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. Author manuscript; available in PMC: 2023 Aug 19.
Published in final edited form as: Curr Biol. 2021 Dec 8;32(2):412–427.e8. doi: 10.1016/j.cub.2021.11.040

Figure 4. Calpain-2-induced talin-1 cleavage-dependent phenotypic conversion to blebbing amoeboid migration.

Figure 4.

A, Talin-1 protein expression, including full length (270 kDa) and cleavage fragment (190kDa), after 48–72 h (4T1) and 96 h (UT-SCC38) of hypoxic culture or HIF-stabilization with DMOG. Representative Western blots with densitometry values (“D”) (top panel) and band intensity by densitometric analysis of the change of the ratio talin-1 cleaved / full-length as the means ± s.d. normoxic or vehicle control culture from n=4–5 independent experiments. * P=0.01, ** P=0.001 (4T1), ** P=0.007 (UT-SCC38) (unpaired t-test, two-sided). B, Full-length and cleaved Talin-1 content in 4T1 cells in hypoxia (48h) or HIF-stabilization (24h) and UTSCC38 cells after HIF- stabilization (72h) alone or combined with calpain inhibition (PD150606, 100 μM). C) Talin-1 cleavage product in 4T1 cells after transient siRNA (100nM)-mediated downregulation of calpain-2. Data in (B,C) show representative Western blots from at least 3–4 independent experiments. Quantification was performed as in (a). * P=0.01–0.03, ** P=0.005 (b), ** P=0.001 (c), **** P<0.0001 (unpaired t-test, two-sided). D, Representative brightfield micrographs of 4T1 tumoroids expressing GFP or calpain-uncleavable GFP-talin (T432L) invading into 3D collagen. Insets show single-cell migration phenotypes after 72 h of culture. Scale bar, 100 μm (overviews), 10μm (insets). E, Morphology of individualized cells expressing calpain-uncleavable talin (L432G) during hypoxia- or DMOG-treatment. Morphometric subtypes: E, elongated; P pseudopodal-amoeboid; B, blebbing-amoeboid. Data represent the means ± s.d. (5 tumoroids/experiment, n=3). *** P < 0.0009, ** P < 0.003 and * P < 0.04 (two-way ANOVA). F, Subcellular localization of uncleavable talin. Representative confocal micrographs of detached 4T1 cells expressing GFP-TalinL432G during migration in 3D collagen 72 h after transfection and simultaneous HIF-stabilization. Arrowheads, focalized clusters. Scale bar, 100 μm (overview), 50 μm (inset).