Journal: Life Science Alliance

Article Title: MAP4K4 regulates forces at cell–cell and cell–matrix adhesions to promote collective cell migration

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: (A) Representative immunoblotting of MAP4K4 and actin using lysates of A431 control cells (sgNT), or A431 cells KO for MAP4K4 with two independent sgRNA (M4K4_sg1, M4K4_sg2). (B) Mean velocity of A431 clusters control or KO for MAP4K4 , tracked over 5 h of migration. (C) Mean velocity of A431 clusters treated with DMSO or GNE-495 at different doses (0.1, 0.5, or 1.0 μM), over 5 h of treatment. Number of clusters analyzed (sgNT: 34, M4K4_sg1: 48, M4K4_sg2: 35, DMSO: 22, GNE 0.1 μM: 34, GNE 0.5 μM: 26, GNE 1.0 μM: 33), from three independent experiments. (D, E) z-scan projection of representative confocal images of F-actin stained A431 clusters, showing the differences in the actin cytoskeleton organization and in the morphology of clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2) or (E) clusters treated with DMSO or GNE-495 at 1.0 μM for 24 h. Arrows represent the actin arches at protrusion bases and arrowheads indicate retraction fibers. (F) Protrusion area of control/ MAP4K4 KO cells or DMSO/GNE-495–treated cells with indicated doses. At least five clusters per experiment, three protrusions per cluster from three independent experiments were analyzed. (G) Circularity of control/ MAP4K4 KO cell clusters, or clusters treated with DMSO or GNE-495 at indicated doses. At least 25 clusters from three independent experiments were analyzed. (H, I) Mean velocity extension (H) or retraction (I) events at the periphery of the clusters before or after treatment with DMSO or GNE-495 at 1.0 μM, over 5 h of treatment. Number of clusters analyzed (DMSO: 28, GNE 0.1 μM: 26, GNE 0.5 μM: 26, GNE 1.0 μM: 28) from three independent experiments. All the data are presented as mean ± s.d. and tested by Kruskal–Wallis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Western Blot, Control, Migration, Staining

Journal: Life Science Alliance

Article Title: MAP4K4 regulates forces at cell–cell and cell–matrix adhesions to promote collective cell migration

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: (A) Representative confocal images of pERM staining, at the substrate z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (B) Quantification of mean intensity of pERM from clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (C, D) Number and (D) length of retraction fibers of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (E) Representative confocal images of pERM staining at the cell–cell junction z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (F) Quantification of mean intensity of pERM of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (G) Representative immunoblotting of moesin and actin from lysates of A431 cells control (Rosa_sg) or KO for MSN using two independent sgRNA sequences ( MSN _sg1, MSN _sg2). (H) Representative confocal images of control or MSN KO clusters stained for zyxin. (I) Number of zyxin-positive focal adhesions in control or MSN KO clusters. (J) Representative confocal z-scan projection of p120 stained cells, control or KO for MSN , showing cell–cell junction morphology. (K) Tortuosity index of cell–cell junction on control or MSN KO cells. At least three junctions of five different clusters per experiment, from three independent experiments were analyzed. All the data are presented as mean ± s.d. and tested by Mann–Whitney test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Staining, Western Blot, Control, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: MAP4K4 regulates forces at cell–cell and cell–matrix adhesions to promote collective cell migration

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: (A, B, C) z-scan projection of representative confocal images of β-catenin stained A431 clusters, stably expressing (A) eGFP–MAP4K4_WT, (B) eGFP–MAP4K4 kinase dead (MAP4K4 D153N ), or (C) deleted for the CNH domain (MAP4K4 ΔCNH ). Line scan indicates the colocalization between β-catenin and MAP4K4. (D) Immunobloting of MAP4K4 or actin for lysates of A431 cells controls (non-infected or sgNT), KO for MAP4K4 (sg_1 or sg_2) alone or expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. (E) Number of zyxin-positive focal adhesions for A431 clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2) and stably expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. (F) Cell–cell junction tortuosity index for A431 clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2), and stably expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. Data on (E, F) are represented as mean ± s.d. and tested by Kruskal–Wallis (ns, nonsignificant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Staining, Stable Transfection, Expressing, Western Blot, Infection, Control