JCS006197 Supplementary Material

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• Supplemental Tables S1 - S3 -

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• Supplemental Figure S1 -

  Fig. S1. VISTA alignment of BCL2A1 and HCCS1 genomic sequences. The genomic DNA segments of BCL2A1 (Bfl-1) and HCCS1 are located on human chromosome 15q24.3 and 15q25.1, respectively. On the schematic structures of BCL2A1 and HCCS1 genes, black and red boxes indicate exons of which location are indicated as shaded area on the graph. Gray boxes indicate 12 conserved genomic segments that are represented in red graph with more than 75% identity.

• Supplemental Figure S2 -

  Fig. S2. ATAP-induced apoptosis does not require Bax and Bak activities, and is not inhibited by Bcl-xL. (A) BMK-D3 cells lack expression of Bax and Bak, as shown by western blot. Potent pro-apoptotic activity of GFP-ATAP is observed in BMK-D3 cells 24 hours after transient expression, such effect is not observed with GFP-mHR7. Cell survival was measured by PI exclusion. (B) CHO cells stably transfected with Bcl-xL (Pan et al., 2000) exhibited similar response to treatment with GFP-ATAP and GFP-mHR7. In both conditions, GFP-ATAP could induce potent apoptosis, whereas GFP-mHR7 is less effective. Mock represents cells transfected with pEGFP plasmid. Data are expressed as the mean ± s.e. Bars, 10 μm.

• Supplemental Figure S3 -

  Fig. S3. External basic residues can function as pseudo-FR-1 for targeting of ATAP to mitochondria. (A) Schematic structure and amino acid sequences of three GFP-ATAP mutants. GFP-mFR7ΔMCS was constructed by deletion of MCS region from GFP-mFR7 construct. GFP-linker-mFR7 was constructed from GFP-mFR7 by replacing one Arg residue coded by multiple cloning site (MCS) with a hydrophobic-rich linker (11 amino acids). (B) Subcellular localization of GFP-mFR7 mutants was observed using confocal microscope. (C) Cell survival was measured by PI exclusion in the HEK293 cells transfected with 1 μg GFP-mFR7 mutants 24 hours after transfection. Bar, 10 μm.

• Supplemental Figure S4 -

  Fig. S4. Expression of BCL2A1 and HCCS1 in normal human tissues and cancer cells. (A) mRNA expression profiles of BCL2A1 (Bfl-1) and HCCS1 in normal human tissues were retrieved from NCBI UniGene microarray database and analyzed using Sigma plot program. (B) The expression of BCL2A1 and HCCS1 mRNA was analyzed by RT-PCR in the normal lung, breast and cervix and cancer cell lines as described in the Materials and methods. Each 1 μg of total RNA was subjected to cDNA synthesis and 25 cycles of PCR amplification were performed. PCR products were analyzed on 2% agarose gel and visualized by ethidium bromide staining.