Fig 1: Analysis of VASP phosphorylation at S239(A–B) VASP phosphorylation at S239 was analyzed in MNT1 cells not treated (NT) and treated with 1 μM PA4 for 24 h. (A) Immunolocalization of S239 pVASP shows increased pVASP after treatment. White arrows indicate the nuclear localization of pVASP. At the right-hand side, confocal analysis shows one optical section at the nuclear level confirming nuclear localization of S239 pVASP in PA4 treated MNT1. (B) Immunoblotting on nuclear extracts and cytoplasmic extracts of pVASP phosphorylated at S239 in MNT1 cells not treated (NT) or PA4 and PA5 treated. Treatment was performed at 1μM concentration for 24 h. The blot was normalized by the analysis of tubulin for cytoplasm extracts and histone H3 for nuclear extracts. (C–D) VASP phosphorylation at S239 was analyzed in SkMel28 cells not treated (NT) and treated with 1 μM PA5 for 24 h and 72 h. (C) Immunolocalization of S239 pVASP shows increased pVASP after treatment. (D) Immunoblotting on nuclear extracts and cytoplasmic extracts of pVASP phosphorylated at S239 in SkMel28 cells not treated (NT) or PA4 and PA5 treated. Treatment was performed with 1μM concentration of the compounds for 24 h. The blot was normalized by the analysis of tubulin for cytoplasm extracts and histone H3 for nuclear extracts. (E) Immunoblotting of RhoA phosphorylated at S188 (pRhoA) in MNT1 cells not treated (NT) or treated with 1 μM PA4 for 24 h and in SkMel28 cells not treated (NT) or treated with 1 μM of PA5 for 24 h or 72 h. The immunoblot was normalized by analysis of actin.
Fig 2: Metastatic peritoneal tumors have increased expression of members of the mevalonate pathway(A) Primary and metastatic tumors from ID8 injected mice (n = 6/group) were collected and stained for HMGCR and small GTPases Rac1 and RhoA. Metastatic secondary tumors had a significantly (*p < 0.05) higher percentage of tissue immunopositive for the enzyme and GTPases than primary tumors. Scale bars are 20 μM (B) Western blot was performed on tumor lysates from primary and metastatic tumors. Metastatic tumors had higher expression of HMGCR, Rac1 and RhoA, compared to primary tumors (*p < 0.05).
Fig 3: p53MUT (28-2, murine; iOvCa147, human) have increased SREBP-2 and RhoA expression compared to p53WT (ID8, murine; iOvCa130, human) EOC cellsIncreased expression of the HMGCR transcription factor SREBP2 was seen in p53MUT compared to p53WT cells (SREBP2 red, nuclei blue). Expression of the small GTPase RhoA, which is a downstream protein in the mevalonate pathway was upregulated in the p53MUT ascites derived cells (RhoA green, nuclei red). Treatment with the mevalonate pathway inhibitor simvastatin (20 μM) caused greater inhibition of RhoA expression in p53MUT metastatic ascites-derived cells (28-2, iOvCa147) than primary tumor cells (ID8) or p53WT (iOvCa130) ascites derived cells.
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