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1 Supporting Information Liew et al. 10.1073/pnas.1323066111 SI Methods RNA Degradation Assay. For degradation experiments, cells were Proinsulin-Processing Assay. For proinsulin-processing assays, incubated with 100 g/mL Actinomycin D to inhibit transcription cells grown in 6-cm plates were lysed in Laemmli sample and harvested at 0, 30, and 60 min. Insulin pre-mRNA and buffer, sonicated, and heated at 80 C for 10 min. After mature RNA were determined by generating specific primer sets electrophoresis through an 816% Trisglycine gel (Invitro- that amplify segments of exons to measure spliced and unspliced gen), proteins were transferred to a nitrocellulose mem- mRNA (i.e., total mRNA). Those primer sets that amplify seg- brane and probed with anti-GFP rabbit polyclonal antibody ments of introns only or intron/exon boundaries we considered (Invitrogen). unspliced mRNA (i.e., pre-mRNAs). Fig. S1. (A) Proinsulin content or proinsulin-to-insulin ratio in islets isolated from insulin receptor knockout in the cells (IRKO) or control mice using ELISA or HPLC (n = 56). *P < 0.05. Data are mean SEM. (B) Proinsulin content (ELISA) in IRKO or control cell lines (n = 4). *P < 0.05. Data are mean SEM. (C) Western blotting for prohormone convertase (PC) 1/3, PC2, carboxypeptidase E (CPE), or tubulin in islets isolated from control or IRKO mice (n = 3). Immunostaining for PC1/3 (green) or CPE (green) and DAPI (blue) in pancreas sections from IRKO or control mice. (Scale bar, 50 m.) (D) Proinsulin content in control, IRKO, or -CPEreexpressing (Re-Exp) cell lines (n = 4). ***P < 0.001. Data are mean SEM. (E) Western blotting for IR or tubulin (loading control) in control (Con), IRKO, mock, or IR Re-Exp -cell lines (n = 3). Liew et al. www.pnas.org/cgi/content/short/1323066111 1 of 6

2 Fig. S2. (A) Real-time quantitative PCR (qPCR) for PC1/3, PC2, or CPE mRNA in control (open bar) or IRKO (gray bar) primary fluorescence-activated cell-sorted (FACS) cells (n = 4 per group). All data are presented as mean SEM. TBP, TATA-binding protein. (B) qPCR for PC1/3, PC2, CPE, or 7B2 mRNA in control, IRKO, mock, or Re-Exp -cell lines (n = 3 per group). All data are presented as mean SEM. (C) mRNA degradation rates for PC1/3, PC2, or CPE in control or IRKO cell lines assessed by real-time qPCR analyses. Data are expressed as the fold change compared with time 0 (n = 3 per group). All data are presented as mean SEM. (D) Protein t1/2 for PC1/3, PC2, or CPE in control or IRKO cell lines assessed by Western blotting (n = 3 per group). CHX, cycloheximide. Liew et al. www.pnas.org/cgi/content/short/1323066111 2 of 6

3 Fig. S3. (A) qPCR for PC1/3, PC2, or CPE mRNA in control or IR knockdown (KD) cells harvested 24, 28, 32, or 36 h after IR shRNA containing lentiviral in- fection (n = 3 per group). All data are presented as mean SEM. (B, Left) Real-time qPCR for mRNA of endoplasmic reticulum (ER) stress markers [immu- noglobulin-heavy-chain-binding protein (BiP), total X-box binding protein-1 (XBP-1), spliced XBP-1, or C/EBP homolog protein (CHOP)] in control (open bar) or IRKO (gray bar) cell lines (n = 4). (B, Right) Western blotting for phosphoinositol-requiring enzyme 1 (p-IRE1), total IRE1, or tubulin (loading control) in control or IRKO cell lines stimulated with 2.5 or 16.7 mM glucose (n = 3). *P < 0.05; **P < 0.01. (C, Upper) Western blotting for p-IRE1, IRE1, or tubulin (loading control) in control or IR KD cells harvested 24, 32, or 44 h after IR shRNA containing lentiviral infection (n = 3 per group). (C, Lower) qPCR for spliced XBP-1, total XBP-1, CHOP, or BiP mRNA in control or IR KD cells harvested 24, 28, 32, or 36 h after IR shRNA containing lentiviral infection (n = 3 per group). All data are presented as mean SEM. Liew et al. www.pnas.org/cgi/content/short/1323066111 3 of 6

4 Fig. S4. (A) Proinsulin content (ELISA) and Western blotting for proinsulin in control, IRKO, or IRKO proinsulin KD cell lines (n = 4 per group). ***P < 0.001 (vs. control); ##P < 0.01 (vs. IRKO). All data are presented as mean SEM. (B) qPCR for mRNA of ER stress markers (BiP, total XBP-1, spliced XBP-1, and CHOP) in control, IRKO, or IRKO CPE Re-Exp cell lines (n = 4 per group). All data are presented as mean SEM. *P < 0.05 (vs. control); #P < 0.05 (vs. IRKO); ##P < 0.01 (vs. IRKO). (C) Western blotting for p-IRE1, total IRE1, BiP, CHOP, and tubulin (loading control) in control, IRKO, or IRKO CPE Re-Exp cell lines (n = 3 per group). (D) Real-time qPCR for mRNA of ER stress markers (BiP, total XBP-1, spliced XBP-1, and CHOP) in control, IRKO, mock, or Re-Exp -cell lines (n = 4 per group). All data are presented as mean SEM. *P < 0.05; **P < 0.01. (E) Western blotting for p-IRE1, total IRE1, BiP, CHOP, and tubulin (loading control) in control, IRKO, mock, or Re-Exp -cell lines (n = 3 per group). Liew et al. www.pnas.org/cgi/content/short/1323066111 4 of 6

5 Fig. S5. (A) Western blotting for eIF4A, eIF4H, eIF3A, 4E-HP, eIF2a, peIF2Be, eIF2Be, p4E-BP1 [threonine (Thr)], 4E-BP1, phospho-mammalian target of ra- pamycin (p-mTOR), mTOR, peIF4B(S), eIF4B, or tubulin (loading control) in control or IRKO cell lines under basal conditions (n = 3 per group). (B) Western blotting for p-mTOR, mTOR, phospho-proline-rich Akt substrate of 40 kDa (pPRAS40), PRAS40, ras-related GTP-binding protein C (RagC), p4E-BP1(Thr), 4E-BP1, peIF4E, eIF4E, eIF4A, peIF4B, eIF4B, polyadenylate-binding protein (PABP), peIF4G1, peIF2a, eIF2a, peIF2Be, eIF2Be, 4E-HP, eIF3A, eIF4H, or tubulin (loading control) in control or IR KD cells harvested 24, 32, or 44 h after IR shRNA containing lentiviral infection (n = 3 per group). Fig. S6. (A) Schematic depicts putative duodenal homeobox protein (Pdx1) binding sites on eIF4G1 promoter. (B) Genome browser views of Pdx1 ChIP- sequencing data for mouse islets (Upper) and human islets (Lower). Liew et al. www.pnas.org/cgi/content/short/1323066111 5 of 6

6 Fig. S7. (A) Schematic depicts putative Sterol Regulatory Element-Binding Protein 1 (SREBP1) binding sites on human, mouse or rat eIF4G1 promoter. (B) Western blotting for phospho-Akt, total Akt, or tubulin (loading control) in control or IRKO cell lines under basal condition (n = 3 per group). Liew et al. www.pnas.org/cgi/content/short/1323066111 6 of 6

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