JCS031070 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Suppression of Sec31 expression does not cause a concomitant loss of Sec13 expression. Cells were transfected with Sec31A siRNA and expression of Sec31A, Sec13, α-tubulin and lamin A/C determined by immunoblotting after 72 hours.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Quantification of collagen deposition in primary fibroblast cultures. (A) Cells were transfected with siRNA duplexes targeting lamin A/C or Sec13. 72 hours after transfection, cells were fixed and immunolabelled with antibodies directed against the C-terminal telopeptide of collagen I and counterstained with DAPI. Montage images were acquired using an automated X-Y scanning stage. Images were thresholded to detect only pixel intensities >200 (of 255) and these thresholded pixels applied as a mask. This was then used to calculate the skeletal length of fibrils within this mask. Panels show the original image, collagen channel alone, masked pixels and a montage of the original image plus mask (where the mask is shown in orange). The upper panels show cells transfected with lamin-A/C siRNA and the larger, lower panels the cells transfected with Sec13 siRNA. Scale bars: 0.5 mm. Note the reduction in orange-coloured masked pixels in Sec13-suppressed cells compared with lamin-A/C-suppressed cells. Quantification of three montage views from each of three independent experiments is shown in Fig. 6B. (B) Cells were labelled with antibodies to detect the N-terminal propeptide of procollagen I (LF-39) and Sec31A, showing intracellular accumulation of procollagen in Sec13 suppressed cells (asterisks).

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Craniofacial-development defects in Sec13 morphant zebrafish embryos. (A,B) Images of live zebrafish embryos microinjected with control or Sec13 morpholino oligonucleotides are shown at (A) 4 days post fertilization using Sec13 MO2 and (B) 5 days post fertilization using Sec13 MO1. Arrows and arrowheads indicate defects in craniofacial and pectoral fin development. These data serve to reinforce those in Fig. 7.