Cancer Cell, Volume 22
Tumor Type-Dependent Function
of the Par3 Polarity Protein
in Skin Tumorigenesis
Sandra Iden, Wilhelmina E. van Riel, Ronny Schäfer, Ji-Ying Song, Tomonori Hirose,
Shigeo Ohno, and John G. Collard
Inventory of Supplemental Information
- Figure S1, related to Figure 1.
- Figure S2, related to Figure 2.
- Figure S3, related to Figure 3.
- Figure S4, related to Figure 6.
- Table S1, related to Figure 6.
Supplemental Experimental Procedures
cleared by centrifugation and pre-cleared with protein A-sepharose for 1hr at 4°C. IP-antibodies
were coupled to fresh protein A-sepharose beads, and immunoprecipitation out of pre-cleared
lysates was performed for 4hrs at 4°C. Immunocomplexes were collected, washed five times
and eluted from the beads using SDS sample buffer. Separation by SDS-PAGE (NuPAGE,
Invitrogen) and immunoblotting was performed according to standard procedures.
Living cells were visualized using a phase-contrast microscope (Axiovert 25, Carl Zeiss
MicroImaging, Inc.). Whole slides of H&E- or DAB-stained tissue sections were scanned at 40x
magnification using automated slide scanners (ScanScope XT, Aperio Technologies, Inc., or
Leica SCN400, Leica Microsystems, Germany), and analysed using corresponding viewer
software provided by the manufacturers (Aperio ImageScope, Leica SCNViewer), or Tissue IA
(SlidePath, Ireland). Images of immunofluorescent samples were captured using a conventional
confocal microscope (TCS SP2, Leica, Germany) or spinning disk confocal microscope (Perkin
Elmer), or alternatively an epifluorescence microscope (Olympus IX81), and analysis was
performed using Image J (NIH, USA), Volocity (Perkin Elmer), and Photoshop (Adobe).
Quantification of protein signals in Western Blot analyses
The band intensity of non-saturated Western Blot signals was determined using the Adobe
Photoshop histogram tool on original digital files of scanned Western Blots. Bar diagrams show
the relative protein or phosphorylation signals after normalization using loading controls.
Quantification of Par3 expression in tissue sections
Tissue sections of healthy skin and different tumor specimens were processed for
immunostaining as described above. Micrographs of Par3 signal were taken using the spinning
disk confocal microscope (Perkin Elmer) at non-saturated detection levels, and identical imaging
conditions among epidermal and tumor areas. The signal intensities in epidermis adjacent to the
tumors or within tumor areas were determined using the automated measurement tool of
Volocity software (Perkin Elmer).
Student's t test was performed for quantifications of cell proliferation, apoptosis, active ERK, Akt
and Bax intermediates, tumor numbers and protein expression using Microsoft Excel software.
The asterisks shown in graphs correspond to the p-values as stated in the figure legends.
Measures of pooled data are represented by mean and standard deviation, SD, or standard
error of the mean, SEM, as indicated in the figure legends.
Gao, L., Macara, I.G., Joberty, G. (2002). Multiple splice variants of Par3 and of a novel related
gene, Par3L, produce proteins with different binding properties. Gene. 294, 99-107.
Michiels,F., van der Kammen,R.A., Janssen,L., Nolan,G., and Collard,J.G. (2000). Expression
of Rho GTPases using retroviral vectors. Methods Enzymol. 325, 295-302.