JCS02752 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Rabbit polyclonal anti-PtdIns4KIIa antibodies are isoform-specific. Total cell lysates from HT1080 cells stably expressing PtdIns4KIIa or PtdIns4KIIb were subjected to western analysis using anti-PtdIns4KIIa and anti-GFP antibodies (A). The same antibody was used to immunostain HT1080 cells transfected with siRNA duplexes targeting either lamin (B, control) or PtdIns4KIIa with oligo-1 (C). Note that the image shown in (C) was obtained at an increased gain setting in order to visualise cells with reduced anti-PtdIns4KIIa immunoreactivity. Scale bars, 20 mm.

• Supplemental Figure 2 -

  Fig. S2. Colocalisation of GFP-PtdIns4KIIa with early endosomal markers EEA1 and transferrin. GFP-PtdIns4KIIa cells were fixed and immunostained for EEA1 (A) as described in the Materials and Methods section. Cells were also incubated with medium containing 5 mg/ml Alexa 568-labelled transferrin for 10 minutes at 37^oC prior to fixation in 4% paraformaldehyde (B). Scale bars and zoom boxes are 10 mm.

• Supplemental Figure 3 -

  Fig. S3. Validation of the siRNA approach. (A) HT1080 cells were either mock or siRNA-transfected with duplex oligonucleotides targeting lamin or with two independent oligonucleotides targeting PtdIns4KIIa (oligo-1 and oligo-2, respectively). Cells were subsequently serum-starved and stimulated with a pulse of Rhodamine-labelled EGF for 120 seconds followed by unlabelled EGFR for the total time indicated. Cells were then examined by confocal fluorescence microscopy as described in the Materials and Methods section. All images are extended focus views representing 12 confocal sections collected 0.14 mm apart.  Images shown are representative of three independent transfections.  Scale bars 10 mm. Cells were also scored for the presence of scattered punctae (B) and the presence of sub-plasma membrane vesicles (C). The data in (B) and (C) are derived from three independent experiments from which three randomly chosen fields of view containing between 43-100 cells were scored.

• Supplemental Figure 4 -

  Fig. S4. PtdIns4KIIa RNAi causes redistribution of lysosomal glycoproteins lamp-1 and CD63. HT1080 Cells were either mock or siRNA transfected with oligo-1 as described in the Materials and Methods section. After 72 hours the cells were fixed and immunostained for lamp-1 (A) or CD63 (B) and imaged by confocal microscopy. The images shown are extended focus views representing 12 optical sections of 0.12 mm thickness.

• Movie 1 -

  Movie 1. Live HT1080 cells stably expressing GFP-PtdIns4KIIa were imaged by confocal laser-scanning microscopy revealing highly dynamic vesicles and tubular structures.

• Movie 2 -

  Movie 2. Live HT1080 cells stably expressing GFP-PtdIns4KIIa were serum-starved and stimulated with rhodamine-labelled epidermal growth factor. GFP-PtdIns4KIIa  fluorescence (green channel) and endocytosed rhodamine fluorescence (red channel) can be seen to co-localise in rapidly moving vesicles.