T H E J O U R N A L O F C E L L B I O L O G Y

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1 T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Rainero et al., Figure S1. The expression of DGK- is reduced upon transfection of an sirna against DGK- in A2780, H1299, and MDA-MB-231 cells, DGK- is not required for the internalization of 5 1 or TfnR, and inhibition of v 3 or expression of mutant p53 did not affect the enzymatic activity or the localization of DGK-. (A C) sirna of DGK- in A2780, H1299, and MDA-MB-231 cells. A2780 (A), vector, and mutant p53 273H -expressing H1299 (B) and MDA-MB-231 (C) cells were transfected with nontargeting sirna (sirna control [si-con]), an sirna targeting DGK- (si-dgk ), or sirna targeting DGK- in combination with sirnaresistant DGK- (si-dgk /DGK (resc)). DGK- protein levels were analyzed by Western blotting, and -actin was used as a loading control. (D and E) DGK- is not required for internalization of 5 1 or TfnR. A2780 cells were transfected with a nontargeting sirna or an sirna targeting DGK-, and the internalization of 5 1 (D) or TfnR (E) was determined. Values are means ± SEM; n = 12 replicates in two independent experiments. (F and G) Assay of DGK- activity. (F) A2780 or vector and mutant p53 273H -expressing H1299 cells were transfected with myc DGK- (G) or left untransfected (F). Cells were treated as indicated with cradfv, 2.5 µm crgdfv, or 100 ng/ml EGF. Endogenous DGK- was immunoprecipitated with the anti DGK- antibody (F), or myc DGK- was immunoprecipitated with an antimyc antibody (G), and the activity of the lipid kinases was assayed in the immunoprecipitates. Data represent means ± SEM; n = 9 replicates in three independent experiments (G); n = 6 replicates in two independent experiments (F). *, P = 0.05; Mann Whitney test. (H) DGK- localization. A2780 cells were plated on CDM, treated with 2.5 µm crgdfv, fixed, and stained for DGK- and phalloidin to visualize F-actin. Bar, 10 µm. S1

2 Figure S2. DGK activity and RCP s C2 domain are not required for the interaction of RCP with 5 1. (A D) A2780 (A and D) or mutant p53 273H -expressing H1299 (B and C) cells were transfected with GFP-RCP (A, B, and D), GFP-RCP (D), or GFP control (A, B, and D), incubated with or without 2.5 µm crgdfv in the absence (DMSO) or presence of 10 µm R59022 for 30 min, and lysed in a buffer containing 0.15% Tween 20. GFP was immunoprecipitated (IP) from lysates by incubation with beads coupled to a monoclonal antibody recognizing GFP, and these immunoprecipitates were analyzed for the presence of 5 integrin by Western blotting. The loading of GFP and GFP-RCP was confirmed by immunoblotting with an anti-gfp antibody. In C, endogenous 5 was immunoprecipitated from lysates by incubation with beads coupled to a monoclonal antibody recognizing 5, and these immunoprecipitates were analyzed for the presence of RCP by Western blotting. S2

3 Figure S3. The effects of DGK- knockdown on speed and persistence of cell migration, the effects of Rho kinase inhibition on random migration induced by inhibition of v 3 or expression of mutant p53, and the role of other DGK isoforms in pseudopod elongation. (A and B) Effects of DGK- knockdown on speed and persistence of cell migration. A2780 (A), empty vector, and p53 273H -expressing H1299 (B) cells were transfected with nontargeting sirna (sirna control [si-con]), an sirna targeting DGK- (si-dgk ), or sirna targeting DGK- in combination with an sirna-resistant DGK- (si-dgk / DGK (resc)), and their migration into scratch wounds in the absence ( ) and presence of 2.5 µm crgdfv was recorded as for Fig. 2. The speed and persistence of cell migration were extracted from the trackplots, and data are represented as box and whisker plots (black lines show medians; whiskers: 5 95 percentile); n = 3 independent experiments. (C and D) Rho kinase activity is required for crgdfv- and p53 273H -induced random migration. A2780 (C), empty vector, and p53 273H -expressing H1299 (D) cells were grown to confluence, and their migration into scratch wounds in the absence ( ) and presence of 2.5 µm crgdfv with and without the Rho-associated protein kinase inhibitor (Y27632; 2 µm) or vehicle control (con) was recorded as for Fig. 2. The forward migration index was extracted from the trackplots, and data are represented as box and whisker plots (black lines show medians; whiskers: 5 95 percentile); n = 3 independent experiments. (E) Role of other DGK isoforms in pseudopod elongation. A2780 cells were transfected with a nontargeting sirna or sirnas targeting DGK-, -, -, -, and - (si-dgk, si-dgk, si-dgk, si-dgk, and si-dgk ) as indicated and plated on CDM, and pseudopod length was measured as for Fig. 3. DGK mrna levels were assessed by RT-PCR. Data are represented as box and whisker plots (black lines show medians; whiskers: 5 95 percentile); n = 4 replicates in two independent experiments. ***, P < ; Mann Whitney test. S3

4 Figure S4. The accumulation of RCP at the tip of pseudopods does not reflect increased cytoplasmic volume at these cellular extremities. (A) RCP accumulation at pseudopod tips does not reflect increased cytoplasmic volume at these cellular extremities. Empty vector and p53 273H -expressing H1299 cells were transfected with GFP-RCP in combination with mcherry, plated onto CDM, and imaged by confocal microscopy. Images were captured at one frame/second over a period of 60 s, and videos were generated from these. Single-section confocal image stills corresponding to individual frames from these videos are presented. Bar, 20 µm. The yellow arrows indicate the direction of migration, and the portion of the cell within the squares is presented in the bottom images. (B) The recovery of GFP after photobleaching is not affected by v 3 inhibition. A2780 cells were transfected with GFP and plated onto CDM, and photobleaching was performed. Recovery curves were generated using the FV10-ASW software, and the immobile fraction (percentage) was extracted from them. (C) sirna/rescue of RCP expression in A2780 cells. A2780 cells were transfected with nontargeting sirna (sirna control [si-con]), an sirna targeting RCP (si-rcp), or sirna RCP in combination with an sirna-resistant GFP-RCP WT (GFP-RCP WT (resc)) or an sirna-resistant GFP-RCP (GFP- RCP (resc)). RCP protein levels were determined by Western blotting. (D) DGK- is required for EGFR recycling induced by inhibition of v 3. The recycling of internalized EGFR1 was determined in A2780 cells transfected with a nontargeting sirna or an sirna targeting DGK- (si-dgk ). 36 nm crgdvn Me V was included during the recycling period. Values are means ± SEM of three independent experiments. si-con versus si-con+crgdfn Me V: **, P = ; ***, P = ; Mann Whitney test. si-con+crgdfn Me V versus si-dgk +crgdfnv: ###, P = ; Mann Whitney test. exp., exposure. S4

5 Figure S5. PLD activity is not required for RCP recruitment to pseudopod tips. (A) PLD activity is not required for RCP recruitment to pseudopod tips. A2780 cells were transfected with GFP-RCP and plated onto CDM in the presence or absence of 36 nm crgdfn Me V. The PLD inhibitor butan-1-ol (0.5%) or secondary alcohol control butan-2-ol (0.5%) was included as indicated, and GFP-RCP was imaged by confocal microscopy. Images were captured at one frame/ second over a period of 60 s, and videos were generated from these. Single-section confocal image stills corresponding to individual frames from these videos are presented. Bar, 20 µm. The arrows indicate the direction of migration, and the portion of the cell within the squares is presented in the bottom images. (B) FRAP of GFP-RCP in vesicular structures or cytoplasmic regions near pseudopod tips. A2780 or mutant p53-expressing H1299 cells were transfected with GFP-RCP and plated onto CDM, and photobleaching was performed with the laser aimed at either vesicular structures or diffuse cytoplasmic regions (as indicated) near the pseudopod tip. Fluorescence time-lapse videos were then collected, and representative stills from these are displayed. Recovery curves were generated using the FV10-ASW software, and the immobile fraction (percentage) was extracted from them the data corresponding to these videos are displayed in Fig. 6 (D and F). Bar, 10 μm. Arrows show direction of cell migration. The circles denote the photobleached area. The arrowheads indicate vesicular structures that recover from photobleaching. S5

6 Video 1. GFP-actin localization in A2780 cells transfected with a nontargeting sirna. A2780 cells were transfected with GFPactin in combination with a nontargeting sirna, and a time-lapse video was recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. sicon, control sirna. Video 2. GFP-actin localization in A2780 cells treated with crgdfn Me V in the presence of a nontargeting sirna. A2780 cells were transfected with GFP-actin in combination with a nontargeting sirna and treated with 160 nm crgdfn Me V, and timelapse videos were recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. sicon, control sirna. Video 3. GFP-actin localization in A2780 cells treated with crgdfn Me V in the presence an sirna against DGK-. A2780 cells were transfected with GFP-actin in combination with an sirna targeting DGK- and treated with crgdfn Me V, and a time-lapse video was recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. Video 4. GFP-RCP localization in A2780 cells treated with crgdfv and transfected with a nontargeting sirna. A2780 cells were transfected with GFP-RCP in combination with a nontargeting sirna, plated onto CDM, and treated with 2.5 µm crgdfv, and time-lapse videos were recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. Video 5. GFP-RCP localization in A2780 cells treated with crgdfv and transfected with an sirna against DGK-. A2780 cells were transfected with GFP-RCP in combination with an sirna targeting DGK- and plated onto CDM. Cells were treated with 2.5 µm crgdfv, and time-lapse videos were recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. Video 6. GFP-RCP localization in p53 273H -expressing H1299 cells transfected with a nontargeting sirna. p53 273H -expressing H1299 cells were transfected with GFP-RCP in combination with a nontargeting sirna and plated onto CDM, and time-lapse videos were recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. sicon, control sirna. Video 7. GFP-RCP localization in p53 273H -expressing H1299 cells transfected with an sirna against DGK-. p53 273H -expressing H1299 cells were transfected with GFP-RCP in combination with an sirna targeting DGK-. Cells were plated onto CDM, and time-lapse videos were recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. S6

7 Video 8. The recovery from photobleaching of GFP-RCP in A2780 cells transfected with a nontargeting sirna or an sirna against DGK-. A2780 cells were transfected with GFP-RCP and a nontargeting si-rna (left) or an sirna targeting DGK- (right), plated onto CDM, and treated with 2.5 µm crgdfv. The areas indicated by the yellow circles were subjected to photobleaching as indicated (frame 4), and the recovery from photobleaching followed by time-lapse fluorescence microscopy was performed using an inverted confocal microscope (FV1000). Frames were captured every second. sicon, control sirna. Video 9. The localization of GFP-RCP in A2780. A2780 cells were transfected with GFP-RCP , plated onto CDM, and treated with crgdfv, and time-lapse videos were recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. Video 10. The localization of GFP-RCP in p53 273H -expressing H1299 cells. p53 273H -expressing H1299 cells were transfected with GFP-RCP and plated onto CDM, and time-lapse videos were recorded using an inverted confocal microscope (FV1000). Frames were taken every second for 1 min. S7