Western blot showing the complete repression of clathrin expression in DKO-S and DKO-R cells when grown in the presence of 0.1 µM doxycycline (dox).

<p>Cells were grown for 72 hours in media with or without doxycycline as indicated. Following development of the blot, the nitrocellulose was stained with amido black to detect total protein.</p

DKO-S cells show a higher apoptotic response to clathrin-depletion than DKO-R cells.

<p>(A) The proportion of apoptotic cells and (B) caspase activity were measured as described in materials and methods for DKO-S and DKO-R cells grown with or without 0.1 µM doxycycline (dox) and in media supplemented with increasing concentrations of chicken serum are as indicated. Values are means of three measurements +/− standard deviation. Statistically significant differences, with p values, are indicated.</p

Microarray analysis of the gene expression profiles of DKO-s and DKO-R cells.

<p>(A) differentially expressed genes ordered in descending significance according to the P value UK: unknown. (B) functional clusters of genes of processes implicated in cell fate. (C) depiction of the CXCR4 signalling pathway.</p

CXCR7 signaling is not responsible for the apoptotic-resistace shown by clathrin-depleted DKO-R cells.

<p>(A) Quantitative RT-PCR for CXCR7 in both cell lines. (B) Caspase activity and percentage of apoptosis of clathrin-expressing and clathrin-repressed DKO-R cells in the presence or absence of 0.5 µM CCX771 and inactive analog CCX704. Cells were grown in standard DT40 medium with 1% chicken serum. Values are means of four determinations +/− standard deviation.</p

RT-PCR for CXCR4, SDF-1 and CXCR7 in DKO-S and DKO-R cells.

<p>(A) standard RT-PCR as described in materials and methods followed by agarose gel migration. Controls (RNA) for genomic DNA contamination in which the RT-PCR reaction was conducted without prior treatment with reverse transcriptase are shown. (B) real time PCR from cDNA obtained from both cell-lines, results are expressed as ΔCp normalising the levels of expression to Cyclophilin A. (C) Caspase activity and percentage of apoptosis of clathrin-expressing and clathrin-repressed DKO-S cells in the presence or absence of 20 nM recombinant human SDF-1α. Values are means of four determinations +/− standard deviation. (D) Caspase activity and percentage of apoptosis of clathrin-expressing and clathrin-repressed DKO-R cells in the presence or absence of 5 µM AMD3100. In both (C) and (D), cells were grown in standard DT40 medium with 1% chicken serum. Values are means of four determinations +/− standard deviation. Statistically significant differences, with p values, are indicated.</p

Identification of the putative chicken survival factor as transferrin.

<p>(A) FPLC separation of the positive fraction from the gel filtration step showing separation of the main bioactivity peak from major protein fractions. In each case, cell growth after 72 hours was determined as described in materials and methods. (B) SDS polyacrylamide gel electophoresis of the fractions from the FPLC column. Fraction 6, which contains the major bioactivity peak contains only two major proteins identified as vitamin D binding protein and transferrin. (C) Western blot of FPLC fraction 6 using anti-(chicken transferrin) monoclonal antibody.</p

Analysis of the expression of transferrin and its receptor.

<p>(A) Quantitative RT-PCT of the transferrin receptor in both cell lines. (B) Western blot for the transferrin receptor in DKO-R and DKO-S cells. (C) Quantitative RT-PCR of transferrin in a control hepatic human cell line (Huh7) and DKO-R and DKO-S cells. Statistically significant differences, with p values, are indicated.</p