Fig. 4

FSCN1 inactivation or pharmacological inhibition reduces in vitro Matrigel invasion of ACC cells. Number of (A) filopodia, (B) lamellipodia/ruffles and (C) area of focal adhesions per cell in control and FSCN1 KO H295R cells treated with either vehicle or Dox (1 μg/ml) for 24 h. n (independent experiments) = 6-9. Mean ± SD is shown. *P < .05; **P < .01, one-way ANOVA with Šidák's multiple comparisons test. (D) In vitro Matrigel invasion of control and FSCN1 KO H295R cells treated with either vehicle or Dox (1 μg/ml). Results are expressed as percentages of control cell invasion. n (independent experiments) = 3-6. Mean ± SD is shown. **P < .01, one-way ANOVA with Šidák's multiple comparisons test. (E) In vitro Matrigel invasion of control H295R cells treated with Dox (1 μg/ml) and with vehicle or G2 (50 μM), G2-044, G2-011 or the inactive G2-112 compound (all 5 μM). n (independent experiments) = 2-4. Mean ± SD is shown. **P < .01, one-way ANOVA with Šidák's multiple comparisons test. (F) Left: immunoblot showing expression of FSCN1 and GAPDH in H295R, CU-ACC2, JIL-2266 and MUC-1 cells. FSCN1 levels (relative to GAPDH) in the CU-ACC2, JIL-2266 and MUC-1 ACC cell lines are indicated as percentages of H295R, which express the highest levels of FSCN1. Right: immunoblot showing expression of SF-1 and GAPDH in H295R, CU-ACC2, JIL-2266 and MUC-1 cells. SF-1 levels (relative to GAPDH) in the CU-ACC2, JIL-2266 and MUC-1 ACC cell lines are indicated as percentages of H295R, which express the highest levels of SF-1. FSCN1 and SF-1 band intensities were quantified by Image J after GAPDH normalization. n (independent experiments) = 3. G) Matrigel invasion of those ACC cell lines treated with vehicle or G2-044 (5 μM). n (independent experiments) = 4-5. Mean ± SD is shown. ***P < .001; ****P < .0001, t test.