mruby lifeact 7  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Loss of RND3/RHOE controls entosis through LAMP1 expression in hepatocellular carcinoma

doi: 10.1038/s41419-024-06420-3

Figure Lengend Snippet: A Dynamics of entosis upon Rnd3 silencing. Hep3B cell lines were transduced with H2B-GFP (yellow) and LifeAct-mRuby (cyan) to mark the nucleus and the actin, respectively. Cells were transfected with siRNA targeting Rnd3 and spinning-disk microscopy analysis was done over 48 h. The gallery corresponds to Video . Time is in hours. Three stages were defined: (i) cell contact, (ii) internalization, arrows show the internalization of the inner binuclear cell 21 h after contact between cells and (iii) degradation of the inner cell; the inner cell degradation starts about 2 h after internalization and the cell degradation takes almost 10 h to be completed. Scale bar, 28 µm. B Analysis of entosis by correlative light-scanning electron microscopy. Entotic cells characterized by the nucleus (H2B-GFP, green) deformation and high concentration of actin (LifeAct-mRuby, red) in the inner cell were chosen using immunofluorescence microscopy (scale bar, 100 µm) and then the same event was analyzed by scanning electron microscopy. Entotic cells were taken with two magnifications 1000x (scale bar, 5 µm) and 2000x (scale bar, 10 µm). C Two populations of Hep3B cells were mixed, one population transduced with H2B-GFP, and another one transduced with H2B-RFP and transfected with siRNA targeting Rnd3. 48 h after mixing, the quantification of entotic cells was performed by evaluating the percentage of different combinations, Green in Green (wild-type in wild-type cells), Green in Red (Wild-type in siRnd3-transfected cells), Red in Green (siRnd3-transfected in wild-type) and Red in Red (siRnd3-transfected in siRnd3-transfected cells). Error bars: SD of three or more independent experiments. Significance was determined with the Mann–Whitney U -test. Examples of events counted after mixing of Rnd3-silencing RFP cells and wild-type GFP cells; G in G = Green in Green, G in R = Green in Red, R in G = Red in Green, R in R = Red in Red. Scale bar, 15 µm.

Article Snippet: The nutrient deprivation was performed by culturing the cells in DMEM 1x glutamax with a very low concentration of glucose (Fisher Scientific) and supplemented with 10% of dialyzed heat-inactivated FBS for 10–13 h. For induction of cell suspension, the adherent cancer cells were trypsinized and cultured in non-treated plastic Petri dishes for 13 h. Stable cell lines with fluorescent nuclei or fluorescent F-actin were generated through transduction with lentiviruses expressing H2B-GFP (Addgene #25999), H2B-RFP (Addgene #26001) or LifeAct-mRuby.

Techniques: Transduction, Transfection, Microscopy, Electron Microscopy, Concentration Assay, Immunofluorescence, MANN-WHITNEY