HDAC7 +/+ or HDAC7 −/−, as indicated. Acini were scored after 8 and 12 days in culture. Data are presented as mean ± SD (n = 3). We marked with ***P(Student) < 0.005. (F) Transwell migration assay of MCF10A/RAS HDAC7 +/+ or HDAC7 −/−. The percentage of migrating cells was calculated as the ratio between the number of invasive cells and the random migrating cells. Data are presented as mean ± SD (n = 3). We marked with ***P(Student) < 0.005. (G) Flow cytometry analysis of CD44 and CD24 markers in MCF10A/HDAC7 +/+ or HDAC7 −/− cells (upper panel) compared to RAS‐transformed cells (lower panel). (H) Quantitative analysis of CD44+/CD24low cell population as performed by FACS. Data are from three independent experiments, ±SD. We marked with ***P(Student) < 0.005. (I) Scatter dot plot illustrating the number of mammospheres generated by MCF10A/HDAC7 +/+ , MCF10A/RAS HDAC7 +/+ or HDAC7 −/− cells, grown in a semisolid medium containing 0.5% methylcellulose. n = 9. We marked with ***P(Dunn) < 0.005. (J) mRNA expression levels of the HDAC7‐repressed genes coding for microenvironmental factors, as measured by qRT/PCR in the indicated cell lines. Data are presented as mean ± SD (n = 3). We marked with *P(Kruskal–Wallis) < 0.05, **P < 0.01, ***P < 0.005. (K) Cell death quantification after incubation of MCF10A/RAS cells for 48 h with 20 ng·mL−1 of IL24. Data are presented as mean ± SD (n = 3). We marked with ***P(Student) < 0.005. (L) Representative images of mammospheres generated by MCF10A/RAS pretreated with 20 ng·mL−1 of IL24 and grown in the presence of 20 ng·mL−1 of IL24 in the MM and the untreated control after 10 days in culture. Mammospheres were stained with MTT. Scale bar, 100 μm. (M) Scatter dot plot illustrating the number of mammospheres generated by MCF10A/RAS cells grown for 48 h in the presence of 20 ng·mL−1 of IL24 before seeding in MM (Pre), growth in control MM (untreated) or pretreated with IL24 (20 ng·mL−1) in MM and next grown for 10 days in MM with IL24 (20 ng·mL−1) (Pre/Post). n = 9, ***P(Dunn) < 0.005.