Fig 1: Effect of LeftyA on actin polymerization in Ishikawa cells.(a) Representative original Western blot of soluble G-actin over F-actin in human endometrial cancer Ishikawa cells after a 2 hour treatment without (−LeftyA) and with (+LeftyA) LeftyA (25 ng/ml). (b) Arithmetic means ± SEM (n = 6; arbitrary units) of soluble G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml). (c) Representative original histogram of DNase1 (G-actin; Left) and Phalloidin (F-actin; Right) binding in Ishikawa cells after a 2 hour treatment without (dark grey) and with (grey) LeftyA (25 ng/ml). (d) Arithmetic means ± SEM (n = 6; arbitrary units) of G-actin over F-actin ratio in Ishikawa cells after a 2 hours treatment without and with LeftyA (25 ng/ml). (e) Original confocal images of eflour660-phalloidin binding to F-actin (red) and SYTOX Green for nuclei (blue) in Ishikawa cells treated with or without LeftyA (white bar 20 μm) (f) arithmetic means ± SEM (n = 6) of actin fluorescence in Ishikawa cells with and without LeftyA treatment. (g) Averaged FRAP curve (n = 21) of control cells or cells treated for 2h with LeftyA. Error bars denote SEM of arithmetic mean of normalized fluorescence intensity at each time point. (h) Mean half-time of recovery for LeftyA-treated (Green) and untreated Ishikawa cells (Control; Blue) obtained by FRAP. *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test.
Fig 2: Effect of LeftyA on actin polymerization in Ishikawa cells in absence or presence of Rac1 inhibitor.(a) Representative original Western blot of soluble G-actin over F-actin in human endometrial cancer Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the Rac1 inhibitor NSC23766 trihydrochloride (100 μM). (b) Arithmetic means ± SEM (n = 3; arbitrary units) of soluble G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the Rac1 inhibitor NSC23766 trihydrochloride (100 μM). (c) Representative original histogram of DNase1 (G-actin; Upper) and Phalloidin (F-actin; Lower) binding in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the Rac1 inhibitor NSC23766 trihydrochloride (100 μM). (d) Arithmetic means ± SEM (n = 5 arbitrary units) of G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the Rac1 inhibitor NSC23766 trihydrochloride (100 μM) *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test.
Fig 3: Effect of LeftyA on actin polymerization in Ishikawa cells in absence or presence of PAK1 inhibitor.(a) Representative original Western blot of soluble G-actin over F-actin in human endometrial cancer Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the PAK1 inhibitor IPA-3 (50 μM). (b) Arithmetic means ± SEM (n = 6; arbitrary units) of soluble G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with Lefty A (25 ng/ml) in the absence and presence of the PAK1 inhibitor IPA-3 (50 μM). (c) Representative original histogram of DNAse1 (G-actin; Upper) and Phalloidin (F-actin; Lower) binding in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the PAK1 inhibitor IPA-3 (50 μM). (d) Arithmetic means ± SEM (n = 5; arbitrary units) of G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the PAK1 inhibitor IPA-3 (50 μM). *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test.
Fig 4: Effect of LeftyA on Rac1 transcript levels and activity in Ishikawa cells.(a) Arithmetic means ± SEM (n = 4) of Rac1 normalized to L19 transcript levels in human endometrial cancer Ishikawa cells following treatment with LeftyA (25 ng/ml) for 2 hours. Data are depicted as fold induction relative to transcript levels of untreated samples. (b) Representative original Western blots showing activated Rac1 and total Rac1 protein abundance in human endometrial cancer Ishikawa cells after 2 hours culture in the absence or presence of LeftyA (25 ng/ml). (c) Arithmetic means ± SEM (n = 4, arbitrary units) of phospho-Rac1 protein ratio normalized to GAPDH in Ishikawa cells after 2 hours culture in the absence or presence of LeftyA (25 ng/ml). *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test.
Fig 5: Schematic showing how stiffness of Ishikawa cells is affected by treatment with LeftyA via Rac1 and PAK1 pathways.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human Lefty-A Protein, CF