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. 2020 Jul 12;12(9):e12050. doi: 10.15252/emmm.202012050

Figure EV2. INSL5 overexpression promotes the progression of NPC via accelerating cell proliferation and invasion depending on GPCR142.

Figure EV2

  • A
    Exogenous expression of INSL5 in normal immortalized NPEC cell lines.
  • B
    Quantitative RT–PCR validation for siRNA knockdown efficiency of GPCR142 in INSL5 stably overexpressing CNE1, CNE2, and HK1cell lines.
  • C–F
    MTT (C), colony formation (D), Brdu incorporation (E), migration, and invasion assay (F) of vector control or INSL5 overexpressing NP69 cell line. The scale bars represent 20 μm in (E) and 100 μm in (F).
  • G
    Invasion assays of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in left. Invaded cells per field of view were plotted in right. The scale bars represent 100 μm.
  • H
    EBV‐positive NPC cell lines CNE2‐EBV and HNE1‐EBV were transfected with siRNA against INSL5 for 36 h, followed by immunoblotting with indicated antibodies (inserted picture). MTT assay of CNE2‐EBV (upper panel) and HNE1‐EBV (lower panel) cells after INSL5 knockdown.
  • I
    Migration and invasion assay of CNE2‐EBV (upper) and HNE1‐EBV (lower) cells with INSL5 knockdown. Representative images are shown in left panels. Migrated or invaded cells per field of view were plotted in right panels. The scale bars represent 100 μm.
  • J
    Colony formation assay of CNE2‐EBV and HNE1‐EBV cells with INSL5 knockdown. Representative images are shown in left panels. Number of colonies was plotted in right panels.
  • K, L
    Xenograft tumor growth of INSL5 overexpression NPC CNE2 stable cell lines in nude mice. Tumor size (K) and tumor weight of two groups (L), n = 7 mice per group.
Data information: In (B and G–L), data are presented as mean ± SEM, in (C–F), data are presented as mean ± SD, from three different experiments, and P‐values were determined by unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P‐values are specified in Appendix Table S4.Source data are available online for this figure.