br Fig Loss of ADD increases transfilter
Fig. 2. Loss of ADD1 increases transfilter migration and Matrigel invasion of lung cancer cells. ADD1 expression was down-regulated H1573 Vorinostat (SAHA, MK0683) using CRISPR/Cas9-mediated gene editing. (A) Immunoblotting analysis shows co-depletion of ADD1 and ADD3 by 4 diﬀerent ADD1 small guide (sg) RNAs. (B) Quantification of the planar migration of the control and ADD1-depleted H1573 cell monolayers at 24 h post-wounding. (C, D) Representative images and quantitative analysis of the DAPI-labeled control and ADD1-deficient H1573 cells after 16 h of transfilter migration in the Boyden chamber. (E, F) Representative images and quantitative analysis of the DAPI-labeled control and ADD1-deficient H1573 cells after 24 h invasion into Matrigel. Data are presented as mean ± SE (n = 3); **p < 0.005, as compared to the control sgRNA-transfected group.
collagen or fibronectin adhesion of either H1573 or 16HBE14o cells (Suppl. Fig. 5 and data not shown). Interestingly, the increased ECM adhesion and decreased transfilter migration of ADD1-overexpressed H1299 cells persisted even when ADD3 was also overexpressed (Suppl. Fig. 6), thereby arguing against a possibility that these are the neo-morphic eﬀects of the selective ADD1 overexpression.
There is a nonlinear relationship between the strength of cell-ECM adhesion and the velocity of cell migration, where migration can be inhibited by both insuﬃcient and very strong cell attachment to ECM [52,53]. Therefore, we rationalized that the observed hyperadhesive-ness of an ADD1-overexpressing H1299 cell is likely to contribute to their impaired motility. Cell adhesion to ECM depends on the assembly of specialized basal structures called ‘focal adhesions’ (FA) [54,55].
Immunofluorescence labeling of a classical FA marker, phosphorylated
(p) paxillin, revealed assembly of small FA localized to the edges of spreading control H1299 cells (Fig. 5C, arrows). ADD1 overexpression stimulated the formation of FA, which became enlarged and localized both at the periphery and the basal surface of spreading H1299 cells (Fig. 5C, arrowheads & Fig. 5D). Furthermore, immunoblotting analysis demonstrated that ADD1 overexpression significantly increased the le-vels of phosphorylated paxillin and phosphorylated (active) Src kinase (Fig. 5E), which is a key upstream regulator of FA assembly [56,57].
Since the formation of FA is known to be regulated by the acto-myosin cytoskeleton, we next examined the cytoskeletal organization in control and ADD1-overexpressing H1299 cells by labeling filamentous
(F) actin and an actin motor, non-muscle myosin IIB (NM IIB). Confocal
Fig. 3. Loss of ADD3 increases transfilter migration and Matrigel invasion of lung cancer cells. ADD3 expression was down-regulated H1573 cells using CRISPR/Cas9-mediated gene editing. (A) Immunoblotting analysis shows co-depletion of ADD3 and ADD1 by 4 diﬀerent ADD3 small guide (sg) RNAs. (B) Quantification of the planar mi-gration of the control and ADD3-depleted H1573 cell monolayers at 24 h post-wounding. (C, D) Representative images and quantitative analysis of the DAPI-la-beled control and ADD3-deficient H1573 cells after 16 h of transfilter migration in the Boyden chamber. (E, F) Representative images and quantitative analysis of the DAPI-labeled control and ADD3-deficient H1573 cells after 24 h invasion into Matrigel. Data are presented as mean ± SE (n = 3); *p < 0.05; **p < 0.005, as com-pared to the control sgRNA-transfected group.
microscopy revealed major morphological changes in ADD1-over-expressing cells, manifested by the formation of long peripheral pro-trusions enriched in F-actin and NM IIB (Fig. 6 arrows). To gain addi-tional insights into the mechanisms that control formation of such protrusions, we performed live cell imaging of migrating cells. Control H1299 cells demonstrated a mesenchymal-type motility with the orchestrated formation of the migrating cellular front and detachment/ forward translocation of the trailing cellular edge (Suppl. Movie 1). By contrast, ADD1 overexpressed cells failed to eﬃciently detach and re-tract the trailing edge resulting in the formation of long tail-like pro-trusions (Suppl. Movie 2). These results are consistent with the ob-served hyperadhesiveness of ADD1-overexpressed H1299 cells (Fig. 5).
Immunofluorescence labeling and confocal microscopy showed lo-calization of exogenous ADD1 in the poorly-retractable F-actin rich protrusions (Suppl. Fig. 7A, arrows). However a significant fraction of the overexpressed ADD1 accumulated in the nuclei of H1299 cells (arrowheads), which was confirmed by the nuclear/cytoplasmic cell fractionation (Suppl. Fig. 7B). This prompted us to investigate whether the observed eﬀects of ADD1 overexpression on H1299 cell adhesion and migration were mediated by its nuclear or non-nuclear fractions. Inhibition of the nuclear export by leptomycin B caused redistribution of peripheral ADD1 into the nucleus (Suppl. Fig. 8, arrows) and