Phospho-MYPT1 (Ser472) Antibody - #AF3779

Fig. 4: ATP levels control plasticity of cell migration through AMPK. a AMP, ATP/AMP and ATP/ADP in A375P cells treated with rotenone (0.5 μM) and antimycin A (0.5 μM) (5 replicates/condition). b Western blot for the same conditions as in (a). c Western blot of the indicated proteins (WM983 n = 6, A375 n = 5). d Western blot upon AMPK knock-down (n = 3). e Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) (n = 3). Scale bar = 5 μm. f–h Quantification of cell morphology (211, 193 cells pooled from n = 3) (f), pMLC2 immunofluorescence signal normalized by cell area (52, 66 cells pooled from n = 3) (g) and representative traction stress (n = 3). Colour bar indicates traction stress magnitude (Pascal, Pa), scale bar = 20 μm (h). i Western blot upon AMPK activation (A769662 10 μM, 30 minutes) (n = 5). j–l Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) after A769662 treatment (10 μM, 24 h) (n = 3), scale bar = 10 μm (j); quantification of cell morphology (317, 283 cells pooled from n = 3) (k) and quantification of pMLC2 immunofluorescence signal normalized by cell area (81, 84 cells pooled from n = 3) (l). m, n Western blot and quantification after DDR1 knock-down and Comp C treatment (2 μM, 24 h) (n = 8 AMPK, n = 6 ACC, n = 9 MYPT1). o–q Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) (n = 3), scale bar = 10 μm (o); quantification of cell morphology (274, 210, 274, 191 cells pooled from n = 3) (p) and quantification of pMLC2 immunofluorescence signal normalized by cell area (93, 84, 94, 93 cells pooled from n = 3) (q). Quantification versus corresponding total protein (b, c, d, i). Graphs (a, n) show mean ± SEM. Violin plots (f, k, p) show median with interquartile range. Box plots (g, l, q) show median (centre line), interquartile range (box) and min-max values (whiskers). p values were calculated using two-tailed tests (f, g, k, l). p value by Mann–Whitney test (f, g, k, l), one-way ANOVA with Dunnett’s correction (a) or Tukey’s multiple test (n) and Kruskal-Wallis with Dunn’s multiple comparisons test (p, q). All n are indicative of independent experiments unless otherwise stated. Source data are provided as a Source Data file.

Fig. 4: ATP levels control plasticity of cell migration through AMPK. a AMP, ATP/AMP and ATP/ADP in A375P cells treated with rotenone (0.5 μM) and antimycin A (0.5 μM) (5 replicates/condition). b Western blot for the same conditions as in (a). c Western blot of the indicated proteins (WM983 n = 6, A375 n = 5). d Western blot upon AMPK knock-down (n = 3). e Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) (n = 3). Scale bar = 5 μm. f–h Quantification of cell morphology (211, 193 cells pooled from n = 3) (f), pMLC2 immunofluorescence signal normalized by cell area (52, 66 cells pooled from n = 3) (g) and representative traction stress (n = 3). Colour bar indicates traction stress magnitude (Pascal, Pa), scale bar = 20 μm (h). i Western blot upon AMPK activation (A769662 10 μM, 30 minutes) (n = 5). j–l Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) after A769662 treatment (10 μM, 24 h) (n = 3), scale bar = 10 μm (j); quantification of cell morphology (317, 283 cells pooled from n = 3) (k) and quantification of pMLC2 immunofluorescence signal normalized by cell area (81, 84 cells pooled from n = 3) (l). m, n Western blot and quantification after DDR1 knock-down and Comp C treatment (2 μM, 24 h) (n = 8 AMPK, n = 6 ACC, n = 9 MYPT1). o–q Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) (n = 3), scale bar = 10 μm (o); quantification of cell morphology (274, 210, 274, 191 cells pooled from n = 3) (p) and quantification of pMLC2 immunofluorescence signal normalized by cell area (93, 84, 94, 93 cells pooled from n = 3) (q). Quantification versus corresponding total protein (b, c, d, i). Graphs (a, n) show mean ± SEM. Violin plots (f, k, p) show median with interquartile range. Box plots (g, l, q) show median (centre line), interquartile range (box) and min-max values (whiskers). p values were calculated using two-tailed tests (f, g, k, l). p value by Mann–Whitney test (f, g, k, l), one-way ANOVA with Dunnett’s correction (a) or Tukey’s multiple test (n) and Kruskal-Wallis with Dunn’s multiple comparisons test (p, q). All n are indicative of independent experiments unless otherwise stated. Source data are provided as a Source Data file.

Figure 4: Energy levels control plasticity of cell migration through AMPK signalling. A-B) Western blot (A) and quantification (B) of AMPK and MYPT1 phosphorylation in rounded-amoeboid versus elongated-mesenchymal cells (n=3). C) Upon AMPK inhibition (Compound C (Comp C) 2 mM, 24 hours) in WM983B and A375M2 cells, western blot showing levels of AMPK and MYPT1 phosphorylation (n=3). Quantification normalized by total AMPK and MYPT1, respectively. D) Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) after Comp C treatment (2 mM, 24 hours). Scale bar = 10 mm. E-F) After Comp C treatment, quantification of cell morphology (>200 cells pooled from 3 experiments) (E) and quantification of pMLC2 immunofluorescence signal normalized by cell area (>90 cells pooled from 3 experiments) (F). G) Upon AMPK activation (A769662 10 mM, 30 minutes) in WM983A and A375P cells, western blot showing levels of AMPK and MYPT1 phosphorylation (n=3). Quantification normalized by total AMPK and MYPT1, respectively. H) Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) after A769662 treatment (10 mM, 24 hours). Scale bar = 10 mm. I-J) After A769662 treatment, quantification of cell morphology (>200 cells pooled from 3 experiments) (I) and quantification of pMLC2 immunofluorescence signal normalized by cell area (>90 cells pooled from 3 experiments) (J). K-L) Western blot (K) and quantification (L) of AMPK and MYPT1 phosphorylation levels after DDR1 knockdown and treatment with Comp C (2 mM, 24 hours) in A375P cells (n=3). M) Cells grown on a collagen I matrix. Immunofluorescence images showing pMLC2 (green) and F-actin (red) after DDR1 knock-down and Comp C treatment (2 mM, 24 hours). Scale bar = 10 mm. N-O) After DDR1 knock-down and Comp C treatment, quantification of cell morphology (>200 cells pooled from 3 experiments) (N) and quantification of pMLC2 immunofluorescence signal normalized by cell area (>90 cells pooled from 3 experiments) (O). Graphs (B, L) show mean ± SEM. Dot plots (E, I, N) show median with interquartile range (each dot represents a single cell). Box plots (F, J, O) show min to max. p value by paired t-test (B), Mann-Whitney test (E, F, I, J), one-way ANOVA with Dunnett’s correction (L) and Kruskal-Wallis with Dunn’s multiple comparisons test (N, O). For all graphs, *p