Figure 7.

Rab11A–RCP moderately regulates Langerin stability and Birbeck granule biogenesis. (A) Immunoblotting was used to assess levels of TfR, Langerin, Rab11, RCP, and Rip11 in Langerin⁺ HeLa cells, mock treated (mock), or treated with siRNA against Rab11A, RCP, or Rip11. The data shown are representative of five independent experiments for Rab11A and RCP siRNA and two experiments for Rip11 siRNA. Proteins were separated by SDS-PAGE in 12% acrylamide gels and blotted onto membranes. Equivalent amounts of total protein were loaded in each lane, and each blot was probed for all the proteins tested. (B) The decreases in Langerin and TfR expression were compared in cells treated with siRNA against Rab11A (5 experiments) or siRNA against RCP (5 experiments). Quantitative data derived from Western blots are presented as the ratio of the decrease in Langerin expression to the decrease in TfR expression and are normalized by the parallel results obtained in mock-treated cells (10 experiments). M10-22E cells, comicroinjected with siRNA against Rab11A (C) or siRNA against RCP (D) and Texas Red-dextran (Dextran TR, C and D, left), were immunolabeled with anti-Langerin (Langerin, C and D, middle) or anti-TfR antibodies (C and D, right) revealed with donkey anti-rabbit A488 or anti-mouse Cy5 secondary antibodies, respectively. Image stacks were acquired and the resulting immunofluorescence signals quantified (E) as described in Materials and Methods. Data are presented as the average fluorescence intensity (±SD) per cell for Langerin (left) and TfR (right) in cells microinjected with siRNA against Rab11A (n = 25) or RCP (n = 31) and compared with results for cells microinjected with Texas Red-dextran only (mock, n = 25). Student's t test was used to analyze the differences in Langerin content between cells mock treated and treated with Rab11A siRNA (p < 0.0001) and between cells mock treated and treated with RCP siRNA (p = 0.015). Quantitative analysis of the number of BG profiles per cell was performed in cells treated with Rab11A siRNA or mock treated, in the presence or absence of chloroquine (F), or in cells treated with siRNA against RCP, Rab4A, or Rab5A or mock treated (G). The number of cells examined per condition was 10 in F (but 25 for cells treated with chloroquine and siRNA against Rab11A) and 15 in G. Note the low specificity of the reduction in BG numbers in RCP-depleted cells. Only cells visible in their totality were counted, considering only sections passing through the centro-cellular region and after tilting each cell to examine it from different angles and check that no BGs had been omitted.