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tra12131-sup-0001-FigureS1.docxWord document264KFigure S1: Western blot analysis of Rab11, Rab9 and Rab8 in CaCo2 controls (Ctr), CaCo2 knock down (KD) as well as CaCo2 cells with a myosin Vb knock down expressing FLAG-tagged myosin Vb (Resc). Tubulin was used as a loading control. Slight reduction of Rab8 in CaCo2 knock down cells.
tra12131-sup-0002-FigureS2.docxWord document328KFigure S2: Western blot analysis of cell lysates probed for the class V myosins, myosin Va and myosin Vc in CaCo2 cells. CaCo2 control cells (Ctr), CaCo2 cells with a myosin Vb knock down (KD) and CaCo2 rescued cells (Resc). Increased expression levels of myosin Va and myosin Vc in CaCo2 knock down cells. IQGAP1 was used as a loading control.
tra12131-sup-0003-FigureS3.docxWord document412K

Figure 3a: Measurement of transepithelial electrical resistance (TEER) in CaCo2 control (CaCo2 CTR) and CaCo2 cells with a myosin Vb knock down (CaCo2 KD). TEER measurements were performed on day 5 and day 10, respectively, at 3 different areas of the filter inserts and out of 5 independent experiments (n = 5). The final p value was p ≤ 0.02 meaning all values under or equal to 0.02 were assumed as being statistically significant. Overall decrease of TEER in CaCo2 cells with a myosin Vb knock down (CaCo2 KD) on both days of measurements (day 5 and day 10).

Figure 3b: Radioactive lipid uptake in CaCo2 cells. CaCo2 control (CaCo2 CTR) and CaCo2 cells with a myosin Vb knock down (CaCo2 KD). Radioactive lipid uptake was measured of 5 different filter inserts cultured with either CaCo2 control (CaCo2 CTR) or CaCo2 cells with a myosin Vb knock down (CaCo2 KD) of 2 independent experiments. Mean lipid uptake in percentage for CaCo2 control = 11.  42, for CaCo2 cells with a myosin Vb knock down = 7.41. About 30 % reduction in lipid uptake in CaCo2 cells with a myosin Vb knock down. The final p value was p ≤ 0.05 meaning all values under or equal to 0.05 were assumed being statistically significant.

tra12131-sup-0004-FigureS4.docxWord document1367K

Figure 4a: Western blot analysis of cell lysates probed for endogenous myosin Vb as well as FLAG-tagged myosin Vb in CaCo2 cells with a myosin Vb knock down expressing FLAG-tagged myosin Vb (Resc). CaCo2 control cells (Ctr), CaCo2 cells with a myosin Vb knock down (KD) and CaCo2 rescued cells (Resc). The endogenous myosin Vb blot showed an upper unspecific band marked by a black star. IQGAP1 was used as a loading control.

Figure 4b: Staining analysis of myosin Vb CaCo2 cells with a myosin Vb knock down expressing FLAG-tagged myosin Vb protein (CaCo2 rescue) in a 3D projection of confocal image z-stack sections of CaCo2 cell monolayers on a Transwell filter. CaCo2 control cells (CaCo2 control), CaCo2 cells with a myosin Vb knock down (CaCo2 knock down) and rescued CaCo2 cells (CaCo2 rescue). Myosin Vb (ALEXA 568, red), actin (Phalloidin ALEXA 488, green) and nuclei (Hoechst, blue). Expression as well as cytoplasmic distribution of myosin Vb along actin filaments in CaCo2 rescued cells. Scale bars = 5 µm.

tra12131-sup-0005-FigureS5.docxWord document799KFigure S5: Effects of myosin Vb depletion in enterocytes. A) Summary table showing cellular alterations caused by a non-functional myosin Vb motor in enterocytes. B) In subpanel (a) polarized enterocytes are characterized by functional tight junctional complexes, organized and appropriate expression and localization of transporter proteins (CD36, Na/K ATPase and TfR) and an intact apical brush border compartment. Thereby trafficking proteins such as Rab GTPases as well as properly localized cell organelles have to be appropriately organized in mature enterocytes. This is shown by subapically localized early, recycling and late endosomes as well as lysosomal compartments, diffuse distribution of Rab11 positive vesicles subapically in the cytoplasm as well as Rab9 distribution perinuclearly in the area of the Golgi compartment and Rab8 localizing either at the apical or at the basolateral membrane and found distributed perinuclearly in the Golgi area. Subpanel (b) shows the effects of myosin Vb depletion in mature enterocytes. Loss of apical microvilli and formation of cytoplasmic inclusions, disorganized actin cytoskeleton, epithelium leakage are because of defective tight junctional complexes, mislocalized transporter proteins (CD36, Na/K ATPase and TfR), which are either from apical or basolateral and basal membrane compartments to the cytoplasm. Furthermore, Rab GTPases, as well as organelles are redistributed, shown by Rab11 clustering subapically in the area of EEA1 clusters or perinuclearly, furthermore perinuclear LAMP2 clusters, as well as perinuclear Rab9 and Rab8 redistribution in the area of the Golgi compartment. EE, early endosome; LE, late endosome; LY, lysosome; RE, recycling endosome; TfR, Transferrin receptor; ER, endoplasmatic reticulum.

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