Fig. 5

Roles of NHE1, NBCn1, MCT1 and MCT4 in MCF-7 spheroid growth. MCF-7 cells were transduced with lentivirus containing plasmids with shRNA constructs targeted against NHE1, NBCn1, MCT1 or MCT4, respectively. Transduction with the empty vector pLKO.1 (pLKO.1) was used as control. a: Western blotting with antibodies directed against the specific transporters was performed to verify knockdown of the respective transporters. b: The transduced cell lines were grown as spheroids, and representative light microscopic images (10×) of the spheroids on day 2, 4, 7 and 9 are shown. Scalebar: 100 μm. 3n. c: Quantification of spheroid diameters shown in b. The dot-plot insert shows diameters on day 9. Mean and SEM are indicated. One-way ANOVA with Dunnett’s multiple comparisons post-test was used to test for statistical significant differences between pLKO.1 and the respective groups. d: NHE1 expression was ablated in MCF-7 cells by CRISPR/Cas9-mediated knockout (KO) and wild-type (WT) and KO cells were grown as spheroids for seven days. Lower panel shows quantification of spheroid areas while the top panel shows representative light microscopic images (10×) of the spheroids on day 2, 4 and 7. Scale bar: 100 μm. 3 n. Error bars denote SEM. A Student’s t-test (unpaired) was used to test for statistical significant difference between the wild-type and NHE1 knockout. * indicates p < 0.05