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. 2020 Sep 10;21(11):e50078. doi: 10.15252/embr.202050078

Figure 6. hMENA/hMENAΔv6 silencing inhibits AXL expression and activity in cancer cells.

Figure 6

  1. Immunoblot of AXL expression in PANC‐1 and A549 cancer cells upon transfection of control siRNA (CNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a reduction of AXL protein expression.
  2. Real‐time qRT–PCR analysis of the relative AXL mRNA expression in PANC‐1 and A549 cancer cells transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a significant reduction of mRNA AXL expression. Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test. **P < 0.01, ***P < 0.001.
  3. Real‐time qRT–PCR analysis of the relative levels of mature AXL mRNA or AXL pre‐mRNA in PANC‐1 (left) and A549 (right) cell lines, transfected with control siRNA (siCNT) or hMENA(t) siRNA, sihMENA(t). Data represent percent of AXL mRNA or pre‐mRNA levels in hMENA(t) silenced cells relative to siCNT control cells (set at 100). Data are presented as the mean ± SD of two biological replicates. P values were calculated by two‐sided Student's t‐test. *P < 0.05, ***P < 0.001.
  4. Immunoblot analysis with the indicated antibodies of PANC‐1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6‐mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (−) or rGAS6 (200 ng/ml), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported.
  5. Quantification of in vitro Matrigel invasion assay of PANC‐1 siCNT cells (siCNT) and hMENA/hMENAΔv6 silenced cells (sihMENA(t)) toward rGAS6 as chemo‐attractant (siCNT + rGAS6 and sihMENA(t) + rGAS6), showing that the knock‐down of hMENA(t) reduced cancer cell invasion toward GAS6. The number of invading cells was counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one‐way ANOVA P < 0.0001, followed by Bonferroni's multiple comparison test. **P < 0.01.
  6. Quantification of in vitro Matrigel invasion assay of PANC‐1 siCNT cells (siCNT) and hMENA/hMENAΔv6 silenced cells (sihMENA(t)), untreated (−) or treated with conditioned media derived from CAFs (P‐CAF#36‐CM) for 48 h. The number of invaded cells was counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one‐way ANOVA P < 0.0001, followed by Bonferroni's multiple comparison test. ***P < 0.001.

Source data are available online for this figure.