|
Display additional annotations [+]
|
| |
Fig. 1. Lipid Rafts in the Pronephric Kidney. (A,B) Whole mount immunostaining of Xenopus pronephroi using the B-subunit of Cholera Toxin (CT-B) or an antibody against Caveolin-1 at stage 42. Proximal tubules were counterstained using the 3G8 antibody (A) or ECL (B). pt, proximal tubules; dt, distal tubules. (C–H׳) Cryostat sections of stage 35, 39 and 42 Xenopus embryos stained with CT-B and either 3G8 (C–F׳) or 4A6 (G–H׳). (I–N׳) Paraplast sections of stage 35, 39 and 42 Xenopus embryos stained with Caveolin-1 and 3G8 (I–L׳) or 4A6 antibodies (M–N׳). The gray-scale panels show CT–B and Caveolin-1 staining only, while the color panels show the merged images. (O–S׳) Paraplast sections of stage 40 Xenopus embryos stained with anti-Caveolin-1 in combination with ECL (O–P׳), Clathrin (Q,Q׳), Rab5 (R,R׳) and Rab7 (S,S׳). In panel P and P׳ co-localization of Caveolin-1 and ECL staining in the apical domain was analyzed using the co-localization module of the LAS AF software and is indicated by white pixels. The panels F,F׳,H,H׳,L,L׳,N,N׳,O׳,P׳,Q׳,R׳ and S׳ are close-ups of the areas indicated by the white boxes. |
|
Display additional annotations [+]
|
| |
Fig. 4. Scp2 and Lipid Rafts. (A–D׳) Immunofluorescence analysis of uninjected controls and scp2-MO1+2-injected embryos at stage 40 using Caveolin-1 (A–B׳) or Rab5 (C–D׳) antibodies with the gray-scale image showing Caveolin-1 or Rab5 staining alone and the color panels showing merged images. ECL was used to identify proximal tubules (green), DAPI (blue) to visualize nuclei. (E) Quantification of the number of Caveolin-1-positive foci identified in (A,B). The number of embryos analyzed is indicated in the individual bars. Data were analyzed by Student׳s t-Test and the three asterisks indicate a significance of p<0.001. (F) Metabolomics analysis of uninjected and scp2-MO1+2-injected embryos at stage 38 showing Box-and-Whisker Plots for cholesterol, 7-dehydrocholesterol and palmitoyl sphingomyelin. |
|
Display additional annotations [+]
|
| |
Fig. 5. Proximal Tubule Phenotype upon Inhibition of Cholesterol Synthesis. (A–B׳) Immunofluorescence analysis of untreated controls and embryos treated with 125 μM Mevinolin at stage 40 using anti-Caveolin-1 (red) antibody and ECL (green) with the gray-scale image showing Caveolin-1 staining alone and the color panels showing merged images. DAPI (blue) was used to visualize nuclei. (C) Quantification of the number of Caveolin-1-positive foci identified in (A,B). The number of embryos analyzed is indicated in the individual bars. Data were analyzed by Student׳s t-Test and asterisk represent a significance of p<0.05. (D) Bar diagram of the number of proximal tubular cells in untreated controls and Mevinolin-treated embryos at stages 40. The number of embryos analyzed is indicated in the individual bars. Data were analyzed by Student׳s t-Test and the three asterisks indicate a significance of p<0.001. (E–I׳) Untreated controls and Mevinolin-treated embryos were processed for 3G8 and 4A6 immunohistochemistry at stage 40 (E–F׳) or whole mount in situ hybridization with β1-Na/K ATPase (G,G׳), Pmp70 (H,H׳) and Catalase (I,I׳) at stage 39. (J,J׳) Model for the role of lipid rafts in proximal tubule elongation in the wild-type situation (J) or in the absence of scp2 or upon treatment with Mevinolin (J׳). See discussion for details. |
|
Display additional annotations [+]
|
| |
Supplementary Fig. S1. Scp2 and Lipid Rafts. (A–D׳) Immunofluorescence analysis of uninjected controls and scp2-MO1+2-injected embryos at stage 40 using a second anti-Caveolin-1 antibody (A–B׳, #3267, Cell Signaling) or anti-Clathrin (C–D׳). Gray-scale image show Caveolin-1 or Clathrin staining alone and the color panels depict the merged images. ECL was used to identify proximal tubules (green). |
|
|
| |
Supplementary Fig. S8. Scp2 Knockdown Effects on Lipid Rafts. (A–D׳) Caveolin-1 immunofluorescence analysis of uninjected controls and scp2-MO1+2-injected embryos at stage 40; neural tube (nt) and notochord (no) (A–B׳) as well as the kidney (C–D׳) were analyzed. Gray-scale image show Caveolin-1 staining alone and the color panels depict the merged images. 3G8 antibody staining (green) was used to identify proximal tubules in (C–D׳), DAPI (blue) to visualize nuclei. Note that scp2 morphants show reduced Caveolin-1 staining not only in the proximal tubules, but also in the notochord, the neural tube and the distal tubules (dt). All the structures are indicated by yellow outlines. |
