Journal: bioRxiv
Article Title: Actin dynamics regulation by TTC7A/PI4KIIIα axis limits DNA damage and cell death during leukocyte migration
doi: 10.1101/2021.10.14.464382
Figure Lengend Snippet: Chemical intervention of HD and TTC7A T cells. (a) Schematic representation of the PI4KIIIα / TTC7A to RhoA / ROCK regulatory pathway, including the chemical modulators used in this study. Inhibitors (red), activators (green) and PI4P (blue). (b-i) Mean instantaneous speed of HD (Black-empty bar) and/or TTC7A deficient (Red-empty bar) T cell treated (Filled bars) or not with different chemical modulators. Migration was assessed in 4µm x 5µm fibronectin-coated microchannels. Boxes include the 80% of the points and bars represent the higher and lower 10% of points. One-way ANOVA or Mann-Whitney test was used to evaluate statistical significance, as appropriate. One experiment representative of three is presented in each case. (b) HD cell treated with inhibitors of PI4KIIIα. 1.7µM of BF738735 (middle) and 100 nM of GSK-A1 (right) were used. (c) HD and TTC7A cell treated (Blue-filled bars) or not with 25 µM of PI4P. (d) Density maps of HD (left panel) and TTC7A (right panel) T cells, treated (bottom panels) or not (top panels) with PI4P, fixed while migrating and stained with actin-phalloidin. n≥30 cell each condition. Scale bar 2µm. (e) Quantification of signal intensity from density maps in d, for actin-phalloidin showed as front / rear ratio for HD (Black-filled point), HD-PI4P (Blue-empty point), TTC7A (Red-filled point) and TTC7A-PI4P (Blue-filled point). T-test was used to evaluate statistical significance. (f) HD cell treated with PI3K inhibitor (Tenasilib, 45 nM). (g) HD cell treated with AKT inhibitor (MK2206-2HCl, 7.5 nM). (h) HD cell treated with inhibitors of SRC. 1µM of SRC-Inhibitor1 1µM (middle) and 10µM of PP2 (right) were used. (i) HD cell treated with inhibitors of PYK2. 1µM of Defactinib (middle) and 10µM of PF-562271 10µM (right) were used. (j) HD and TTC7A T cells treated with AKT activator SC79 (16.5 µM). (k) Immunofluorescence of T cell blast fixed while migrating and stained with pAKT and Hoechst. HD (top panel), TTC7A (middle panel), TTC7A-PI4P (bottom panel). Representative image from 2 experiments, ≥24 cells analyzed in each condition and experiment. (l) Quantification of intensity of fluorescence / area of cells in (J). HD n=62, TTC7A n= 66 and TTC7A-PI4P n=70. Fluorescence intensity determined using Icy. Scale bar 2µm.
Article Snippet: BF738735 (1.7 µM), Tenasilib (45 nM), MK2206-2HCl (7.5 nM), SC79 (16.5 µM), Src Inhibitor1 (1µM), PP2 (10µM), Defactinib-HCl (1µM), PF-562271 (10µM) from Medchemexpress.
Techniques: Migration, MANN-WHITNEY, Staining, Immunofluorescence, Fluorescence
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