Journal: bioRxiv
Article Title: Plastin-3 membrane recruitment drives cell-in-cell invasion during entosis
doi: 10.64898/2026.03.17.709257
Figure Lengend Snippet: (A) Schematic overview of ROCK1 construct transfection used to assess entosis induction. (B) Representative immunoblots showing ROCK1, phosphorylated MLC2 (pMLC2), and tubulin (loading control) in MCF7 cells transfected with GFP, GFP–ROCK1, or constitutively active GFP–ROCK1 Δ3. Immunoblot analysis of pMLC2 was performed 6 h post-transfection, whereas ROCK1 expression was assessed at 24 h. (C) Representative 3D confocal images of live MCF7 cells expressing GFP–ROCK1 Δ3 and stained with SiR-Actin (red) and Hoechst (blue). White arrows indicate entotic structures. Boxed regions showing representative entotic structures are magnified, with corresponding orthogonal z-stack views shown alongside each image. (D) Quantification of entotic events in MCF7 cells expressing GFP–ROCK1 or GFP–ROCK1 Δ3. (E) Distribution percentages of GFP-positive cells participating in CIC structures as outer cells, inner cells, or both. (F) Representative time-lapse imaging (phase contrast, Hoechst, and GFP) capturing an entotic event in MCF7 cells expressing GFP–ROCK1 Δ3. Yellow dotted lines outline inner cells, and red dotted lines outline host cells. Experiments were performed in biological triplicate (n = 3). Data are presented as mean ± SEM. Statistical analysis in panel D was performed using a paired two-tailed Student’s t-test (**p < 0.01), whereas panel E was analyzed using two-way ANOVA followed by Bonferroni post hoc test (*p < 0.05, **p < 0.01, ****p < 0.0001).
Article Snippet: Membranes were blocked in 5% non-fat milk in TBS-T or in 5% BSA in TBS-T when detecting phospho-specific antibodies and incubated with primary antibodies against ROCK1 (rabbit monoclonal, 1:1000; Cell Signaling Technology, #28999S), RhoA (rabbit monoclonal, 1:1000; Cell Signaling Technology, #2117S), PLS2 (rabbit monoclonal, 1:1000; Atlas Antibodies, #HPA019493), PLS3 (mouse monoclonal, 1:1000; Invitrogen, #MA5-27772), α-tubulin (rabbit polyclonal, 1:3000; Cell Signaling Technology, #2144S), phospho-MLC2 (Ser19) (rabbit, 1:1000; Cell Signaling Technology, #3671S), and GFP (chicken polyclonal, 1:1000; Rockland Immunochemicals, #600-901-215).
Techniques: Construct, Transfection, Western Blot, Control, Expressing, Staining, Imaging, Two Tailed Test
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