JCS007948 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Two different siRNAs can transiently knockdown TI-VAMP in Caco2 cells. Caco2 cells were electroporated with 60 pmol of two different siRNAs against human TI-VAMP or against human beta-globin and grown on filters for four days. Cell extracts (40 μg) from control (siRNA beta glob) and TI-VAMP transient-knockdown (siRNA8 TI-VAMP and siRNA7 TI-VAMP) cells were analysed by SDS-PAGE and western blotting with antibodies against human calreticulin (top panel, internal control for protein loading) and human TI-VAMP (bottom panel). TI-VAMP expression decreased almost equally using the two different siRNAs.

• Supplemental Figure S2 -

  Fig. S2. Analysis of the effect of TI-VAMP and VAMP8 knockdown in Caco2 cells by indirect immunofluorescence in non-permeabilized conditions. Caco2 cells were electroporated with 60 pmol of siRNA against human TI-VAMP and VAMP8 or against human beta-globin and grown on filters for four days. After fixation, cells were labelled by adding an antibody against PLAP (A) or against DPPIV (B) and a secondary antibody coupled to FITC to the apical and to the basolateral domain so that only the plasma membrane was labelled. Confocal Z and X-Y sections (top and bottom) of control (siRNA beta glob), TI-VAMP transient-knockdown (siRNA TI-VAMP) and VAMP8 transient-knockdown (siRNA VAMP8) Caco2 cells were taken. Bars, 10 μm.

• Supplemental Figure S3 -

  Fig. S3. Rescue of the transient knockdown of TI-VAMP in Caco2 cells. Caco2 cells were electroporated with 60 pmol of siRNA against human beta-globin or human TI-VAMP alone or together with 2 μg of rat TI-VAMP cDNA and grown on filters for four days. (A) Cell extracts (40 μg) from control (siRNA beta glob) and ‘rescued’ TI-VAMP transient-knockdown (siRNA plus cDNA TI-VAMP) Caco2 cells were analysed by SDS-PAGE and western blotting using antibodies against human calreticulin (top panel, internal control for protein loading) and human TI-VAMP (bottom panel). (B) Confocal Z and X-Y sections (top and bottom) of TI-VAMP transient-knockdown (siRNA TI-VAMP) Caco2 or ‘rescued’ TI-VAMP transient-knockdown (siRNA plus cDNA TI-VAMP1/3 and 2/3) Caco2 cells were taken. Cells were labelled with antibodies against PLAP and secondary antibodies coupled to FITC. Note that the apical localization of PLAP is affected by the transient knockdown of TI-VAMP (left lane), whereas, in the rescue, its apical localization is unaffected (middle lane in 1/3 of the monolayer) or poorly affected (right lane in 2/3 of the monolayer). Bars, 10 μm.

• Supplemental Figure S4 -

  Fig. S4. Recycling of DPPIV to the apical plasma membrane is impaired by VAMP8 transient knockdown in Caco2 cells. Caco2 cells were electroporated with 60 pmol of siRNA against human VAMP8 or against human beta-globin and grown on filters for four days. Cells were then incubated with an antibody against human DPPIV for 30 minutes at 4°C (pulse). After warming the cells at 37°C to allow DPPIV internalization for different indicated times (0, 5 or 10 minutes chase), the monolayers were fixed and incubated with the FITC-conjugated secondary antibody to label surface DPPIV. In order to distinguish the internalized DPPIV from the surface protein, we quenched the secondary antibody and permeabilized with saponin (0.075%). The cells were then incubated with a TRITC-conjugated secondary antibody, fixed again and processed for indirect immunofluorescence. Confocal Z and X-Y sections (top and bottom) of control (siRNA beta glob) and VAMP8 transient-knockdown (siRNA VAMP8) Caco2 cells were taken. Note that, in control conditions, the basolateral labelling of DPPIV disappears rapidly and after 10 minutes the staining is mainly intracellular, showing the occurrence of internalization. By contrast, in VAMP8 transient-knockdown cells, DPPIV staining persists on the basolateral membrane, indicating that VAMP8 is required in the recycling of DPPIV from the basolateral to the apical plasma membrane. (B) Cells were then incubated with an antibody against human DPPIV for 30 minutes at 4°C (pulse) and warmed at 37°C for 30 minutes to allow DPPIV to transcytose to the apical membrane. Monolayers were fixed and incubated with the FITC-conjugated secondary antibody to label surface DPPIV. Bars, 10 μm. Note that, in control conditions, all the signal disappears from the basolateral surface and accumulates on the apical surface, whereas, in VAMP8 transient-knockdown conditions, DPPIV staining persists on the basolateral membrane.

• Supplemental Figure S5 -

  Fig. S5. The localization of syntaxin 3 and syntaxin 4 is not affected by transient RNAi both of TI-VAMP and of VAMP8. Caco2 cells were electroporated with 60 pmol of siRNA against human TI-VAMP, VAMP8 or human beta-globin and grown on filters for four days. Cells were then labelled with antibodies against human Stx3 (A) or against human Stx4 (B), and secondary antibodies were coupled to FITC. Confocal Z and X-Y sections (top and bottom) of control (siRNA beta glob), TI-VAMP transient-knockdown (siRNA TI-VAMP) Caco2 or VAMP8 transient-knockdown (siRNA VAMP8) Caco2 cells were taken. Note that the apical localization of Stx3 and basolateral localization of Stx4 are both unaffected by the transient knockdown of TI-VAMP or VAMP8. Bars, 10 μm.