Fig. 2
The tumor suppressive effects of RCAN1.4 in breast cancer cells by inhibition of CaN-NFATC1 signaling. a, b Immunoblot in MDA-MB-231 and BT549 cells expressing sgRNAs targeting human RCAN1.4 (a), and in HCC1806 cells stably overexpressing RCAN1.4 (b). Blue star, RCAN1.1; Red closed circle, RCAN1.4. c The transwell cell migration and invasion assay of the indicated MDA-MB-231, BT549 and HCC1806 cells was shown. The migration or invasion of control cells was set as 100%. d 2 × 105 luciferase-tagged WT or RCAN1.4-knockout MDA-MB-231 cells were injected intracardially. BLI images showed representative mice in each group (upper). Normalized metastasis BLI signals from mice (n = 8, bottom). e Kaplan-Meier survival curve of mice were calculated (n = 8). f-g Tumour growth of the indicated BT549 cells in BALB/c nude mice (n = 7 mice per group). Tumor volumes (f) and tumour weights upon autopsy on day 21(g) were calculated. h The indicated MDA-MB-231, BT549 and HCC1806 cells were stained with fluorescent antibodies against NFATc1 (green) or with DAPI (blue). Statistics of the percentage of NFATc1 in the nucleus were determined in the indicated samples (n = 30 cells per group). i-j qRT-PCR monitoring target gene expression in RCAN1.4-knockout MDA-MB-231 and BT549 cells or RCAN1.4-overexpression HCC1806 cells (n = 3 biological independent samples). Expression levels were normalized for GAPDH. Average KO/WT ratios of genes expression in MDA-MB-231and BT549 cells (i), and average OV/vector ratios of genes expression in HCC1806 cells (j) were quantified. k The indicated MDA-MB-231 and BT549 cells were treated with 5 μM CsA for 24 h and the nuclear levels of NFATC1 were detected by Immunoblot. l-n MDA-MB-231-RCAN1.4 KO cells or BT549-RCAN1.4 KO cells were transfected with CaN siRNAs and NFATC1 siRNAs for 48 h. qRT-PCR was used to monitor IGF1 expression (l, n = 3 biological independent samples). Quantification of migratory and invasive cells of images per group was shown (m-n, n = 3 biological independent samples). Error bars represent mean ± SD. **P < 0.01. The P value in c was determined by one-way ANOVA with Dunnett’s multiple comparisons test, the P value in l, m, n was determined by one-way ANOVA with Tukey’s multiple comparisons test, no adjustments were made for multiple comparisons. The P value in d, f, g, h was determined by a two-tailed unpaired Student’s t test. The P value in e was assessed using the log-rank test. All data are representative of three independent experiments