Journal: eLife

Article Title: G-protein-coupled receptor signaling and polarized actin dynamics drive cell-in-cell invasion

doi: 10.7554/eLife.02786

Figure Lengend Snippet: ( A ) Immunolabeling of endogenous mDia1 (green) and phalloidin staining of F-actin (red) of a MCF10A cell undergoing entosis. Nuclei are labeled by DAPI (blue). Scale bar 5 μm. ( B ) Visualization of mDia1-GFP (green) and mCherry-LifeAct (red) localization at the invading cell rear in fixed and non-permeabilized HEK293 cells co-transfected with LPAR2 to trigger cell-in-cell invasion events. Merged image including bright-field and DAPI (blue) is shown in the right panel. Scale bar 5 μm. ( C ) Immunolabeling of endogenous Ezrin (green) and F-actin (red) of control and mDia1 siRNA-treated MCF10A cells. ( D ) MCF10A cell population after incomplete siRNA treatment against mDia1 showing mDia1 knockdown of the upper two cells (red only) and endogenous mDia1 detection of the lower three cells were labeled for mDia1 (green) and F-actin (red). Note the presence of mDia1 on cellular blebs, while the two upper mDia1-negative cells fail to bleb. 2 frames are shown from a confocal z-scan using a LSM 700 (Zeiss). ( E ) MCF10A cells treated with indicated siRNAs were analyzed for the number of blebbing cells (n = 3 ± SD, p<0.007, t test). ( F ) MCF10A cells pretreated for 40 min with 20 μM of the LPAR inhibitor Ki16425 before analysis of the number of blebbing cells (n = 3 ± SD, p<0.001, t test). ( G ) MCF10A cells expressing LifeAct-GFP (green) or LifeAct-mCherry (red) silenced for control or mDia1 respectively. White arrowheads in the first frame indicate red (siDia1) and green (siMOCK) cell in contact with a host cell. Red arrowhead indicates addition of 100 nM Latrunculin B (LatB) at time frame 104 min. ( H ) MCF10A cells treated with indicated siRNAs for 48 hr were analyzed for entosis (n = 3 ± SD analyzed by one way ANOVA followed by Dunnett's post-tests compared with siMOCK group). ( I ) HEK293 cells expressing Flag-LPAR2 to trigger cell-in-cell invasion events were treated with indicated siRNAs for 48 hr before analyzing entosis rates (n = 3 ± SD analyzed by One way ANOVA followed by Dunnett's post-tests compared with Flag-LPAR2 expressing siMOCK group). DOI: http://dx.doi.org/10.7554/eLife.02786.012

Article Snippet: Poly (2-hydroxyethyl methacrylate) (PolyHEMA) was purchased from Polysciences Inc. Antibodies against EDG4 were from Assay Biotechnology; LPAR receptors, Ezrin and LARG from Santa Cruz Biotechnology; PDZ-RhoGEF from IMGENEX; pMLC2 from Sigma and mDia1 from BD Biosciences. pCMV6-XL5 LPAR2 expression vector were purchased from OriGene (SC117226). pWPXL-based lentiviral expression vectors for H2B, LPAR2, Gα12, and Gα12Q/L were generated using standard PCR-based procedures or in the case of LifeAct-GFP were a kind gift from Oliver Fackler.

Techniques: Immunolabeling, Staining, Labeling, Transfection, Control, Knockdown, Expressing