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1 Supplemental Materials, Nishimura et al, Page of 5 Quantitative assessment of Pdx promoter activity in vivo using a secreted luciferase reporter system Wataru Nishimura, Koki Eto, Atsushi Miki, Motohito Goto, Miho Kawaguchi, Takao Nammo, Haruhide Udagawa, Masaki Hiramoto, Yukiko Shimizu, Tadashi Okamura, Toshiyoshi Fujiwara, Yoshikazu Yasuda and Kazuki Yasuda. 0 Supplemental Materials Supplemental Materials and Methods. Supplemental Figure Legends. Supplemental Tables (). Supplemental Figures (0) 9

2 Supplemental Materials, Nishimura et al, Page of SUPPLEMENTAL MATERIALS AND METHODS Cell Culture Rat β-cell line INS- was cultured in RPMI medium (Sigma, St. Louis, MO), with 0% fetal bovine serum (FBS; Thermo Scientific, Logan, UT), mm sodium pyruvate, 0 mm Hepes, 00 U/ml penicillin, 00 mg/ml streptomycin and 55 µm β-mercaptoethanol (Life Technologies, Carlsbad, CA). NIH-T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 0% FBS, 00 U/ml penicillin and 00 mg/ml streptomycin. Mouse β-cell line MIN6 cells were cultured in DMEM with 5% FBS, 00 U/ml penicillin, 00 mg/ml streptomycin and 55 µm β-mercaptoethanol. All cell lines were cultured at 7 C under 5% CO Genotyping Genotyping of the mice was performed by NaOH extraction methods. Briefly, mm tails of the mice were cut and plated into the 96-well plate. 75µl of 5mM NaOH, 0.mM EDTA was added to each well, followed by placing the plates in the thermocycler at 98 for one hour, then the temperature was reduced to 5. 75µl of 0mM of Tris/HCl ph5.5 was added to each well, and was centrifuged at,000rpm for 5 minutes. µl of supernatant was used for PCR reaction with the primers (see Supplemental Table for information on the primers and see Supplemental Figure A for the results of PCR). In parallel, the plasma GLuc activity was measured as mentioned below (Supplemental Figure BC). The plasma GLuc activity of genotype positive mice varies even within the same line, which can be divided into the two groups (Supplemental Figure C), and transgenic mice with high GLuc activity were analyzed in this study. 5 Islet Isolation Mice were deeply anesthetized followed by cervical dislocation. A laparotomy was performed, and the pancreas was exposed. One milliliter of collagenase solution (Liberase-TL; Roche,

3 Supplemental Materials, Nishimura et al, Page of Indianapolis, IN) was injected into the pancreas via the common bile duct from the ampulla of Vater after the ligation of the distal common bile duct. The pancreas was removed and incubated in a water bath for 0 min at 7 C. The islets were separated by a density gradient (Histopaque-08 and 9; Sigma) and centrifuged at 60g for 0 min. The islets were handpicked and cultured overnight in RPMI-60 supplemented with 0% FBS, mm sodium pyruvate, 0 mm Hepes, 00 U/ml penicillin and 00 mg/ml streptomycin. Islets of similar size were handpicked and cultured in 6-well dishes, followed by performance of the reporter assay, imaging or transplantation Preparation of Samples from Medium, Plasma or Organ Homogenates for Gaussia Luciferase Assay INS-, MIN6 and NIH-T cells were transfected with the indicated reporter plasmids and the psv-β-gal (Promega) or the pgl. vector (Promega) for the internal controls using Lipofectamine 000 (Life Technologies) according to the manufacturer's protocol. Forty-eight hours after the transfection, the culture medium was analyzed for Gaussia luciferase activity as described in the Materials and Methods, and the cells were examined for Firefly luciferase activity with the One-Glo luciferase assay system (Promega) or the β-gal assay as reported previously (8). The culture medium of the transgenic islets was also used for the assay. For both of these assays, 00 µl of culture medium was briefly centrifuged, and 0 µl were used for the luciferase assay. Blood from a tail snip was taken via heparinized tubes followed by centrifugation, and the plasma was taken, frozen with liquid nitrogen and kept at -80 C. Thawed samples were used after brief centrifugation. For the homogenates of organs or isolated islets, various organs from the mice were removed and frozen with liquid nitrogen after the injection of collagenase solution from the pancreatic duct followed by pancreatectomy for islet isolation. Samples were weighed and homogenized in a solution of 50 mm Tris/HCl at ph 8.0, 00 mm NaCl, and % Triton-X with the homogenizer Shakeman and.0 mm zirconia beads (Bio Medical Science, Tokyo, Japan), followed by centrifugation at 6,00g for

4 Supplemental Materials, Nishimura et al, Page of 5 minutes. Supernatants were diluted to a tenth of their original concentration in PBS and used for the reporter assay. The results of the luciferase activity derived from organ homogenates were normalized using protein concentrations as determined from a BCA assay Analysis of metabolic parameters Blood glucose values were measured on blood from tail snip using Glutest Ace-R and Glutest Sensor (Sanwa Kagaku Kenkyusho, Nagoya, Japan). For intraperitoneal glucose tolerance testing, mice were deprived of food for to 6 hours and blood glucose and body weight were measured prior to peritoneal injection of g / kg body weight glucose. Blood glucose was measure at the time point of 5, 0, 60 and 0 minutes after glucose loads. For insulin tolerance testing, mice were fasted for three hours, followed by the measurement of blood glucose and body weight. U/kg body weight of human regular insulin (Humalin R, Eli Lilly, Indianapolis) was injected and blood glucose levels were measured at the time point of 5, 0 and 60 minutes Assay of insulin secretion from isolated islets. 5 isolated islets from - to -month-old mice were manually selected, incubated in Krebs- Ringer solution, and stimulated at 7 C with indicated concentrations of either glucose or KCl for the indicated time period. The supernatants were then collected by centrifugation and were assayed for GLuc by luciferase assay or for insulin by ELISA (Shibayagi, Gunma, Japan). 0 5 The detailed information of immunohistochemistry and morphometric analysis for quantification Pancreatic samples were isolated in ice-cold PBS. Tissues were transferred to 0% sucrose in PBS at C overnight. Subsequently, tissues were transferred to a : 0% sucrose/oct compound solution for hours at C, followed by 00% OCT for hour at C, then were placed in OCT and kept at -80 C until sectioning. After cryosectioning, immunostaining analyses were performed after

5 Supplemental Materials, Nishimura et al, Page5 of incubation in % paraformaldehyde in 0.% Sorenson s Phosphate buffer as described previously (8) and in the Materials and Methods. See Supplemental Table for the list of the primary antibodies used in this study. For the quantification of the proportion of insulin positive cells expressing GLuc, totally 7 or 5 insulin positive cells in randomly selected sections of the transgenic pancreas or the transplanted islets (n=) were counted for the expression of GLuc. The data was presented as the percentage of GLuc positive cells per insulin positive cells. For the quantification of insulinexpressing or GLuc-expressing areas, sections of transgenic pancreas with or without surgery (n = for each group) were immunostained at 00-µm intervals to avoid measurement of the same islets twice. The images of entire pancreas were obtained by automatic scanning of BZ-9000 and analyzed with BZ-HC (Keyence, Osaka) Bioluminescence (BLI) A total of 00 µg of CTZ (500 µl of 00 µg/ml CTZ; approximately mg per kg body weight) was injected into the tail vein of 8 week-old mice anesthetized with isoflurane (% in 98% O ) (,8). For cultured islets, 5 µg of CTZ (5 µl of mg/ml CTZ) was added directly to -well plates containing 500 µl of islet culture medium. The mice and medium were analyzed using an IVIS Lumina II equipped with a charge-coupled device (CCD) camera (Xenogen/Caliper, Alameda, CA). BLI intensity was quantified using Living Image software (Xenogen) and by performing photoncounting measurements in equal-area regions of interest centered over the bioluminescent region. The stock solution of mg/ml CTZ in methanol with 0.M HCl was stored at 80 C, which was diluted in PBS with 5mM NaCl (ph7.) for the working solution and used after incubation for 0 minutes at room temperature with protection from light. 5 6 Islet Transplantation Mouse islets were transplanted under the kidney capsule of the male SCID mouse. The isolated islets were counted and aliquoted into groups of islets. The islets were then

6 Supplemental Materials, Nishimura et al, Page6 of centrifuged in PE50 polyethylene tubing (BD Bioscience, San Jose, CA) to form pellets. Male SCID mice were deeply anesthetized by intraperitoneal injection of 65 mg/kg body weight of the pentobarbital solution Somnopentyl (Kyoritsu Seiyaku, Tokyo, Japan), and an incision was made in the kidney capsule. The islets were gently deposited in the subcapsular space through a polyethylene tube. The incision in the kidney capsule was then closed, and the surgical wound was sutured. After wound closure, mice were allowed to recover on a warm plate. Blood glucose values were measured in blood from a tail snip using a Glutest Ace-R and Glutest Sensor (Sanwa Kagaku Kenkyusho), plasma samples for the GLuc assay were taken and frozen, and body weights were measured at the indicated time points after the surgery Partial Pancreatectomy Blood glucose and body weights of -week-old transgenic or 0-week-old wild type mice were measured, and blood samples for the GLuc activity were taken from snipped tails and frozen prior to the surgery. Mice were deeply anesthetized using Somnopentyl (Kyoritsu Seiyaku). Partial pancreatectomy (approximately 50%) was performed by resection of the body and tail of the pancreas and the spleen, leaving the pancreatic head and duodenum intact. Blood glucose values, plasma GLuc activity and body weight were measured at the indicated time points after the surgery. 8

7 Supplemental Materials, Nishimura et al, Page7 of SUPPLEMENTAL FIGURE LEGENDS Supplemental Fig. : Dynamic state of GLuc in vivo and in vitro (A) The medium with (GLuc) or without (Control) GLuc was injected into the tail vein of wild-type mice, followed by the analysis of plasma GLuc activity at the indicated time points. GLuc is quickly cleared from the circulation within twenty minutes (n=). (B) GLuc activity in the urine of Pdx-GLuc and wild-type mice collected overnight revealed secreted GLuc is eliminated mainly by renal excretion (n=). (C) Culture medium of INS- cells two days after the transfection with ppdx-gluc-enhancer vector (Figure BC) were stored at, and an aliquot of medium was frozen at the indicated time points. The GLuc activities were measured, which revealed GLuc is extremely stable at. (D) The plasma of Pdx-GLuc mice were incubated at 7, and an aliquot of the plasma was frozen at the indicated time points. The GLuc activities were measured, which revealed that the half life of GLuc is more than days at Supplemental Fig. : Identification, establishment and genotype of Pdx-GLuc mice (A) The results of PCR for genotyping founders # to #7. Founders #, # and #5 were genotypepositive. (B) Plasma GLuc activity in each founder relative to that in the wild-type control (n = ). Lines #, #, # and #7 were subjected to the screening using BLI. (C) The averages of the plasma GLuc activity in Pdx-GLuc (line #) and wild-type mice. Pdx-GLuc mice could be divided into two groups, GLuc high group (n=) and low group (n=7), both of which are higher than wild-type mice (n=85) in the plasma GLuc activity. (D, E, F) Body weight (D), fed blood glucose (E) and plasma GLuc activity (F) in Pdx-GLuc mice were measured for weeks. The relaive plasma luciferase activities were stable over time. n=0. Each bar and dot represent the mean ± SEM. 5 Supplemental Fig. : Metabolic characterization of Pdx-GLuc mice

8 Supplemental Materials, Nishimura et al, Page8 of (A) Body weight, (B) fasting blood glucose and (C) the results of intraperitoneal glucose tolerance testing of Pdx-GLuc mice (n = 5) and wild-type mice (n = ). (D) The results of insulin tolerance testing of Pdx-GLuc mice (n = ) and wild-type mice (n = ). Each bar and dot represent the mean ± SEM Supplemental Fig. : Batch incubation assay of Pdx-GLuc islets (A) 5 islets of Pdx-GLuc and wild-type mice were incubated in 500 µl of Krebs-Ringer buffer (KRB) with.8mm glucose for 60 minutes, followed by the measurement of GLuc activity. The results are presented as the fold increase relative to the wild-types. n = for Pdx-GLuc and n= for wild-type mice. (B) 5 islets of Pdx-GLuc mice were incubated in KRB with.8mm glucose, 6.7 mm glucose or 60 mm KCl for 60 minutes, followed by the measurement of GLuc activity. The results are presented as the fold increase relative to the values of.8mm glucose. The assay # (n=) used the islets from two mice and the assay # (n=) and # (n=) used the islets from a single mouse. (C) Insulin secretion measured in parallel with the experiments (B). Each bar represent the mean ± SEM Supplemental Fig. 5: Analysis of the islets of Pdx-GLuc mice line # (A) The reporter assay performed in homogenates of various organs from Pdx-GLuc line # (C; red) or wild-type mice (blue) reveals GLuc activity in islets that is greater than 00-fold higher than that in non-islets of the pancreas. n = for both genotypes. The plasma GLuc activity is also presented in RLU. (B, C) Immunohistochemistry of the Pdx-GLuc pancreas line #. (B) GLuc (green) colocalizes with insulin (red). (C) GLuc (green) is not colocalized with glucagon (red). DAPI (blue). Bar: 0 µm. (D-F) Isolated Pdx-GLuc islets line # were cultured overnight, and the indicated number of islets of similar sizes were handpicked and cultured for two hours. (D) The results of a reporter assay performed in culture medium demonstrate that the secreted luciferase activity correlated with the number of islets. (E) Analysis of the medium demonstrates a graded photon emission in response to an increasing number of islets. (F) The quantification of the BLI presented in (E) demonstrates a

9 Supplemental Materials, Nishimura et al, Page9 of correlation between GLuc activity and the number of islets. n =. Each bar in A and dot in D and F represent the mean ± SEM Supplemental Fig. 6: Immunohistochemistry of the Pdx-GLuc pancreas for GLuc and glucagon Colocalization of GLuc (green) with glucagon (red) is rare. Quantification of immunohistochemistry revealed that less than % of the glucagon positive cells expressed GLuc in the Pdx-GLuc pancreas (n = ); 07 glucagon positive cells were counted. DAPI (blue). Bar: 0 µm Supplemental Fig. 7: Secreted GLuc from Pdx-GLuc islets correlated with the number of islets (related to Figure C) Islets isolated from Pdx-GLuc line # or wild-type mice were cultured overnight, and the indicated number of Pdx-GLuc islets of similar sizes were handpicked and cultured for two hours. Native CTZ was added to 500 µl of medium, followed by analysis of the medium using BLI (presented in Figure C). The quantification of the BLI with natve CTZ demonstrates a correlation of GLuc activity with the number of islets. n = for both genotypes. Each dot represents the mean ± SEM Supplemental Fig. 8: BLI for Pdx-GLuc mice after partial pancreatectomy BLI after the injection of CTZ reveals reduced bioluminescent signals in the Pdx-GLuc mice after partial pancreatectomy (A; Line # PPx) compared to that in the conventional transgenic mice (B; Line #). No signals were observed in wile-type mice (C). The reduction in the signals in the pancreatomized mice may be partially due to the interference of the bioluminescence by the black wound. Supplemental Fig. 9: Exendin- increased the Pdx-GLuc activity in islets culture medium

10 Supplemental Materials, Nishimura et al, Page0 of (A) 50 pancreatic islets were isolated from Pdx-GLuc or wild-type mice, and cultured overnight in RPMI-60 supplemented with.mm glucose, 0% FBS, mm sodium pyruvate, 0 mm Hepes, 00 U/ml penicillin and 00 mg/ml streptomycin. Subsequently the islets were preincubated in.5mm glucose for ten hours, followed by incubation with RPMI-60-based medium supplemented with () 00nM exendin- and 0mM glucose, () 0mM glucose and ().5mM glucose for hours. Analysis of culture medium revealed the increased luciferase activity secreted from Pdx-GLuc islets cultured with exendin-, suggesting the effect of GLP- on the Pdx promoter activity in mouse islets. n = 8 for transgenic islets, n = for wild-type islets. (B) The experiments same as (A) were performed with 75 Pdx-GLuc islets, followed by analysis of mrna expression in the islets by TaqMan qrt- PCR in addition to the secreted GLuc activity. n =. Each bar represents the mean ± SEM Supplemental Fig. 0: Batch incubation assay of the Pdx-GLuc islets with various glucose concentrations 5 islets of Pdx-GLuc or wild-type mice were incubated in 500 µl of Krebs-Ringer buffer with the indicated concentrations of glucose for the indicated time period, followed by the measurement of GLuc activity in time-course manner. The dots represent the mean ± SEM. n= for each concentration of glucose. 8

11 Supplemental Materials, Nishimura et al, Page of SUPPLEMENTAL TABLE The oligonucleotide primer sets to detect the presence of the transgene by PCR amplification of genomic DNA. Forward primer Reverse primer Product size PCR-a 5 -ATTAAGCGCGGCGGGTGTGGT- 5 -TTGGGGTCGAGGTGCCGTAAAG- 86 bp PCR-b 5 -AAGGGGGCCGGTGGTGACTAAGC- 5 -CGCCCCTAACTCCGCCCATCC- 75 bp PCR-c 5 -CAAGGGGGCCGGTGGTGACTA- 5 -ACGCGGCCTTTTTACGGTTCCTG- 856 bp PCR-d 5 -GGACGCTGCCACACCTACGAA- 5 -CACCACCGGCCCCCTTGAT- 80 bp 5

12 Supplemental Materials, Nishimura et al, Page of SUPPLEMENTAL TABLE The antibodies used in this study. Antigen Species Maker Catalogue number Dilution Gaussia Mouse Nanolight technologies, Pinetop, 0M :00 luciferase AZ Pdx Rabbit Trans Genic Inc., Japan KR059 :00 Insulin Guinea pig Takara Bio Inc., Japan M78 :00 Glucagon Guinea pig Takara Bio Inc., Japan M8 :00 Somatostatin Rabbit Chemicon International, AB59 :50 Billerica, MA

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