Fig. 4: ITSN1-S in nucleus inhibited breast cancer cells proliferation in vitro and in vivo.

A 3×flag-labeled vector, 3×flag-labeled NLS-mutant ITSN1-S, and 3×flag-labeled wild-type ITSN1-S were transfected into KOITSN1/MDA-MB-231 cell clones and tested with anti-flag and anti-ITSN1-S antibodies in Western blot, respectively. β-actin was used as a loading control. B Proliferation ability was examined by EdU incorporation assay in KOITSN1–3×flag-vector/MDA-MB-231, KOITSN1–3×flag-ITSN1-S-NLS-mutant/MDA-MB-231, and KOITSN1–3×flag-ITSN1-S-WT/MDA-MB-231 cells. Scale bars, 25 μm. Quantitative results were analyzed in the lower panel. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t-test, ^*P < 0.05, ^***P < 0.001). C 3×flag-labeled vector, 3×flag-labeled NLS-mutant ITSN1-S (which accumulated in the cytoplasm), and 3×flag-labeled wild-type ITSN1-S (which accumulated in both cytoplasm and nucleus) were transfected into shITSN1-S #2/MDA-MB-231 cell clones and tested with anti-flag and anti-ITSN1-S antibodies in Western blot, respectively. β-actin was used as a loading control. D Proliferation ability was examined by EdU incorporation assay in shITSN1-S-3×flag-vector/MDA-MB-231, shITSN1-S-3×flag-ITSN1-S-NLS-mutant/MDA-MB-231, and shITSN1-S-3×flag-ITSN1-S-WT/MDA-MB-231 cells. Scale bars, 25 μm. Quantitative results were analyzed in the right panel. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t-test, ^*P < 0.05, ^**P < 0.01). E Orthotopic xenograft models were performed in vivo. The representative images of tumor size of shITSN1-S-3×flag-vector/MDA-MB-231 (n = 18), shITSN1-S-3×flag-ITSN1-S-NLS-mutant/MDA-MB-231 (n = 16), and shITSN1-S-3×flag-ITSN1-S-WT/MDA-MB-231 (n = 18) mice groups were presented in the left panel. Quantitative results were analyzed in the right panel. Values were expressed as mean ± SEM (two-way ANOVA, ^*P < 0.05, ^***P < 0.001).