Fig 2: Membrane association of membrane anchoring proteins is lost during cleavage regression in endomitosis. (A–D) Representative stills of IF experiments showing DAPI, α-tubulin, E-cadherin, and membrane-anchoring protein (A) RacGAP1, (B) Anillin, (C) SEPT9, or (D) CIT-K in late anaphase and late telophase, with either ingressed or regressed cleavage furrows. Hep-Org 1 line expressing E-cadherin-tdTomato was used for IF stainings. Scale bars represent 3 µm. Close-ups show single- or double-channel images of marked regions of interest (white box). Dashed lines represent membrane outline. All images show one plane in the middle of the midbody. Numbers in the top right show quantification of displayed localization. More detailed quantifications of RacGAP1, Anillin, SEPT9, and CIT-K localizations during the different stages are shown in Fig. 4.

Fig 3: Loss of cell motility in MINC1 treated cells. A) MINC1 treated A549 cells lose the ability to move freely in 2D culture. Cells were imaged every 5 min, and images were used to track cell movement over 24 h. B-C) Cells treated with increasing concentrations of MINC1 display a decreasing ability to migrate, and siRNA transfected cells show increased wound-healing times. MDA-MB-231 cells were treated with an increasing dose of MINC1 (B) or two pooled siRNAs targeting MgcRacGAP (C). Error bars represent SEM (n=4) (color image is available online).

Fig 4: Clinical significance of the expression levels of seven genes targeted by miR-204-5p (RACGAP1, DHRS9, AP1S3, FOXC1, PRP11, RHBDL2 and MUC4) based on data from The Cancer Genome Atlas (TCGA) database. (A) Kaplan-Meier plots of overall survival and (B) disease-free survival with log-rank tests comparing the survival of PDAC patients with high (red lines) versus low (blue lines) expression levels of each of the abovementioned genes, based on data from TCGA database.

Fig 5: Directed transport of ALIX and CHMP4B to the midbody. a ALIX transport into the ICB and recruitment to the midbody. Selected frames at indicated time points from time-lapse imaging of cytokinetic cells stably expressing ALIX-mCherry (magenta) upon addition of SiR-tubulin (green). Arrowheads indicate transport of individual vesicles at given time points. Scale bar = 5 µm. b Detection of endogenous ALIX in the cytokinetic bridge. SIM micrograph of fixed cells stained for ALIX (magenta), RacGAP1 (green) and tubulin (blue) showing ALIX in vesicular structures in the ICB, at the abscission site and at the midbody (arrowheads from right to left). Scale bar = 1 µm. c ALIX co-transport with CHMP4B into the ICB. Selected frames at indicated time points from time-lapse imaging of cytokinetic cells stably expressing CHMP4B-GFP (green) and ALIX-mCherry (magenta) upon addition of SiR-tubulin (blue). Arrowheads indicate transport of individual vesicles at given time points. Scale bar = 2 µm. d Detection of endogenous ALIX/CHMP4B proximity by fluorescent microscopy using Duolink® PLA. Fluorescent dots (red) represent ALIX in close proximity with CHMP4B. In control cells dots can be detected in the cell body as well as in the ICB, stained by tubulin (grey), and at the midbody. ALIX knockdown in cells leads to a substantial decrease in the number of dots. Scale bars = 10 µm. e Co-transport of ALIX and Rab11. Selected frames from a time-lapse microscopy of cytokinetic cells expressing Rab11-GFP (green) and ALIX-mCherry (magenta) with SiR-tubulin (blue). Arrowheads of same color indicate transport of individual vesicles at given time points. Scale bar = 3 µm. f Detection of endogenous ALIX/Rab11 proximity by fluorescent microscopy using Duolink® PLA. Fluorescent dots (red), which represent ALIX in close proximity with Rab11, can be detected in the cell body as well as in the ICB, stained by tubulin (grey), and at the midbody. Scale bars = 10 µm. g Co-transport of ALIX and TSG101. Selected frames from a time-lapse microscopy movie of cytokinetic cells expressing GFP-TSG101 (green) and ALIX-mCherry (magenta) with SiR-tubulin (blue). Arrowheads indicate transport of an individual vesicle at given time points. Scale bar = 3 µm. h Detection of endogenous ALIX/TSG101 proximity by fluorescent microscopy using Duolink® PLA. Fluorescent dots (red), which represent ALIX in close proximity with TSG101, can be detected in the cell body as well as in the ICB, stained by tubulin (grey). Scale bars = 10 µm. i Scatter plot showing the time interval between bridge formation and first appearance of GFP-TSG101 at the midbody in control (ctrl.) cells or upon ALIX knockdown (KD) as indicated (n ≥ 26 cells per treatment from four independent experiments; control: 53 ± 1.6 min; ALIX KD: 67.7 ± 3.2 min [± SEM]; P < 0.01)