Supplemental data. Supplemental Materials and Methods

Transcription

1 Supplemental data Supplemental Materials and Methods Transfection of plasmid. Transfection of plasmids into FRTL5 cells was performed using Lipofectamine LTX with Plus reagent (Invitrogen) according to the manufacturer s protocol. Briefly, FRTL5 cells were cultured on coverslips in 6well plates for 45 days and then 200 l of OptiMEM containing the DNAPlus reagentlipofectamine LTX mixture (0.16 g pflagpi3kap/xb130 or vector, 0.04 g pegfp, 1 l Plus reagent, 2.5 l Lipofectamine LTX) were added to the cells cultured in 1 ml of growth medium. After 3 h incubation, the medium was replaced by fresh growth medium, and cell culture was continued for 24 h. Thereafter, cells were serumstarved for 24 h, followed by treatment with the indicated hormones/chemicals. Immunofluorescence analysis. FRTL5 cells were washed once with PBS, fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X100 in PBS for 5 min at room temperature. Cells were then washed with PBS and incubated with 3% bovine serum albumin in PBS for 1 h at room temperature, and primary antibodies (1:100 for antipi3kap/xb130 antibody) were added overnight at 4C. The samples were again washed with PBS, incubated with 40 M tetramethylrhodamine B isothiocyanate (TRITC)labeled phalloidin (Sigma) and a secondary antibody conjugated to Alexa Fluor 488 (1:1000 dilution, Invitrogen) for 1 h and washed with PBS. Fluorescent images were obtained using a confocal laser scanning microscopy FV500 (Olympus, Tokyo, Japan). Bromodeoxyuridine (BrdU) incorporation assay Quiescent FRTL5 cells plated on coverslips were pretreated with camp and then treated with IGFI in the presence of BrdU. Cells were fixed with 70% ethanol containing 15 mm glycine (ph 2.0) at 20 C for 30 min, and then washed with PBS three times. After incubation with antigfp rabbit polyclonal antibody overnight at 4 C, cells were stained with antibrdu mouse monoclonal antibody using BrdU labeling and detection kit I (Roche Diagnostics, Basel, Switzerland). AntiGFP antibody and antibrdu antibody were labeled with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen), respectively. GFPpositive cells were identified using a fluorescence microscope IX81

2 (Olympus, Tokyo, Japan), and then BrdUpositive cells were counted. Approximately 300 cells per coverslip were examined. Supplemental Figure Legends Fig. S1. PI3KAP/XB130 localizes to actin filaments. Quiescent FRTL5 cells were treated with or without 1 mm camp for 24 h. Fixed and permeabilized cells were subjected to immunostaining with antipi3kap/xb130 antibody. As a negative control, antipi3kap/xb130 antibody was preincubated with an antigen peptide (blocking peptide) prior to immunostaining. Factin was stained with TRITClabeled phalloidin. PI3KAP/XB130 protein was visualized by Alexa488 in green, and Factin was visualized in red in the confocal microscopy. Representative photographs are shown. Scale bar, 10 m. Fig. S2. Assessment of PI3KAP/XB130 knockdown effects on its expression and DNA synthesis at a single cell level. A, FRTL5 cells were transfected with control sirna or sirna against PI3KAP/XB130, serumstarved, and then treated with 1 mm camp for 24 h. Fixed and permeabilized cells were subjected to immunostaining with antipi3kap/xb130 antibody. Factin was stained with TRITClabeled phalloidin. PI3KAP/XB130 protein was visualized by Alexa488 in green, and Factin was visualized in red in the confocal microscopy. Representative photographs are shown. Scale bar, 10 m. B, FRTL5 cells were transfected with control sirna or sirna against PI3KAP/XB130.followed by transfection with control vector or plasmid encoding PI3KAP/XB130 in combination with GFP plasmid. GFP plasmid was used to detect plasmidtransfected cells. To avoid unintended knockdown of transfected PI3KAP/XB130, sirna targeted to 3 UTR of PI3KAP/XB130 was used. The cells were serumstarved, pretreated with 1 mm camp for 24 h, and then.treated with 100 ng/ml IGFI for 24 h in the presence of 10 M BrdU. Fixed cells were subjected to immunostaining with antigfp and antibrdu antibodies. GFPpositive cells and BrdUpositive cells were visualized by secondary antibodies conjugated to Alexa488 or Alexa594, respectively. BrdUpositive cells were counted using the fluorescent microscopy, and were presented as a percentage of GFPpositive (transfected) cells. The means ± SEM of three replicate wells are shown. There are significant differences between values with different superscript characters (p<0.05). Similar results were obtained in two independent experiments. Fig. S3. Inhibition of PI3KAP/XB130 tyrosine phosphorylation does not alter its

3 intracellular localization. Quiescent FRTL5 cells were treated with 1 mm camp for 24 h in the presence or absence of 3 M PP1 or PP2. Fixed and permeabilized cells were subjected to immunostaining with antipi3kap/xb130 antibody. Factin was stained with TRITClabeled phalloidin. PI3KAP/XB130 protein was visualized by Alexa488 in green, and Factin was visualized in red in the confocal microscopy. Representative photographs are shown. Scale bar, 10 m.

4 Fig. S1 camp blocking peptide api3kap/xb130 Factin + + +

5 Fig. S2 A. sirna api3kap/xb130 Factin control camp PI3KAP #1 PI3KAP #2 B. 8 b bc BrdUpositive cells (% of transfected cells) 4 c a 0 camp IGFI PI3KAP plasmid sirna control + PI3KAP #3

6 Fig. S3 api3kap/xb130 Factin No additivves camp 3 mm PP1 3 mm PP2