JCS03302 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Sequence specificity of siRNA SNX5-D1 and SNX6-D2. Alignment of protein sequences for SNX1, SNX5 and SNX6 using the ClustalW algorithm and the encoding nucleotide sequences. Identical amino acids are highlighted in black, similar ones in grey. Nucleotides exactly matching the corresponding base of the siRNA are highlighted in red. Note the differences in sequence between the siRNA and the non-target genes, e.g. between SNX5-D1 siRNA and SNX1/SNX6, strongly arguing for a specific knock-down of the target gene.

• Supplemental Figure 2 -

  Fig. S2. Each of the single RNAi duplexes constituting the SNX5- or SNX6-SMARTpools provokes an effect similar to that of the pool itself. The fact that a similar phenotype although varying slightly in severety is produced by knocking down either SNX5 or SNX6 with four different siRNAs strongly argues for the specificity of the phenotypic effect, excluding the possibility of off-gene targeting. Bars, 20 μm.

• Supplemental Figure 3 -

  Fig. S3. RNAi of SNX6 strongly depletes the cellular levels of SNX6 protein as shown by immunofluorescence. Endogenous SNX6 is stained in green, nuclei in blue using DAPI. Scr, scrambled control siRNA; SNX6, siRNA D2.

• Movie 1
• Movie 2
• Movie 3 -

  Movies 1, 2 and 3. GFP-SNX6 and RFP-SNX1 co-expressing HeLa cells. GFP-SNX6-labelled tubules (Movie 1) can be observed that emanate from brightly stained vesicles, pinch off and move through the cytoplasm. This sequence of events can also be seen in the RFP channel for visualization of RFP-SNX1 (Movie 2). Note the perfect colocalization on vesicles and tubules of SNX1 and SNX6. (Movie 3) Overlay of GFP-SNX6 and RFP-SNX1.

• Movie 4
• Movie 5 -

  Movies 4 and 5. GFP-SNX5 and RFP-SNX1 co-expressing HeLa cells. GFP-SNX5 (Movie 4) and RFP-SNX1 (Movie 5) colocalize on vesicles and pinching off tubules. Note the tubule emerging from the brightly stained vesicle in the lower right part of the frame and its very rapid movement through the cytoplasm.