Fig 2: LAPTM5 promotes tumorigenesis of breast cancer in vivo. (A and B) Tumor volume of xenografts in mice injected with LAPTM5-overexcpressed T-47D or sh-LAPTM5 MDA-MB-231 was measured every 3 days until day 27. (C) Images of xenografts cultured for 27 days. (D) The expression of KI67 and LAPTM5 in xenografts was detected by immunohistochemistry. Scale bar, 50 µm. (E) Protein expression of LAPTM5, CyclinD1 and MMP9 in xenografts was evaluated by western blot analysis. Data originated from three independent experiments and are presented as the mean ± SD. *P<0.05 and **P<0.01. LAPTM5, lysosomal protein transmembrane 5; MMP, matrix metallopeptidase; ns, not significant; sh-, short hairpin; NC, negative control.

Fig 3: LAPTM5 promotes cell proliferation of breast cancer. (A and B) Cell Counting Kit-8 was used to evaluate cell proliferation of the two breast cancer cell lines at 0, 24, 48, 72 and 96 h. (C and D) The effects of LAPTM5 upregulation or downregulation on cell cycle were detected by flow cytometry. The percentage of cells at different stages of cell cycles was statistically analyzed. (E and F) The protein expression of CyclinD1, CyclinA, CDK2 and CDK4 was examined by western blot analysis. β-actin served as loading control. Data originated from three independent experiments and are presented as the mean ± SD. **P<0.01. LAPTM5, lysosomal protein transmembrane 5; ns, not significant; sh-, short hairpin; NC, negative control.

Fig 4: β-catenin mediates breast cancer development induced by LAPTM5. (A) Nuclear and total protein expression of β-catenin were detected by western blotting. Histone H3 was used as a loading control for the nucleoplasm protein. β-actin was used as a loading control for the total protein. (B) The location and activation of β-catenin was revealed by immunofluorescence. Scale bar, 50 µm. (C) The inhibition efficiency of β-catenin in the T-47D cells was detected by reverse transcription-quantitative PCR and western blotting. (D) Cell proliferation affected by overexpressed LAPTM5 and si-β-catenin was evaluated by Cell Counting Kit-8 assay. (E) The effect of si-β-catenin on LAPTM5 induced cell migration was revealed by wound healing assay. The relative migration ratio was statistically analyzed. Scale bar, 200 µm. (F) The effect of si-β-catenin on LAPTM5 induced cell invasion was detected by Transwell assay. Invasive cell number was statistically analyzed. Scale bar, 100 µm. Data originated from three independent experiments and are presented as the mean ± SD. *P<0.05 and **P<0.01. LAPTM5, lysosomal protein transmembrane 5; si-, small interfering; OE, overexpressing; ns, not significant; NC, negative control.

Fig 5: Overexpression of LAPTM5 is associated with a poor outcome of breast cancer. (A) LAPTM5 mRNA level in healthy tissue, tumor-adjacent tissue and tumor was detected by bc-GenExMiner. (B) Reverse transcription-quantitative PCR was used to examine the mRNA expression level of LAPTM5 on breast cancer tissues (n=40) and para-tumor tissues (n=40). Data are presented as the mean ± SD. (C-K) Raincloud plot indicating the association between LAPTM5 and clinical parameters. (L) The relationship between LAPTM5 and overall survival of patients with breast cancer. **P<0.01. LAPTM5, lysosomal protein transmembrane 5.