Fig. 6
Effect of stable knockdown of NHE1, NBCn1 or MCT4 on growth of MDA-MB-231 spheroids. MDA-MB-231 cells were transduced with lentivirus containing plasmids with shRNA constructs targeted against NHE1, NBCn1 or MCT4. 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 (10Ă—) 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 dotplot 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 was transiently knocked down in MDA-MB-231 cells by siRNA interference and cells were grown as spheroids. Representative light microscopic images (10Ă—) of the spheroids on day 2, 4 and 7 are shown on the right while quantification of spheroid diameters is shown on the left. Scalebar: 100 μm. 3n. Error bars denote SEM. A two-tailed, paired Student’s t-test was used to test for statistically significant difference in means between the two groups on day 7. e: NHE1 expression was ablated in MDA-MB-231 cells by CRISPR/Cas9-mediated knockout (KO) and grown as spheroids for nine days. Left panel shows quantification of spheroid areas while the right panel shows representative light microscopic images (10Ă—) of the spheroids on day 2, 4, 7 and 9. Scalebar: 100 μm. 3 n. Error bars denote SEM. WT: wild-type. A two-tailed, unpaired Student’s t-test was used to test for statistically significant difference in means between the two groups on day 9. * and ** indicate p < 0.05 and p < 0.01, respectively