The sources of chemicals and other materials used in the study are described in an Additional File 4
Cells and Cell Culture
All experiments were performed using a normal rat (Fischer strain) fibroblast cell line, F111, the origin of which has been described earlier, and primary cultures of skin fibroblast (PC) were made from skin grafts of neonatal rat pups (1-2 days old). F111 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (50 mg/ml) and they were used within five passages after revival from cryo-preservation. PC cells were maintained Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum, which were used within 2 to 3 passages after plating.
Preparation of A and NA cells
Adherent (A) and non-adherent (NA) cells from F111 and PC cells were collected at different time points. The method is described briefly in Additional File 4
. The type of A and NA cells collected is shown by the suffixed number to these letters which represents the time point (in hours) at which they were collected from the agarose/plastic surface.
Cell Viability Assay
The cell viability for adherent and non-adherent cells at different time points was measured by using the MTT assay as described earlier [13
Soft Agar Colony formation Assay
1×105 cells in DMEM containing 10% FCS from both F111 and PC cells in both adherent and non-adherent conditions were suspended in 0.3% agarose. Then, this mixture was layered onto a solidified 1%agarose layer and incubated at 37°C, 5% CO2. The assay was repeated 5-6 times, and colonies each containing > 50 cells, were counted after a week of incubation.
Tumour formation in nude mice
5×106 A16 and NA16 cells from PC and F111 cells were collected at different time points in PBS were injected subcutaneously in to a 4-6 week old, homozygous nude mice (NIH strain, Nu/Nu). The size of the tumor was measured at weekly intervals. All experimental procedures were approved by the Institutional Animal Ethical Committee (IAEC 132/2007 dated May 19th 2008).
Isolation of colony cells and tumour cells
The colonies embedded in soft agar were collected in DMEM containing 10% FCS by disrupting the agar; the cells were allowed to adhere on a 70 mm petri dish for a short period (approximately 4 hr). The attached cells were processed for further experiments. Cells from tumors were collected by treatment of small tumor explants with 0.1% trypsin-EDTA for 30-40 minutes and layered in 20% serum containing DMEM medium with 5% CO2. Cells from the explants were released after a week was used as source of tumour cells.
Excised tumours and the corresponding lung and liver tissues were collected from nude mice and fixed in 10% formalin. The tissues were dehydrated gradually in alcohol; embedded in paraffin and were sectioned at a thickness of 5 μm. The tissue sections were stained with Haematoxylin and Eosin and Masson-Trichome for collagen. The details of the methods were described in Additional File 4
Karyotyping of chromosomes by using Rat SKY probes
Metaphases from PC cells, normal F111, NA16, Colony and tumour cells were dropped on a clean glass slide (Fisher scientific, USA) was which was used for chromosomal painting. The detailed method is described in Additional File 4
Side Population (SP) cell analysis
One million single cells from all cell types were incubated with 2.5 μg/mL Hoechst 33342 (Sigma) for 90 minutes at 37°C. Cells were collected by centrifugation at 2000× g and washed once in HBSS (Hank's balanced salt solution) and re-suspended in ice-cold HBSS, Propidium iodide (50 μg/ml) was added just before analysing the cells on a MoFlo cell sorter (Dako Cytomation Glostrup, Denmark). Inhibitory action on the Hoechst efflux was checked with 50 mM Verapamil.
Immunoflurescence and immunoblotting
Single cell suspensions, of all cell types were plated on coverslips and fixed in 70% ethanol followed by three washes with PBS. Mild cell permeabilization was done with 0.1% triton-X 100 and staining for Hif 1α (1:100), VEGFa (1:150), Stc1 (1:200), ABCG2 (1:50) and CD-133 (1:100) was done by incubating cells at 4°C for 1 hr with the corresponding primary antibodies. After washing, the cells were treated with appropriate fluorescent-labelled secondary Ig antibodies. Stained cells were observed under a Leica confocal microscope with a 63 X, Plan Apo objective. Immunoblotting was performed by analysing equal amount of protein lysates (prepared in lysis buffer containing 50 mM Tris buffer, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 Mm EGTA, 1 mM Na3VO4.H2O) on a 10% SDS-PAGE proteins were blotted on to nitrocellulose paper and probed with different antibodies such as Hif-1α, VEGFa, Stc1 which were visualized by enhanced chemi-luminescence.
RNA extraction and Microarray Analysis for differential gene expression
Total RNA collected from F111-A16, NA16, Colony and Tumour cells was used to screen for differential gene expression patterns The method for the extraction of RNA and microarray analysis has been provided in Additional File 4
. The files for the microarray analysis of all the four cell types has been deposited in the Gene Expression Omnibus (GEO) database and can be publically accessed via the accession number GSE24893.