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Fig. 3 | Cancer Cell International

Fig. 3

From: Aberrant accumulation of NIK promotes tumor growth by dysregulating translation and post-translational modifications in breast cancer

Fig. 3

NIK upregulation facilitated inherent tumor growth but not the lung metastatic potential of LM05 cells. A Cell growth curves of LM05-shGFP, shNIK no.1 and shNIK no.2 cells on planar culture (n = 3, two-way ANOVA followed by Tukey’s multiple comparison test). B Representative images (upper) and quantification data (lower) (n = 5, one-way ANOVA followed by Tukey’s multiple comparison test) of the soft agar colony formation assay in LM05-shGFP, shNIK no.1 and shNIK no.2 cells. The scale bar is 5 mm. C Representative images of primary tumors (upper) and quantification data of the primary tumor weights (lower) (n = 4, one-way ANOVA followed by Tukey’s multiple comparison test) in LM05-shGFP, shNIK no.1 and shNIK no.2 cells. The scale bar is 1 cm. D Representative in vivo bioluminescent images of LM05-shGFP and shNIK no.2 cells (upper). Tumor growth curves (lower) (n = 6 per group, two-way ANOVA followed by Tukey’s multiple comparison test) of NOD-SCID mice orthotopically injected with LM05-shGFP, shNIK no.1 and shNIK no.2 cells. E Representative ex vivo bioluminescent images (upper) and quantification data of the lung metastasis tissue (lower) (one-way ANOVA followed by Tukey’s multiple comparison test) derived from NOD-SCID mice orthotopically injected with LM05-shGFP (n = 5), shNIK no.1 (n = 4) and shNIK no.2 cells (n = 5). F Representative HE and IHC staining images (upper) and quantification data (lower) (one-way ANOVA followed by Tukey’s multiple comparison test) of NIK protein production in lung metastasis tissue derived from NOD-SCID mice orthotopically injected with LM05-shGFP, shNIK no.1 and shNIK no.2 cells. The average CAM5.2-positive percentages were calculated from 5 fields of view from n = 4 individual lung metastasis slides. The scale bar is 100 μm. G Representative HE and IHC staining images (upper) and quantification data (lower) (one-way ANOVA followed by Tukey’s multiple comparison test) from the TUNEL assay and α-SMA protein production in primary tumor tissues derived from NOD-SCID mice orthotopically injected with LM05-shGFP, shNIK no.1 and shNIK no.2 cells. The average number of TUNEL- and α-SMA-positive cells were calculated from 5 fields of view from n = 4 individual primary tumor slides. The scale bar is 100 μm. H Representative images (upper) and quantification data (lower) (n = 5, one-way ANOVA followed by Tukey’s multiple comparison test) of the Boyden chamber assay with TIG-3 cells that were co-cultured with LM05-shGFP, shNIK no.1, and shNIK no.2 cells. The scale bar is 500 μm. I Western blotting (upper) and immunofluorescence staining (lower) of α-SMA expression in TIG-3 cells that were co-cultured with LM05-shGFP, shNIK no.1 and shNIK no.2 cells or no cells (NC). The scale bar is 50 μm. All data are shown as the mean ± SEM. n.s. not significant. * P < 0.05

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