Figure S1
Internalisation and degradation of ErbB2 after flotillin depletion and GA treatment. (a)Quantification of co-IP signals. The signal intensities of ErbB2 and Hsp90 from five different co-IP experiments have been quantified and analysed. The mean intensities (+/-SEM)of Hsp90 and ErbB2, pulled down by flotillin-1 or -2 are reproducibly increased to 200-250% (**, P < 0.01, Mann-Whitney Rank Sum Test), compared to control samples. (b) GA-induced internalisation of ErbB2. SKBR3 cells were treated with 3µM GA for 2h and subsequently immunostained with antibodies against ErbB2, nuclei were stained by DAPI. Administration of GA induces ErbB2 internalisation as visualized by intracellular dotted structures in confocal imaging (orthogonal views). (c) Localisation of flotilin-1/-2 and ErbB2 after GA treatment. SKBR3 cells treated with 3 µM GA for 2h, were fixed, permeabilised, immunostained with antibodies against ErbB2 and flotillin-1 or-2 and visualized by confocal microscopy. Treatment of cells with GA resulted in internalisation of ErbB2 receptors, which are not co-localising with flotillin-1 or -2. GA treatment did not change the vesicular localisation of flotillins. Scale bar, 10 µm.(d)GA treatment induces the degradation of ErbB2 in SKBR3 cells. Cells were treated with 3µM GA for the indicated time periods. Protein levels were detected by Western blot analysis. GA treatment dramatically reduced ErbB2 levels but did not affect levels of flotillin-1 or -2.(e)Quantification of ErbB2 internalisation. Cells depleted for flotillins or treated with GA (2h) were quantified for internalized ErbB2. Compared to control cells, flotillin depletion significantly increases (***,P < 0.005, Student’s t-test)the percentage of cells with intracellular ErbB2 (n = 3, mean +/-SEM). (f) Intracellular ErbB2 localises to Lamp-1 positive vesicles. Cells were depleted for flotillin-1 or -2, fixed and stained for ErbB2, Lamp-1 and DAPI. Internalised ErbB2, triggered by flotillin depletion, localises to Lamp-1 positive vesicles. Scale bars, 5 µm).
Figure S2
Specificity of PLA assays and flotillin depletion. (a) Quantification of ErbB2/Par-2 proximity by PLA. Treatment of GA has no influence to ErbB2/Par-2 proximity. Detection of flotillin-2/Par-2 proximity shows 70% reduced number of signals (mean +/- SEM) compared to ErbB2/Par-2 (- GA). (b) Representative pictures of ErbB2/Hsp90 proximity, analysed by PLA and confocal microscopy. Treatment of GA or depletion of flotillins leads to a dramatically reduced ErbB2/Hsp90 proximity. Scale bar, 20 µm.(c)Quantification of ErbB2/Hsp90 proximity by PLA experiments (n = 5, mean + SEM). Knockdown of flotillin-1 or -2 results in strongly reduced proximity signals from ErbB2 and Hsp90. Rescue of flotillin-1 or -2 levels, by transfection of siRNA resistant flotillin DNA, results in a restoration of the ErbB2/Hsp90 proximity comparable to control cells transfected with an empty expression vector (n = 3, flotillin-1: P = 0.8, flotillin-2: P = 0.87). Analysis of > 70 cells per condition and experiment. (d) Representative Western blot of flotillin-1 or -2 protein levels in rescue experiments described in (c).(e) Co-Immunoprecipitation of ubiquitinylated ErbB2. Cells, transfected with control siRNA (-/+ GA treatment) or depleted for flotillin-1/-2 (-GA treatment), were lysed, ErbB2 was immunoprecipitated and ubiquitin was detected by SDS-PAGE. GA treatment leads to strong reduction of total ErbB2 and significant ubiquitinylation of ErbB2, whereas in flotillin-depleted cells no or very weak ubiquitinylation occurs.
Figure S3
Allred scoringof TMA cores. The tumour tissue cores were given scores for intensity of the membrane antibody staining and the relative number/proportion of epithelial cells that were stained. Intensity was categorised as negative (0), low (1), intermediate (2) or strong (3). Representative cores showing the different intensities are shown in the upper line. Proportion was scored as either no stained cells (0), which is similar to negative intensity, more than 0 to less than 1/100 stained cells (1, note that there is cytoplasmic staining in this core, but very slight membranous staining), more than 1/100 to 1/10 stained cells (2), more than 1/10 to 1/3 stained cells (3), more than 1/3 to 2/3 stained cells (4) and more than 2/3 stained cells (5). Representative cores showing the different proportion scores are shown in the lower line.
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