The red dotted lines mark the arbitrary border of the LF and DZ as in Fig.?1A. to four cell layers immediately distal from the leaders of the group. Talin-1 (hereafter referred to as talin) and paxillin, both markers of FAs, mainly localize within 10?m of the free edge of leader cells in small puncta (immature FAs, sometimes termed focal complexes) and variably sized FAs (Fig.?1A). There are no obvious differences in staining observed with anti-talin and -paxillin antibodies in these cells (Fig.?1A). FAs are relatively rare both at the rear of the leader cells and in follower cells although there are occasional groups of FAs away from cell edges (Fig.?1A). The overall FA organization in HaCaT cells we describe is consistent with observations by others (Stehbens et al., 2014). We quantified FA staining in our images and compared FA density within a zone, 10?m thick, of the free Rgs5 surface of leader cells (leading front, LF) as well as FA density within a zone of 120?m thickness, distal to the LF (for convenience we term this the distal zone, DZ). The DZ consists of both the rears of leader cells and several (up to four) layers of follower cells (Fig.?1A). There is a significant decrease in FA density in the DZ compared with the density in the LF (Fig.?1A,B). It should be noted that talin, paxillin and F-actin distribution in the DZ are similar, if not identical, to that in keratinocytes in intact monolayers (Fig.?S1A). Open in a separate window Fig. 1. FA protein localization in HaCaT cells. (A) Talin, paxillin and F-actin (labeled with phalloidin as indicated) localization in a scratch-wounded monolayer cell sheet of HaCaT cells at 4?h after wounding. The red dotted line indicates an arbitrary border between the leading front (LF) and distal zone (DZ) of the wounded monolayer (bracketed at the right of the images). Scale bar: 20?m. (B) FA density at the LF and DZ in scratch-wounded monolayers of HaCaT cells as in A (means.e.m.; n=4). (C) -PIX and talin localization in a scratch-wounded monolayer at 4?h after wounding. The third panel from the left shows overlays of the two stains (green, -PIX; red, talin). The boxed area is shown at a higher magnification in the fourth panel. Scale bars: 20?m (first panel); 2?m (fourth panel). (D) Extracts of parental HaCaT cells (HaCaT WT), HaCaT cells expressing control Prodigiosin shRNA (HaCaT conshRNA), and two cloned lines of HaCaT cells expressing -PIX shRNA (-PIX KD HaCaT C9 and C19) were processed for immunoblotting using antibodies against -PIX. Reactivity of a lamin antibody with lamin C was used as a loading control. (E) Immunoblots as in D were quantified and the levels of -PIX protein normalized to the lamin C protein level in extracts are presented relative to those in HaCaT WT cells (set at 1) (meanss.e.m.; n=3 independent samples). **P<0.01, ***P<0.001 (Student's t-test). -PIX colocalizes with talin in FAs in both the LF and in the DZ (Fig.?1C). In Prodigiosin some Prodigiosin FAs in the LF of a wounded monolayer, -PIX and talin staining in individual FAs does not completely overlap (Fig.?1C). In addition to its FA association, -PIX can be seen distributed as small puncta throughout the cytoplasm of both leader and follower cells. These puncta are most obvious in the high-power image shown in Fig.?1C. Likewise, in intact cell sheets, -PIX localizes to small puncta but is also co-distributed with Prodigiosin talin in small FA-like structures (Fig.?S1B). In single cells, -PIX associates with talin-positive FAs, but the staining generated by talin and -PIX antibodies does not necessarily overlap within individual FAs (Fig.?S1C). -PIX also localizes to small cytoplasmic puncta (Fig.?S1C). In both single HaCaT cells and HaCaT cell sheets, -PIX does not co-distribute with the hemidesmosome protein 4 integrin, which is found in puncta distinct from both FAs and the puncta stained by anti-talin and -paxillin antibodies (Fig.?S1D; localizations of -PIX and 4.