Fig 8. Actin and Tubulin stabilization contribute to Ifnγ induced aggregation of APECs.
Fluorescence microscopic images acquired at a magnification of 63X with the scale bar representing 10 μm of F-Actin (green) and α-Tubulin (red) in C57BL/6 APECs treated with 10 μM Cyt D and 1 μg/ml of Col respectively for 6 h (A). Yellow and white arrows indicate cortical arrangement of F-Actin and α-Tubulin respectively. Thin white arrows indicate the cell boundary while the fatter white arrows are used to highlight the shrinkage of the α-Tubulin network upon Col treatment when compared to the untreated control C57BL/6 APECs. The amount of nitrite in the supernatant (B) produced by APECs from C57BL/6 mice upon treatment with 25 U/ml of Ifnγ in the absence or presence indicated doses of inhibitors Cyt D (μM) and Col (μg/ml). The number of cell aggregates (C) of APECs from C57BL/6 mice upon treatment with 25 U/ml of Ifnγ in the absence or presence of indicated doses of inhibitors Cyt D (μM) and Col (μg/ml). The amounts of E-Selectin (D) and CD11b (E) on the cell surface of C57BL/6 APECs treated with 25 U/ml of Ifnγ for 36 h, post 6 h of pretreatment without or with Cyt D (10 μM) and Col (1 μg/ml). The data is represented as mean ± S.E from three independent experiments. The velocity of APECs from C57BL/6 and Nos2 -/- mice treated without or with 25 U/ml of Ifnγ between 18–24 h of addition (F). The velocity of APECs from C57BL/6 pretreated for 6 h with Cyt D (10 μM) and Col (1 μg/ml) before tracking them from 6–12 h post treatment. Also, the velocity of Nos2 -/- APECs pretreated in the presence of 5 μM of SNAP (SNAPlo) and 100 μM of SNAP (SNAPhi) for 12 h and without or with 25 U/ml of Ifnγ before tracking cells 18–24 h post Ifnγ addition. The velocity for each of the above mentioned conditions are represented as percentage velocity with respect to average velocity exhibited by untreated C57BL/6 APECs. The data is representative of two independent experiments. Significance is represented as * when compared to untreated controls and # when compared to Ifnγ treated C57BL/6 APECs respectively. The significance with respect to untreated controls, Ifnγ treated C57BL/6 APECs controls. untreated Nos2 -/- and SNAPlo alone Nos2 -/- APECs controls are represented as *, θ, Δ, and # respectively.