SUPPLEMENTAL MATERIALS SIRTUIN 1 PROMOTES HYPEROXIA-INDUCED LUNG EPITHELIAL DEATH INDEPENDENT OF NRF2 ACTIVATION

Size: px
Start display at page:

Download "SUPPLEMENTAL MATERIALS SIRTUIN 1 PROMOTES HYPEROXIA-INDUCED LUNG EPITHELIAL DEATH INDEPENDENT OF NRF2 ACTIVATION"

Transcription

1 SUPPLEMENTAL MATERIALS SIRTUIN PROMOTES HYPEROXIA-INDUCED LUNG EPITHELIAL DEATH INDEPENDENT OF NRF ACTIVATION Haranatha R. Potteti*, Subbiah Rajasekaran*, Senthilkumar B. Rajamohan*, Chandramohan R. Tamatam, Narsa Machireddy, and Sekhar P. Reddy $

2 MATERIALS AND METHODS (ADDITONAL DETAILS) Gene Expression Analysis: Cells and lungs were harvested immediately after hyperoxia for mrna and protein expression analysis. Total RNA was isolated, cdna synthesized, and realtime RT-PCR was performed using β-actin as a reference. Values from control samples set as one unit. For Immunoblot analysis, total protein was extracted from cells and lung tissues, and comparable amount of total protein was separated on a % SDS-PAGE, and probed with the antibodies. The bands were detected with appropriate horseradish peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence detection reagent. Immunoprecipitation and immunoblot analysis: Cells were exposed to hyperoxia and lysed in immune precipitation assay buffer (5 mm Tris-HCl, ph 7.; % Nonidet P-;.5% sodium deoxycholate; 5 mm NaCl; mm EDTA; mm PMSF; and protease inhibitor mixture ( µl/ml, Sigma)). Supernatants were incubated with anti-acetylated lysine antibodies ( µg) for h at C, and the immune complexes were precipitated with Protein A-agarose beads. The immunoprecipitates were then washed twice with radioimmune precipitation assay buffer before separating the proteins on a SDS-PAGE, and immunoblotting with anti-nrf antibody. After incubation with secondary antibodies, proteins were detected by ECL chemiluminescence. Cell Viability Assays: Cells in equal number were plated and exposed to hyperoxia as described above. Cell viability was quantified by CellTiter-Glo kit (Promega, Madison, WI) kit. LDH release was measured by CytoTox 9 Non-Radio Cytotoxicity assay kit (Promega, Madison, WI). Viability and LDH release was calculated as a percentage of increase or decrease over their respective room air controls. E

3 Transfections and Reporter Gene Analyses: Cells were transfected with the NQO (NADPH: quinone oxidase reductase-) or HMOX (Heme oxygenase ) promoter reporter (luciferase) construct (). To normalize transfection efficiency, the cells were co-transfected with 5 ng of the Renilla luciferase plasmid, prl-tk (Promega Corp., Madison, WI). At h after transfection, cells were treated with either DMSO or sirtinol, and extracts were assayed for firefly and the Renilla luciferase activities using a dual luciferase kit (Promega Corp., Madison, WI). Firefly luciferase activity was normalized to that of Renilla. Hyperoxia Exposure in vivo: Mice (8 weeks old, C57BL/ background) were exposed to room air or hyperoxia for 8 h or h. Lungs were harvested and total lysates were subjected to immunoblot analysis. All experimental animal protocols were approved and performed in accordance with guidelines of the Animal Care and Use Committee at University of Illinois at Chicago (Chicago, IL). Sirtinol (5 mg) was dissolved in. ml DMSO and 5 µl of this solution (5 µg) was further diluted to µl with PBS. As vehicle, 5 µl DMSO was diluted to µl PBS. Mice were given sirtinol or vehicle in µl volume prior to hyperoxia exposure and one more dose after h by intraperitoneal (i.p.) injection. E

4 Table : PCR primers used for qrt-pcr analysis Human Primer sequence Gene name Nrf Forward: 5 GAGAGCCCAGTCTTCATTGC Amplicon (bp) Reverse: 5 TGCTCAATGTCCTGTTGCAT SIRT Forward: 5 AAATGCTGGCCTAATAGAGTGG 78 Reverse: 5 TGGTGGCAAAAACAGATACTGA NQO Forward: 5 CCATTCTGAAAGGCTGGTTTG Reverse: 5 TACTCCGGAAGGGTCCTTTGT HMOX Forward: 5 GCCTGGAAGACACCCTAATGTG Reverse: 5 GGCCGTGTCAACAAGGATACTT GCLC Forward: 5 ATGGAGGTGCAATTAACAGAC Reverse: 5 ACTGCATTGCCACCTTTGCA GCLM Forward: 5 TGATGCCACCAGATTTGACTG Reverse: 5 GTGCGCTTGAATGTCAGGAAT BCL-XL Forward: 5 GACTGAATCGGAGATGGAGACC Reverse: 5 GCAGTTCAAACTCGTCGCCT BAD Forward: 5 AGGGAGGGCTGACCCAGAT Reverse: 5 GGCGGAAAACCCAAAACTTC Bcl Forward: 5 TGGGATGCCTTTGTGGAACT Reverse: 5 GAGACAGCCAGGAGAAATCAAAC BAX Forward: 5 GGACGAACTGGACAGTAACATGG Reverse: 5 GCAAAGTAGAAAAGGGCGACAAC β-actin Forward: 5 GCTGTGCTACGTCGCCCTG Reverse: 5 GGAGGAGCTGGAAGCAGCC E

5 Table : List of antibodies Antibody Company Cat # Sirt Nrf (H-) Lamin A/C () Hmox (H-5) Nqo BCL Caspase Cleaved Caspase PARP Cleaved PARP Acetyl Lysine Proteintech --AP) Santa Cruz Biotechnology SC-) Santa Cruz Biotechnology SC-79) Santa Cruz Biotechnology SC-789) NeobioLab A58) NeobioLab A8) Cell Signaling #9 Cell Signaling #9S Cell Signaling #95 Cell Signaling #95 Cell Signaling #9S E5

6 Figure E Cell viability (%) 8 Vehicle Sirtinol % LDH release 5 5 DMSO Sirtinol h h Figure E: The effect of sirtinol on chronic hyperoxia-induced cell death in mouse lung epithelial cells, MLE-. Following h pre-treatment with sirtinol or vehicle, cells were exposed to hyperoxia or room air, and cell viability was measured. Quantification of cell viability evaluated by MTT and LDH assays. LDH levels from vehicle-treated hyperoxia-exposed samples are considered as %. Data are mean ± SEM (n = -). Values from the vehicletreated room air-exposed group are considered as one unit or %. *p.5, room air vs. hyperoxia; p.5, vehicle vs. sirtinol. E

7 A % Cell viability 5 5 HSAE cells LDH release 8 Figure E h h B % Cell Viability 5 5 Vehicle Vehicle RVT RVT MLE- cells LDH release Vehicle Vehicle RVT RVT h Figure E: The effect of resveratrol (RVT) on chronic hyperoxiainduced cell death in HSAE (panel A) cells and MLE-. cells (panel B) Following h pre-treatment with RVT ( µm) or vehicle, cells were exposed to hyperoxia or room air, and cell viability was measured. Quantification of cell viability evaluated by MTT and LDH assays after h and h hyperoxia. LDH levels from vehicle-treated hyperoxiaexposed samples are considered as %. Data are mean ± SEM (n = -). Values from the vehicle-treated room air group are considered as one unit or %. *p.5, room air vs. hyperoxia; p.5, vehicle vs. RVT. E7

8 Sirtinol h h NRF β-actin NQO β-actin HMOX β-actin Figure E Figure E: SIRT inhibition enhanced NRF and NRF-target gene expression under basal condition. HSAE cells were treated with sirtinol for indicated time points and cell extracts were prepared. Equal amount of protein was separated on SDS-PAGE and probed with the antibodies as indicated. A representative blot of two independent experiments is shown. E8

9 A Relative mrna expression NQO HMOX GCLC GCLM Vehicle RVT Vehicle RVT Vehicle RVT Vehicle RVT Figure E h h B Relative mrna expression NQO Vehicle RVT HMOX 5 8 GCLC GCLM 5 5 Vehicle RVT Vehicle RVT Vehicle RVT h h Figure E: The effect of resveratrol (RVT) on acute (panel A) and chronic (panel B) hyperoxia-induced Nrf- target gene expression in HSAE cells. Data are mean ± SEM (n = -). Values from the vehicle-treated room air group are considered as one unit or %. *p.5, room air vs. hyperoxia; p.5, vehicle vs. RVT. E9

10 A Rel. mrna levels Rel. mrna levels Rel. mrna levels B BAD BAX BCL BCLXL 8 BAD BAX BCL BCLXL 8 Vehicle Sirtinol Scr-Si SIRT-Si Vehicle Sirtinol Vehicle Sirtinol Vehicle Sirtinol Scr-Si SIRT-Si Scr-Si SIRT-Si Scr-Si SIRT-Si C BAD BAX BCL BCLXL Scr-Si NRF-Si Scr-Si NRF-Si Scr-Si NRF-Si Scr-Si NRF-Si.7.7 Figure E5 Figure E5: The effect of sirtinol (panel A), SIRT-- (panel B) or NRF (panel C) depletion on and chronic hyperoxia-induced Nrf-target gene expression in HSAE cells. Data are mean ± SEM (n = -). Values from the vehicle-treated room air group are considered as one unit or %. *p.5, room air vs. hyperoxia; p.5, vehicle vs. sirtinol; Scr-Si vs SIRT or NRF sirna. E h h h h h h

11 D Relative mrna levels BAD Scr-Si SIRT-Si BCL Scr-Si SIRT-Si Figure E Figure E: The effect of SIRT- depletion on acute hyperoxia-induced pro- and anti-apoptotic gene expression in HSAE cells. Data are mean ± SEM (n = -). E

12 Cytosol Nuclear h 8h h 8h Figure E7 NRF Maf F/G/K Lamin B Poncieu staining Figure E7: HSAE cells were exposed to room air () or hyperoxia for indicated time points and cytoplasmic and nuclear extracts were prepared using kit. Equal amount of protein ( µg cytosolic, and µg nuclear extracts) was separated on SDS-PAGE and probed with the antibodies as indicated. E

13 DMSO Sirtinol Ig G h h h h h MW 5 kd 5 kd kd 75 kd 55 kd 7 kd 5 kd min exposure DMSO Sirtinol Ig G Figure E8 h h h h h EL MW 5 kd 5 kd kd 75 kd 55 kd 7 kd kd 5 kd kd 75 kd 55 kd 7 kd 5 kd min exposure kd 5 kd 5 kd kd 75 kd 55 kd 7 kd Figure E8: HSAE cells were pretreated with vehicle or Sirtinol for h then exposed to hyperoxia. Whole cell lysates (5 ug) were prepared and immunoprecipitated with anti acetyl lysine and immunoblotted with anti-nrf antibody (see Fig. for more details). Immunoblots developed at different time points are shown. E 5 kd