JCS038513 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression of MTGM in human brain tumors and in normal tissues analyzed by RT-PCR. (A) Expression of MTGM in three indicated human tumor cell lines. (B) Expression of MTGM in six medulloblastoma (Mb) (left panel) and three astrocytoma (A) tumor tissues (right panel). (C) Expression of MTGM in eight normal adult human tissues. The ratios of MTGM to β-actin levels are indicated in each panel. PCR reactions were run for 35 cycles (in A and C) or 42 cycles (in B) to detect MTGM and for 26 cycles (in A and C) or 28 cycles (in B) to detect β-actin.

• Supplemental Figure S2 -

  Fig. S2. Structural organization of the human MTGM gene and transcript variants. (A) Nucleotide and deduced amino acid sequences of MTGM-α (GenBank accession no: AM397244), MTGM-β (GenBank accession no: AM397245) and MTGM-γ (GenBank accession no: AM397246) transcript variants. The exon 2b is distinguishable from exon 2a by a 103 bp extension at the 5′ end. Exon-exon junction sites are pointed out by short arrows and the predicted stop codons are marked by asterisks. Long arrows show primers used in this work. (B) Comparison of exon 1a in variant α and exon 1b in variant β of the human MTGM mRNA transcripts. Exon sequences are in upper case and intron sequences are in lower case. Exon 1b contains an extension of five nucleotides in its 3′ end (underlined), compared with exon 1a. Alternative splice sites of exons 1a and 1b with introns are indicated by arrows. (C) Schematic representation of the MTGM gene on chromosome 20 (top). Each box represents an exon and lines represent introns. The solid grey area in exons 2 and 3 represents the coding region. The three alternative transcripts contain the same open reading frame but differ in the 5′ untranslated region.

• Supplemental Figure S3 -

  Fig. S3. Multiple amino acid sequence alignment of MTGM orthologs in different species. The putative TM domain is marked. A tetrad of the GXXXG motif was identified in the TM domain of all species examined and is marked at the bottom. Abbreviations for the species and GenBank numbers included in the alignment analysis are: H. sapiens (NM_080748); M. musculus (NM_025946); R. norvegicus (XM_001053332); B. taurus, (XM_870863); M. mulatta (CO774585); G. gallus (XM_001231645); S. salar (BT046576); D. rerio (NM_200797); D. melanogaster (NM_140500); C. elegans (CAB02486); S. cerevisiae (AY558335); and A. thaliana (NM_111670).

• Supplemental Figure S4 -

  Fig. S4. The effect of overexpression of MTGM-V5 on the levels of cyclins and Cdk inhibitors. 293T cells were transfected with MTGM-V5 and empty vector for 24 hours and analyzed by immunoblotting.

• Supplemental Figure S5 -

  Fig. S5. Inhibition of caspase activity by the inhibitor Z-VAD-FMK had no effect on mitochondrial fragmentation induced by MTGM. (A) 293T cells were transfected with MTGM-V5 vector, and 2.5 hours after transfection the cells were incubated with culture medium in the presence and absence of the pan-caspase inhibitor Z-VAD-FMK (100 µM) for 24 hours. (B) Percentages (mean ± s.d.) of MTGM-V5 expressing cells with indicated mitochondrial morphologies are shown for the cells treated with Z-VAD-FMK (n=440) and for non-treated cells (n=422).

• Supplemental Figure S6 -

  Fig. S6. Reduction of endogenous MTGM by siRNA resulted in mitochondrial elongation. (A) Representative images of 293T cells transfected with the negative control siRNA (upper panel) and siMTGM-7 (lower panel) and stained with MitoTracker red to reveal mitochondrial morphology. (B) Representative images of HeLa cells transfected with the negative control siRNA (upper panel) and siMTGM-10 and siMTGM-7 (lower panel) and stained with MitoTracker red to reveal mitochondrial morphology. Two cells pointed by arrows were highly magnified in each panel.

• Supplemental Tables S1 - Adobe PDF

• Supplemental Tables S2 - Adobe PDF

• Supplemental Tables S3 - Adobe PDF