Fu L , Sun G , Fiorentino M , Shi YB .

Intramural NIH HHS

	Figure 1. There are two X. laevis TIMP2 genes that are highly homologous to the TIMP2 gene in other vertebrates.A) Nucleotide sequence comparison of X. laevis TIMP2A and TIMP2B. Dot (.): identical sequence; dash (-):a gap introduced for better alignment. Arrowed lines indicate the locations of the primers used to distinguish the two isoforms in RT-PCR analysis. B) Comparison of the protein sequences of putative X. laevis TIMP2A (XlTIMP2A, AAH74452), X. laevis TIMP2B (XlTIMP2B, NP_001087748), and X. tropicalis TIMP2 (XtTIMP2, NP_001015760) with their ortholog from chick (ChTIMP2, NP_989629), mouse (mTIMP2, P25785) and human (HuTIMP2, NP_003246). The conserved cysteine sequences are in bold and the signal peptide sequence is underlined. Dot (.): identical sequence; dash (-): a gap introduced for better alignment.
	Figure 2. Comparison of the genomic locus and organization of X. tropicalis and human TIMP2.X. tropicalis, chicken, mouse and human TIMP2 genomic organizations were obtained from NCBI database. The lines represent the genomic sequences and the boxes with arrowed lines on top represent transcribed regions and the direction of the transcription. The lines and boxes were not drawn to the same scale for better visualization. The TIMP2 gene in all four species has the same intron/exon organization with 5 exons total as shown for X. tropicalis TIMP2 (not shown). Note that mouse and human TIMP2 loci are identical. In the chicken, CNTNAP1 gene is present on a different chromosome (shown in a darker shade).
	Figure 3. TIMP2 expression during X. laevis development.Total RNA was isolated from whole animals during X. laevis development and subjected to One-Step RT-PCR analysis with a pair of primers amplifying both TIMP2A and TIMP2B (TIMP2) or stromelysin-3 (ST3). The expression of the house-keeping gene histone H4 was similarly determined as a control.
	Figure 4. Co-regulation of TIMP2A and TIMP2B expression during intestinal metamorphosis.A) Validation of primer specificity for X. laevis TIMP2A or TIMP2B. The TIMP2A and TIMP2B cDNA were cloned into pCR2.1 vector, sequenced and serially diluted to serve as template DNA for PCR. Note that each primer pair specifically amplified only the intended TIMP2 gene. B) Total RNA was isolated from intestines of tadpoles during X. laevis metamorphosis and subjected to One-Step RT-PCR with a pair of primers recognizing both TIMP2 genes (TIMP2), specific for either TIMP2A or TIMP2B, respectively. The expression of the house-keeping gene rpL8 was similarly determined as a control.
	Figure 5. Tissue-specific regulation of TIMP2 expression correlates with metamorphosis.Total RNA was isolated from intestines, hindlimbs and tails of metamorphosing tadpoles at the indicated stages and subjected to qRT-PCR analyses with a primer/probe set specific for TIMP2 or rpL8 (RNA loading control), respectively, and the TIMP2 expression level was normalized to that of rpL8 expression level and plotted in arbitrary units. *: p<0.05 when compared to stage 52.
	Figure 6. T3-inducktion of TIMP2 expression in different tissues of premetamorphic tadpoles.Tadpoles at premetamorphic stage 54 were treated with 10 nM T3 at 18°C and collected every other day for isolating total RNA from different tissues. The RNA was analyzed by qRT-PCR analysis as in Fig. 5. *: p<0.05 when compared to the group without T3 treatment.

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