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Files in this Data Supplement:
Fig. S1. Analysis of caveolin-1 and galectin-3 protein expression, and quantitative PCR of different KDs in the cyst cultures. Caveolin-1 (rabbit anti-cav-1 antibodies, N20; Sigma) expression in the control (A) compared with Cav-1 KD (B), and galectin-3 (Mac-2 antibody; Cedarlane laboratories) expression in the control (C) compared with Gal-3 KD (D) cysts are shown by immunostaining with the specific antibodies and Alexa Fluor 488 secondary antibodies in green. Nuclei DAPI staining is visualized in gray pseudocolour. The z-sections shown are from the middle of the cysts. Scale bars, 30 micrometer. In E-G western blot analysis of the control and the Cav-1 KD cysts and Gal-3 KD cysts are shown. Total protein extracts were harvested after collagenase treatment as described (O’Brien et al., Nat Cell Biol, 2001). Twenty, five or one microgram of the total protein extracts (1×, diluted 1:4 or 1:20, respectively) were then separated on 12% SDS-PAGE and transferred onto nitrocellulose membranes, which were treated with specific antibodies, the same that were also used for the immunostaining of the cysts. The proteins identified by primary antibodies were detected with secondary HRP-conjugated antibodies, and SuperSignal West Pico Chemiluminiscent Substrate (Pierce) finally applied on the membranes. Caveolin-1 appears as a protein band of slightly over 20 kDa (E) and galectin-3 as a band of close to 35 kDa (F) as expected (indicated with arrowheads). The blots were also incubated with the mouse anti-gamma-tubulin antibody (Sigma). The image insets of the membranes below (G) show the amount of the 48 kDa gamma-tubulin (indicated with an asterisk) in the different protein extracts (1x, 1:4 or 1:20 per lane, respectively) as a loading control. Finally, results of a quantitative PCR analysis showing KD efficiencies at the time of cysts fixation (H).
Fig. S2. Inverted polarity in cysts of Crb3-KD and Stx-3 KD cells. Unlike the situation in the normal cysts, where the actin meshwork is facing the lumenal membrane, the inverted actin meshwork (TRITC-phalloidin; in red) of Crb-3 KD (A) and Stx-3 KD (B) cysts is facing the collagen I matrix. Nuclei are in blue. Staining of podocalyxin, pcx (A) and acetylated tubulin (cilia; B) appear in green. Scale bars, 30 µm.
Fig. S3. Stx-2 KD cysts. Overlay staining of actin (TRITC-phalloidin) and the nuclei (DAPI), in red and blue (A, B, C, and D), and podocalyxin (Pcx), CEA, E-cadherin (E-cad) or ZO-1, in green with the nuclei, respectively are shown. Scale bars, 30 micrometer.
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