C G K D H L BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 100000 10000 1000 100 10 1 ce 10000 n e c 1000 s e r o 100 u l f y5 10 C 1 10000 1000 100 10 A BSF v PCF E DTT (1 hr) v BSF I B F J 30%FBS v 10%FBS 10%FBS v 0%FBS + BSA 10%FBS v 0%FBS + BSA/Tfn DTT (4 hr) v BSF Tunicamycin (4 hr) v BSF Tunicamycin (24hr) v BSF 1 1 VSG RNAi (1 day) v BSF 10 100 1000 10000 1 VSG RNAi (3 days) v BSF 10 100 1000 10000 1 CLH RNAi v BSF 10 100 1000 10000 1 BSF v MITat1.1 10 100 1000 10000 100000 Cy3 fluorescence SFcigatuterer p4lots of transcriptome data from trypanosomes subjected to altered culturing conditions and other challenges Scatter plots of transcriptome data from trypanosomes subjected to altered culturing conditions and other challenges. In each case, Cy5 fluorescence is plotted on the Y-axis and Cy3 fluorescence on the X-axis. Spots with a Cy5/Cy3 or Cy3/Cy5 ratio above two, i.e. significantly differentially expressed are highlighted in black, spots with a ratio below two are shown in grey. Panel A: BSF (Cy5) vs PCF (Cy3); data are identical to Figure 1 and reproduced for comparison. Panel B: BSF in 30% FBS (Cy5) vs BSF (Cy3). Panel C: BSF (Cy5) vs BSF in 0% FBS, 5 mg/ml BSA (Cy3). Panel D: BSF (Cy5) vs BSF in 0% FBS, 5 mg/ml, BSA 0.3 mg/ml Tfn (Cy3). Panel E: BSF in 1 mM DTT for 1 hr (Cy5) vs BSF (Cy3). Panel F: BSF in 1 mM DTT for 4 hr (Cy5) vs BSF (Cy3). Panel G: BSF in 5 μg/ml tunicamycin for 4 hr (Cy5) vs BSF (Cy3). Panel H: BSF in 5 μg/ml tunicamycin for 24 hr (Cy5) vs BSF (Cy3). Panel I: VSG RNAi in BSF, induced for 24 hr (Cy5) vs uninduced (Cy3). Panel J: VSG RNAi in BSF, induced for 72 hr (Cy5) vs uninduced (Cy3). Panel K: CLH RNAi in BSF, induced for 24 hr (Cy5) vs uninduced (Cy3). Panel L: BSF (Cy5) vs MITat1.1 grown in vivo in rats (Cy3). The scatter plots for most experiments (panels B-K) indicate few transcripts that fall outside of the region considered as constitutive, in marked contrast to the scattergrams obtained for developmental changes (panel A) and in the comparison between in vitro versus in vivo BSF cultures (panel L). significant augmentation of fluid phase and receptor- mediated endocytic activity [116]. The expression level of CLH was increased, providing a mechanistic explanation for augmented endocytic activity (Figure 5A). These data extend earlier work showing loss of CLH protein follow- ing Rab5A or 5B knockdown in BSF [117], and demon- strating coordinated expression of clathrin and Rab5. Very limited differential expression was seen between Rab5 over-expressing and control cells (Figure 5B, Table 2). Analysis by array, qRT-PCR and Western blot con- firmed that the cell lines were overexpressing Rab5A or Rab5B but only detected significant upregulation for RabX3, adenylate kinase 3 (Tb10.70.5150), and COPIε mRNAs in the Rab5AWT, Rab5AQL and Rab5BQL overex- pressor lines (Table 2, Figure 5C), none of which are expected as specific changes related to early endosome activity. Further, several mRNAs implicated as altered by array analysis failed to be validated by qRT-PCR, suggest- ing small or insignificant changes only (Table 2). Impor- tantly, we were unable to detect a significant increase in CLH mRNA in any of the cell lines by qRT-PCR, despite Page 16 of 28 (page number not for citation purposes)