Journal: Journal of Cell Science

Article Title: α5β1 integrin trafficking and Rac activation are regulated by APPL1 in a Rab5-dependent manner to inhibit cell migration

doi: 10.1242/jcs.207019

Figure Lengend Snippet: APPL1 alters α5 integrin dynamics. (A) HT1080 cells expressing α5-PA-GFP and either mCherry or APPL1-mCherry were subjected to live-cell photoactivation experiments. Images from selected time points are shown and are pseudocolored for intensity. Circle demarcates ROI used for photoactivation and subsequent average fluorescence intensity measurements. Dotted lines indicate the boundaries of the cell. Scale bar: 15 μm. (B) Quantification of experiments from A as average fluorescence intensity, normalized to the amount of signal within the ROI at time=0. (C) HT1080 cells expressing APPL1 gRNA#3 or NT gRNA were transfected with α5-PA-GFP and mCherry-paxillin and subjected to live-cell photoactivation experiments. White boxes in top images indicate regions shown at higher magnification in bottom panels. Bottom left, zoomed images from top panel showing mCherry-paxillin. Arrows indicate adhesions chosen as ROI for photoactivaiton. Bottom right, images showing α5-PA-GFP fluorescence from selected time points, pseudocolored for intensity. White circles represent ROI used for quantification. Scale bars: 15 μm (top) and 5 μm (bottom). (D) Quantification of photoactivation experiments from C. Error bars represent the s.e.m. of 14–15 cells (B) or 27–28 cells (D) from each condition from three separate experiments. A nonparametric regression analysis was used to fit the curves (using natural cubic splines with three knots) and then the difference in the curves compared between two groups using the likelihood ratio test (**P<0.001).

Article Snippet: The average fluorescence intensity was quantified by dividing the background-corrected, integrated fluorescence intensity of individual cells by the cell unit area using MetaMorph software.

Techniques: Expressing, Fluorescence, Transfection

Journal: Journal of Cell Science

Article Title: α5β1 integrin trafficking and Rac activation are regulated by APPL1 in a Rab5-dependent manner to inhibit cell migration

doi: 10.1242/jcs.207019

Figure Lengend Snippet: APPL1 decreases PAK activity. (A,C,E,K,M) HT1080 cells expressing GFP or APPL1-GFP (A,C) and APPL1-AAA-GFP (K) or APPL1-N308D-GFP (M), APPL1 gRNA#3 or NT gRNA control cells (E) were plated on FN (A,E,K,M) or ColI (C) and immunostained for PAK phosphorylated at threonine 423 (p-PAKThr423). Representative images are pseudocolored for intensity. Scale bars: 15 μm. (B,D,F,L,N) Quantification of p-PAKThr423 levels from A,C,E,K and M, respectively, as a percentage of control. Error bars represent the s.e.m. from at least 58 cells total from each condition from three separate experiments [*P<0.05, **P≤0.007, n.s, not significant, P=0.81, determined by Student's t-test (B,D,F) or one-way ANOVA followed by Tukey's post hoc test (L,N)]. (G) HT1080 cells expressing APPL1 gRNA#3 or NT gRNA (control) were lysed and western blots were performed for phosphorylated PAK (pPAK), total PAK, or β-actin (loading control). A representative image is shown. M, molecular weight marker. (H,I) Quantification of pPAK (normalized to total PAK levels) (H) or total PAK (I) levels from G as a percentage of control. Error bars represent the standard error of the mean (s.e.m.) from three separate experiments. (**P<0.005, n.s., not significant, P=0.51, determined by Student's t-test). (J) Box plot for migration assays performed for cells co-transfected with APPL1-GFP or GFP and either constitutively active PAK (CA-PAK) or vector control. At least 25 cells total were analyzed from each condition from three separate experiments (***P<0.0001, n.s., not significant, P=0.63, determined by Student's t-test). (O) Cells expressing APPL1-GFP or GFP immunostained for F-actin (phalloidin, red) and p-PAKThr423 (pseudocolored for intensity). Red boxes in the second set of panels indicate regions shown at higher magnification in the third and fourth panels. White boxes represent a 5 μm region corresponding to the leading edge of the cell used for linescan analysis in this example. Scale bars: 15 μm (left) and 5 μm (right). (P) Linescan analysis performed at the leading edge of cells (identified by intense phalloidin staining) expressing APPL1-GFP or GFP. Quantification of the average fluorescence intensity is represented as a percentage of control (GFP at 0 μm). Error bars represent the s.e.m. from 45–50 cells total from each condition from three separate experiments (Z=−5.012, P<0.0001, determined by Wilcoxon signed-rank test).

Article Snippet: The average fluorescence intensity was quantified by dividing the background-corrected, integrated fluorescence intensity of individual cells by the cell unit area using MetaMorph software.

Techniques: Activity Assay, Expressing, Control, Western Blot, Molecular Weight, Marker, Migration, Transfection, Plasmid Preparation, Staining, Fluorescence