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Files in this Data Supplement:
Fig. S1. shRNAs and siRNAs targeting mouse, rat or human IKAP. (A) Graphic presentation of mouse, rat and human IKAP with localizations and sequences of the shRNAs and siRNAs used in the study to downregulate IKAP together with the scrambled control sequences. Schematic representation of the FD-IKAP used for reintroduction experiment is included.
Fig. S2. HeLa cells display the same phenotypes as MEFs upon IKAP depletion. (A) Western blot analysis of HeLa cells transfected with IKAP siRNA (H1) or scramble (scr) siRNA for 72 hours. The % IKAP values represent the quantification of IKAP depletion (n=1). (B) Plating efficiency of HeLa cells treated with designated oligos for 48 hours after which they were re-plated and calculated after 24 hours inoculation (means ± s.d.; n=3; triplicates; ***P<0.001). (C) Cell cycle analysis of retrovirally transduced IKAP-depleted (m1 and m2) and corresponding vector control MEFs. Cells were fixed and stained with propidium iodide (PI) and the DNA content of the cells was analyzed by flow cytometry (FACSCalibur, Becton Dickinson) and analyzed by Cell Quest Software (Becton Dickinson). Figure is a representative of four individual experiments. (D) Cell death was investigated by measuring the percentage of LDH release 24 hours after re-plating or 48 hours after re-plating with a change of media after first 24 hours (means ± s.d.; n=3; **P<0.01). (E) The adhesion capability of HeLa cells was analyzed by inoculating 5×104 cells/ml in serum-free media on non-supporting adherent-surface 24-well plates (Sarstedt) for 3 hours and analyzed as in Fig. 1E. The absorbance at 570 nm was measured (means ± s.d.; n=1; triplicates; ***P<0.001). (F) Cell spreading was evaluated as described for Fig. 1F, measuring the average cell areas and using the ImageJ software and dividing with the number of cells present (means ± s.d.; n=3; triplicates; **P<0.01). (G) To evaluate migration, cells were seeded at a density of 2×105 cells/ml and allowed to adhere over night. A wound was inflicted and the cells were incubated for 24 hours followed by imaging as described for Fig. 1G. Average wound area was quantified in the picture using the ImageJ software (means ± s.d.; n=3; *P<0.01).
Fig. S3. RT-PCR array analysis of human fibroblasts with depletion of IKAP. (A) Comparison of the transcriptional level of 84 genes important for cell-cell and cell-matrix interactions of IKAP-depleted and vector-control cells analysed by RT-PCR array. (B) Western blot analysis of NCAM levels in IKAP-depleted (m1 and m2) and vector-control cells.
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