Pancreatic beta cells lack a low glucose and O2-inducible mitochondrial protein that augments cell survival -- Wang et al. 103 (28): 10636 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Wang et al. 10.1073/pnas.0604194103.

Supporting Information

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Fig. 6. Sketch of HIMP1 gene structure and cluster analysis of HIMP1 homologues. (A) HIMP1 gene structure. Exons are shown as rectangles and numbered. Introns are shown with lines. The intron-like part of exon 3 is shadowed. (B) Cluster analysis of HIMP1 homologues. Columns "(N)" and "Identity" indicate AA number, and percent of identical AAs compared to HIMP1-a, respectively. Dashes indicate AA gaps. Asterisks indicate the two predicted transmembrane helices.

Supporting Text

Cell Culture, Islet Isolation, and RNA Preparation.
Normal culture of aTC1.6, MIN6, and bTC3, cells, islet isolation, and RNA preparation were described previously (Wang et al., 2003a). Construction of a aTC1.6 cDNA Library and Molecular Cloning of HIMP1 cDNAs.
Poly(A)+ RNA was prepared from aTC1.6 cells by using the Oligotex mRNA Mini KIT (Qiagen). A aTC1.6 cell cDNA library in the Uni-ZAP XR vector was constructed by following the instruction manual of the ZAP Express XR library construction kit (Stratagene). For molecular cloning of HIMP1 cDNAs, 1 ´ 10⁶ clones were screened using a cDNA probe found by mRNA differential display to be expressed well in aTC1.6 cells, but not in MIN6 cells. The corresponding phagemids were produced from purified positive lambda phage clones by in vivo excision. Sequencing was performed by using an ABI PRISM sequencing kit (Perkin-Elmer). Characterized nucleotide sequences for the mouse HIMP1 cDNAs have been deposited in the GenBank database (accession nos. AY028386 and AY028387). RT-PCR, Northern Blot, and In Vitro Synthesis.
The procedures have been described (1, 2). Total RNA (1 mg) from cell lines and islets was subjected to RT-PCR. 10 mg of total RNA was used for Northern blot. HIMP1-a was in vitro synthesized with the TnT Coupled Transcription/Translation system (Promega). The cDNA of HIMP1-a were amplified by PCR with primers (5'-CTTAAGCTTGCGGCCAGAAACCGGCAGGAC-3' and 5'-AGTGGATCCAGCTCTTCTAAGGCTTAGGGC-3', digested with HindIII and BamHI, and then cloned into the pcDNA3.1(+) vector for In vitro synthesis and transfection. [³⁵S]methionine was purchased from Amersham Pharmacia. Antibodies, Immunoblot, Immunoprecipitation, and Histology.
Antiserum against the N-terminal 1-17 residues of HIMP1 was raised in rabbits (Covance, Richmond, CA) for immunoblot and immunoprecipitation. For histological studies, the antiserum was further purified by incubation with a PVDF membrane bound with proteins extracted from MIN6 cells. Antibodies against mtHsp70, VDAC (Affinity BioReagents), COX IV subsunit I (Molecular Probes), GM130 (BD Transduction Laboratories), glucagon or C-peptide (Linco Research), and tubulin (Sigma) were purchased commercially. DAPI was purchased from Roche Molecular Biochemicals.

The procedures for immunoblot, immunoprecipitation, and immunofluorescent double or triple staining were basically similar with those described by Wang et al. (1, 2). Pancreas sections were double-stained with antibodies against HIMP1 (1:400) and glucagon or C-peptide (1:500). Staining of cells cultured on cover glass was performed as the procedure for tissue section (1) besides adding a step of treatment with 0.1% Triton X-100/PBS for 5 min after fixation. Nuclear staining with DAPI (1 mg/ml) is for cell number counting in some cases. For TUNEL staining, cells treated with Triton X-100 were first incubated with biotin-conjugated TdT mix and enzyme from DeadEnd colorimetric TUNEL system (Promega) at 37°C for 30 min, then carried out immunofluorescent staining with Texas Red dye-conjugated straptavidin (1:500). Second antibodies (Cy2 AffiniPure Donkey Anti-Guinea IgG, Cy2 or Cy3 AffiniPure Donkey Anti-Rabbit IgG, Texas Red dyr-conjugated straptavidin, and Texas Red AffiniPure Donkey Anti- Mouse IgG) were purchased from Jackson ImmunoResearch. Fluorescent and DIC images were examined with an Olympus BX51 microscope or Fluoview 200 laser scanning confocal microscope.

For immunoelectron microscopy, pellets of aTC1.6 or MIN6 cells and a slice of mouse heart or testis were fixed with a solution (2% formaldehyde and 0.05% glutaldehyde/PBS, pH 7.4) for 30 min at room temperature and 30 min on ice, then embedded in Lowicryl K4M (EM sciences). After 10 days, polymerization by UV light inside a freezer, and the samples were sectioned and mounted on carbon-coated nickel grids. The grids were rehydrated with PBS for 30 min, blocked with 1% BSA/PBS for 30 min, and then in anti-HIMP1 serum (1: 150) for 3.5 h. After washing, the second antibodies (goat anti-Rabbit IgG conjugated with 10 nm gold particles, Ted Pella) were applied for 45 min. After washing, the grids were fixed for 5 min with 1% glutaldehyde followed by rinsing, staining briefly with uranyl acetate and lead citrate, and drying for examination at accelerating potential of 120 kV.

Subcellular Fractionation and Topology Studies.
Membrane and cytosol fractions of aTC1.6 cells were separated as previous described (3) with modifications as follows: 2 ´ 10⁷ harvested cells were incubated in 1.0 ml of 100 mM sodium carbonate solution (pH 11.5) with proteinase (10 mg/ml of leupeptin and aprotinin) on ice for 30 min, and then homogenized by 30 strokes with a Wheaton homogenizer. The postnuclear supernatant (PNS) prepared by centrifugation at 750 ´ g for 10 min, was centrifuged at 40,000 ´ g for 60 min to separate the membrane and cytosol fractions for immunoblotting. Sucrose gradient fractionation was carried out using the method described by Wendland and Scheller (4). Separated fractions were subject to immunoblots and assays for glucagon by RIA (Linco Research Inc.). For separating the outer and inner membranes of mitochondria, a mitochondrial pellet was initially obtained by centrifuging at 7,000 ´ g for 15 min from a PNS fraction in homogenization buffer (0.25 M sucrose, 5 mM Hepes-KOH, pH 7.4, and 10 mg/ml each of leupeptin and aprotinin). The mitochondrial suspensions were treated by digitonin (1.2 mg/ 10 mg of mitochondrial protein), and diluted as previous described (5). The inner membrane fraction (mitoplasts) was pelleted by centrifuging at 9,500 ´ g for 10 min from the digitonin-treated mitochondrial suspensions, then the outer membrane fraction was obtained by centrifuging at 144,000 ´ g for 30 min from the supernatant. The topology studies were carried out with a method described by Blobel and Dobberstein (6) using in vitro synthesized HIMP1-a protein and membrane fractions of mitochondria with trypsin and chymotrypsin (Sigma) treatment. Expression of HIMP1-a Stably in MIN6 Cells, and Transiently in bTC3 Cells.
The HIMP1-a or vector plasmid DNA was transfected into MIN6 or bTC3 cells using LipofectAMINE (Invitrogen). Clones of MIN6 cell expressing HIMP1-a permanently, termed MIN6^HIMP1-a, were selected with G418-resistant and immunoblots with anti-HIMP1 serum. bTC3 cells were used only for transient transfection because of their relatively high transfection efficiency (~10-15%). Apoptosis and Viability Examination of b Cells with Ectopic Expression of HIMP1-a Under Different O₂ or Glucose Conditions.
At 48 h after culture in 12-well plates, equal numbers of clone 10 MIN6^HIMP1-a and control cells were subjected to hypoxia (5% O₂/5% CO₂/90% N₂) and normal culture condition (95% air/5% CO₂) for 24 h, or subjected to either low (2.5 mM) and high (25 mM) glucose DMEM culture in 95% air/5% CO₂ incubator for 1, 2, 3 or 7-days. In bTC3 cells, at 48 h after transfection, cells were subjected to the same treatments as for the MIN6^HIMP1-a cells. Total cell numbers were counted by DAPI staining. Apoptotic cells were determined by TUNEL staining. The number of viable cells was calculated by subtracting the number of apoptotic cells from total cells. The percent apoptotic cells was calculated as no. of apoptotic cells/no. of total cells ´ 100. Percent of viability at low glucose was calculated as the no. of viable cells at low glucose/no. of total cells at high glucose for the same time point ´ 100. The number of HIMP1-positive cells in bTC3 transfected cultures was determined by HIMP1 immunofluorescent staining. Cell number of each was determined by counts from 10 image fields of each of duplicates, and at least 5 independent experiments were carried out for each treatment.

Data are present as the means ± SE. Statistical significance was assessed by the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0005.

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