Volume 8 issue 11 November 2007
Annexin A6-induced alterations in cholesterol transport and caveolin export from the Golgi complex
Laia Cubells, Sandra Vilà de Muga, Francesc Tebar, Peta Wood, Rachael Evans, Mercedes Ingelmo-Torres, Maria Calvo, Katharina Gaus, Albert Pol, Thomas Grewal and Carlos Enrich

Supplemental Figure 1: (A-C) Western blot analysis of Sucrose gradients (A), Percoll gradients (B) and Golgi membrane fractions (C) from CHOwt cells. (A) Early (EE) and late (LE) endosomal membrane fractions (20 µg protein/fraction) are characterized by the presence of Rab5 and Rab7 and are well separated from the bulk membrane (Na⁺/K⁺ ATPase) at the bottom of the gradient (8, 16). (B) Plasma membrane (PM) enriched membrane fractions (20 µg protein/fraction) are characterized by the enrichment of Ras (15). Incubation of cells overnight with ³H-Cholesterol predominantly labels the Ras-containing fractions 4 and 5. (C) Analysis of Golgi membrane fractions (Golgi, 100 µg protein) and whole cell lysates (WCL, 100 µg protein) shows the enrichment of Golgi markers GM130, TGN38 and GMAP210 (79, 80). No significant amounts of EE, LE and PM markers (Rab5, Rab7, KDEL, Na⁺/K⁺ ATPase) were detected. Gradients from CHOanx6 cells showed similarly well separated endosomal, PM and Golgi membrane fractions (not shown).

Figure 1 (.jpg)

Supplemental Figure 2: Cholesterol efflux is decreased in AnxA6 expressing cells. (A) CHOwt and CHOanx6 were incubated with [³H]-Chol (2 x 10⁶ cpm/ml) for 24 h. Cells were washed with PBS and incubated ± 1 % methyl-β-cyclodextrin (CD) for 120 min. The ratio of released and cell-associated radioactivity was determined, normalized to total cell protein and the amount of efflux is given in (%). The background efflux obtained from CHOwt and CHOanx6 was equivalent to 2.0 – 8.0 x 10⁵ cpm/mg cell protein, respectively. Mean values ± S.D. of 3 independent experiments with triplicate samples are given. *, p<0.05 for student’s t test. Alternatively, CHOwt and CHOanx6 cells were labeled overnight with [³H]-Chol-LDL (1 x 10⁶ cpm/ml) and efflux was induced with 5 - 100 µg/ml HDL3 for 8 h as indicated. The percentage of [³H]-Chol efflux was calculated as described above and represents the mean ± S.D. of three independent experiments with triplicate samples. (B) A431wt, A431anx6, BT20wt (express low amounts of AnxA6, unpublished data) and BT20anx6 were incubated with 2 µg/ml U18666A together with [³H]-Chol (2 x 10⁶ cpm/ml cpm/ml) for 24 h. Cells were washed with PBS and incubated ± 1 % methyl-β-cyclodextrin (CD) for 120 min. The ratio of released and cell-associated radioactivity was determined, normalized to total cell protein and the amount of efflux is given in (%). The background efflux in U18666A-treated cells was equivalent to 3.0 – 6.0 x 10⁵ cpm/mg cell protein), respectively. Mean values ± S.D. of 3 independent experiments with triplicate samples are given. **, p<0.01 for student’s t test. (C) 4 x 10⁵ CHOwt and CHOanx6 cells (in triplicate) were grown in media containing 5 % lipoprotein-deficient FCS and incubated overnight with 1 µCi/ml [1-¹⁴C]-acetic acid. Lipids were extracted and newly synthesized cholesterol was separated from its major precursors including desmosterol by thin layer chromatography (TLC) as described (35). For visualization and quantification, TLC plates were exposed to X-ray film, and the amount of radioactive cholesterol was quantified using ImageJ (NIH). Relative intensities were normalized for total cellular protein (78). The mean values for a representative experiment are given. In 2 independent experiments with triplicate samples an overall reduction of cholesterol synthesis in CHOanx6 cells by 36 ± 18 % (p<0.05) was observed.

Figure 2 (.jpg)

Supplemental Figure 3: Caveolin accumulates in a perinuclear compartment of AnxA6 expressing cells. (A) CHOwt (a) and CHOanx6 (b) cells grown on coverslips were fixed, permeabilized and stained with polyclonal anti-caveolin. Arrowheads point at the perinuclear accumulation of caveolin in CHOanx6 cells. Images are representative for 90% of cells analyzed. The fluorescence intensity of the perinuclear cav-1 staining in CHOwt and CHOanx6 cells was quantified. Values represent the mean ± S.D. of 5 independent experiments with 50 cells per cell line in each experiment. ***, p<0.001 for student’s t test. (B) CHO wildtype cells (a,b) were transiently transfected with GFP-Anx6 and stained with polyclonal anti-caveolin. Arrowheads point at the accumulation of cav-1 in the perinuclear Golgi region of the transfected cell. The protein levels of cav-1, GFP-Anx6 and endogenous AnxA6 in transfected (+) and non-transfected (-) CHOwt cells are given. (C) Western blot analysis of cav-1, Scavenger receptor BI (SR-BI), H-Ras and AnxA6 from total cell lysates of CHOwt and CHOanx6 cells.

Figure 3 (.jpg)

Supplemental Figure 4: Cholesterol restores the cellular distribution of cav-1 in CHOanx6 cells. CHOwt (a, c) and CHOanx6 cells (b, d) were grown on coverslips, incubated ± cholesterol for 90 min, fixed, permeabilized and immunolabeled with anti-caveolin as indicated. Images are representative for 3 independent experiments. Arrows point at the localization of endogenous cav-1 at the PM in CHOwt and CHOanx6 cells upon addition of cholesterol (c and d). Bar is 10 µm. (E) CHOwt (lane 1 - 4) and CHOanx6 cells (lane 5 - 8) were incubated with cholesterol and Cyhx for 90 min as indicated. Then the Golgi-associated cav-1 was immunoprecipitated and analyzed by Western blotting as described (40). The amount of immunoprecipitated cav-1 from the Golgi was quantified and normalized to actin. The relative amount of cav-1 in the Golgi is given and is representative for 3 independent experiments. ***, p<0.001 for student’s t test.

Figure 4 (.jpg)

Supplemental Figure 5: Cholesterol restores the ability of CHOanx6 cells to re-establish cholesterol sorting events of VSV-G. CHOwt (a, c) and CHOanx6 cells (b, d) were grown on coverslips and transfected with VSVG-GFP. 24 h after transfection, cells were incubated ± cholesterol for 90 min, fixed, permeabilized and analyzed by epifluorescence microscopy. The arrow points at the perinuclear localization of GFP-tagged VSV-G in CHOanx6 cells upon addition of cholesterol (d). Results are representative for 3 independent experiments. Bar is 10 µm.

Figure 5 (.jpg)