Reagents and Plasmids. Arsenite (As3+) was bought from Aldrich (Milwaukee, WI, USA). Actinomycin D (Act D), proteasome chemical inhibitor MG132, and protein synthesis inhibitor cyclohexamide (CHX) were purchased from Calbiochem (San Diego, CA, USA), Bafilomycin A1 was purchased from Santa Cruz (St. Louis, MO, USA). TRIzol reagent, the dual luciferase assay kit, and SuperScript™ First-Strand Synthesis system were bought from Promega (Madison, WI, USA) and Invitrogen (Grand Island, NY, USA), respectively. Poly Jet™ DNA In Vitro Transfection Reagent was obtained from SignaGen Laboratories (Rockville, MD, USA). The plasmid of human ATG7 promoter (from − 1398 to -227)-driven luciferase reporter was constructed with KpnI and BglII using genomic DNA based on NCBI database. Human ATG7 promoter mutant luciferase reporter (the binding site of Sp1 was mutated) was cloned into the pGL3 basic luciferase reporter vector. The GFP-LC3B and its control vector were a kind gift from Dr. Gang Chen (University of Kentucky, Lexington, KY, USA).41 The shRNA plasmids specifically targeting p50, NCL, Sp1, Beclin1 and ATG7 were bought from Open Biosystems Inc. Construct expressing miR-494 was a gift from K. Yoshida (Department of Life Sciences, Meiji University, Kanagawa, Japan).42 GFP-NCL expression vector was a generous gift from Dr. Michael B. Kastan (Duke University School of Medicine, Duke Cancer Institute, Durham, NC).43 Human NCL 3’-UTR luciferase reporter was cloned into the pMIR luciferase assay vector. All plasmids were prepared by the Plasmid Preparation/Extraction Maxi kit from QIAGEN (Valencia, CA, USA).
Cell Culture and Transfection. Beas-2B cells were cultured at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; 11995065) supplemented with 10% fetal bovine serum (FBS; 26140079), 1% penicillin/streptomycin (15140163), 2 mM L-glutamine (25030164), all were purchased from Life Technologies.44 The p50-/- and p65-/- MEFs and their corresponding wild type (WT) MEFs were cultured as described in our previous reports.10 The stable cell lines of p50-/-(p50) and p65-/-(p65) were established and described in our previous publications.10 Mouse epidermal JB6 Cl41 cells were cultured in MEM with 5% FBS. Cell transfections were performed by using PolyJet DNA In Vitro Transfection Reagent, according to the manufacturer's instruction. For stable transfection, cultures were subjected to either puromycin (Alexis, Plymouth, PA, USA) or G418 (Invitrogen, Carlsbad, CA, USA) selection. The surviving cells that from the drug selection were pooled as stable mass culture.
Antibodies and Western Blot Analysis. Antibodies against HIF-1α were purchased from Novus Biologicals, Inc. (Littleton, CO); The antibodies against GADD45α, p65, p50, NCL, Sp1 were obtained from Santa Cruz Biotechnology Inc. Anti-COX2 antibody was purchased from Cayman Chemical. The antibodies specific against ATG3, ATG5-ATG12, ATG7, Beclin1, LC3A, LC3B, ELAVL1, Elk1, Ets1, STAT5 and GAPDH were bought from Cell Signaling Technology (Beverly, MA, USA). The antibody specific against HNRNPD was purchased from Aviva Systems Biology (San Diego, USA), while antibodies against β-Actin were purchased from Sigma (St. Louis, MO, USA). Western blotting was performed as described in our previous publication.45
Quantitative RT–PCR for mRNA and miRNA Assay. The indicated cells were treated with arsenite for 24h, and the cells were then used for total miRNA extraction using the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA (2.0 µg) was used for reverse transcription. The analysis of miRNA expression was carried out using the miScript PCR system (Qiagen, Valencia, CA, USA) and the 7900HT Fast Real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The initial activation was performed at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 15s, annealing at 55°C for 30s and extension at 72°C for 30s. Cells were cultured as described same as for miRNA extraction, 5.0 µg total RNA was used for first-strand cDNA synthesis with oligod T (20) primer by SuperScriptTM First-Strand Synthesis system (Invitrogen, 11904018). The analysis of mRNA expression was performed by using the Fast SYBR Green Master Mix kit (Applied Biosystems, 4385614) in the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers used in this study were: human HIF-1α (Forward, 5’-TGG TCA AAT CGG CCT CAG CA-3’; Reverse, 5’-CCC TGA ACG GAG GCA TTG GC-3’) and human β-Actin (Forward, 5’-CTC CAT CCT GGC CTC GCT GT-3’; Reverse, 5’-GCT GTC ACC TTC ACC GTT CC-3’), human ATG7 (Forward, 5’-ACC CGG CTC ACC CTG GTT TGT-3’; Reverse, 5’-ACC CCA GTC CTG TAG GTG TGC TG-3’), human Sp1 (Forward, 5’-AAA TTG AAT GGG AAG GTG AT-3’; Reverse, 5’-GAA CCC ACG CCT CTT ATT G-3’), human NCL (Forward, 5’-ACC CGG CTC ACC CTG GTT TGT-3’; Reverse, 5’-ACC CCA GTC CTG TAG GTG TGC TG-3’). Data were analyzed as described in the previous publication.35
Luciferase Reporter Assay. ATG7 promoter driven-luciferase, ATG7 mutated promoter driven-luciferase (Sp1 binding site was mutated) or NCL 3’-UTR luciferase reporter plasmids were transiently transfected into cells. The transfectants were seeded into each well of 96-well plates (8×103 cells per well) and subjected to the various treatments, as described in our previous study.34 Luciferase activities were determined with the Dual-Luciferase Reporter Assay System using a luminometer as described previously.46
Fluorescence Microscopy. p50-/- and its WT cells were cultured on cover slides in 10% FBS DMEM medium for 48 h. The cells were exposed to 20 µM arsenite for the indicated time and fixed with 4% paraformaldehyde (Sigma Aldrich Corporation, 158127) in PBS (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2.0 mM NaH2PO4, pH 7.4) at room temperature for 15 min, and then stained with 0.1 mg/ml DAPI (Sigma Aldrich Corporation, 9542) for 1 min. The slides were washed 3 times with PBS and mounted with anti-fade reagent (Molecular Probes, P36930). All the cell images were captured using an inverted Leica fluorescence microscope (Wetzlar, Germany). For quantification of autophagic cells, GFP-LC3B puncta were determined by counting at least 30 cells per slide.47, 48
RNA-IP Assay. 293T cells were cultured in 10-cm dishes. When cell confluence reached 70 ~ 80%, cells were transiently transfected with GFP-NCL and its vector control. Twenty-four hours after the transfection, the cells were extracted with polysomelysis buffer (10 mM HEPES pH7; 100 mM KCl; 5 mM MgCl2; 25 mM EDTA; 0.5% IGEPAL; 2 mM DTT; 50 units/ml RNase OUT; 50 units/ml Superase IN; 0.2 mg/ml heparin; and complete proteinase inhibitor). The cell lysates were centrifuged at 14,000×g for 10 min at 4°C. The anti-GFP agarose beads A/G (Vector laboratories, Burlingame, CA, USA) were added into the supernatant and rotated overnight at 4°C in NET2 buffer (50 mM Tris–HCl, pH 7.4, 150 mM sodium chloride, 1 mM magnesium chloride, 0.05% IGEPAL, 50 U/mL RNase OUT, 50 U/mL Superase IN, 1 mM dithiothreitol, and 30 mM EDTA). The beads were washed three times, and resuspended in 100 µL NET2 and 100 µL SDS-TE (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 2% sodium dodecyl sulfate) and then incubated at 55°C for 30 min, mixing occasionally. The RNAs in the buffer of the beads were extracted by phenol-chloroform- isoamyl alcohol and RT-PCR was performed to identify the mRNA presented in the immune-complex.
The Construct of Human ATG7 Promoter-driven Mutant Luciferase Reporter and NCL mRNA 3’-UTR Luciferase Reporter. The plasmid of ATG7 promoter (from − 1398 to -227)-driven luciferase reporter was constructed by amplifying from genomic DNA isolated from Beas-2B cells based on NCBI database, using primers: forward, 5’-ACTGGTACCACTGACACACACAACCCCTACT GAG-3’, and reverse, 5’-ACTAGATCTGAGAGGCGGCATCAAACGCAGCACA-3’. A three-point mutation was introduced into the seed region of Sp1/ATG7 promoter putative interacting sequence using primers: MIRMUTFOR, 5’-TCC TGA CCT CGT GAT CCA TAC GCC TCG GCC TCC CAA-3’ and MIRMUTREV, 5’-TTG GGA GGC CGA GGC GTA TGG ATC ACG AGG TCA GGA-3’, according to the site directed mutagenesis protocol (Quick-Change Site Directed Mutagenesis, Stratagene), for producing the pGL3-ATG7 promoter mutant plasmid. The plasmid of NCL 3’-UTRluciferase reporter was constructed by amplifying from cDNA isolated from Beas-2B cells based on NCBI database, using primers: forward, 5’-ACT GGT ACC ACT GAC ACA CAC AAC CCC CTA CTG AG-3’ and Reverse, 5’-ACT AGA TCT GAG AGG CGG CAT CAA ACG CAG CAC A-3’), and then subcloned into the KpnIand BglII sites into pMIR vector, thus originating the pMIR-NCL 3’-UTR luciferase reporter plasmid. Constructs were all sequence verified by GENEWIZ (South Plainfield, NJ).
Statistical Analysis. The Student’s t-test was utilized to determine significant differences. The differences were considered to be significant at a P ≤ 0.05.