Journal: Biology Open

Article Title: Dynamic microtubules drive fibroblast spreading

doi: 10.1242/bio.038968

Figure Lengend Snippet: The length of MT tracks in normal Vero cells and after MT stabilization with nocodazole and paclitaxel. Scale bars: 10 µm. (A) Maximum intensity projection of MT tracks visualized by EB3 protein in an untreated cell, (MIP of 15 frames, the time interval between frames is 2 s). MT tracks are organized into a radial array. (B) Maximum intensity projection of MT tracks visualized by EB3 protein in a cell with stabilized MTs, (MIP of 15 frames, the time interval between frames is 2 s) MT tracks are shortened compared to tracks in the untreated cell. (C) Maximum intensity projection of MT tracks visualized by EB3 protein in an untreated 3T3 cell, 20 min after initial attachment (MIP of 15 frames, the time interval between frames is 2 s). In the radially spreading cell, most plus ends grow into the nascent lamellae, the distance between MTs' plus ends and cell margin is short. (D) Maximum intensity projection of MT tracks visualized by EB3 protein in a 3T3 cell with stabilized MTs, 20 min after initial attachment (MIP of 15 frames, the time interval between frames is 2 s). Tracks of MTs' plus ends lose their radial organization, and the distance between MTs' plus ends and cell margin increases dramatically. (E) Length distribution of tracks in cells with normal and stabilized MTs. MT tracks are shorter in cells with stabilized MTs (gray bars) compared to untreated cells (black bars).

Article Snippet: EB3 shRNA probe [Pubmed Probe 3104666, described in ( )] was cloned into pGPV lentiviral vector containing copGFP as a reporter and puromycin as drug selection marker (Eurogene, Russia), lentiviral vector was cotransfected into HEK293 packaging cells (ATCC #CRL-11268) with X-tremeGENE HP DNA transfection reagent according to the manufacturer's protocol, viral harvest was collected at 36 and 72 h after transfection and then used for transduction of Vero cells.

Techniques:

Journal: Biology Open

Article Title: Dynamic microtubules drive fibroblast spreading

doi: 10.1242/bio.038968

Figure Lengend Snippet: Effects of EB3 depletion in Vero cells. (A) Life histories of single MTs in 3T3 cells transfected with alpha-tubulin, MTs in untreated cells demonstrate dynamic instability. (B) Life histories of MTs in 3T3 cells, expressing shEB3 and transfected with alpha-tubulin. In EB3-depleted cells dynamic instability of MTs was suppressed. (C–D) Immunofluorescent staining with EB3 antibodies, in EB3-depleted cells (D) comets are almost absent compared to control cells (C). (E) Spreading of Vero cells in normal conditions (white circles), in EB3-depleted cells (black circles) and in cells with stabilized MTs (black triangles). (F) Spreading of EB3-depleted cell is mainly anisotropic, the cell fails to form a large lamellum and spreads through formation of short lamellipodia. Scale bars: 10 µm.

Article Snippet: EB3 shRNA probe [Pubmed Probe 3104666, described in ( )] was cloned into pGPV lentiviral vector containing copGFP as a reporter and puromycin as drug selection marker (Eurogene, Russia), lentiviral vector was cotransfected into HEK293 packaging cells (ATCC #CRL-11268) with X-tremeGENE HP DNA transfection reagent according to the manufacturer's protocol, viral harvest was collected at 36 and 72 h after transfection and then used for transduction of Vero cells.

Techniques: Transfection, Expressing, Staining, Control