Cell Culture and Transfection

Methods

Cell culture and Transfection

HeLa and HeLa S3 cells (ATCC) were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37°C and 5% CO2. Cells were transfected with DNA and siRNA as described6. Stealth siRNAs (Invitrogen Inc.) directed against human PARP-16 mRNA coding region 5’- GACUUGAGCCUGGCCCUCAUAUACA-3’ (siRNA 16.3) and 5’-CCCAAGUACUUCGUGGUCACCAAUA-3’ (siRNA 16.4) were used to knock down PARP-16. siRNA 16.3 was used for all knock-down experiments, and 16.4 and 16.3 for Fig. 2d. Control siRNAs (Qiagen Allstars Negative Control siRNA) were used in parallel. All experiments involving overexpression of PARP-16 were performed at 16 h post-transfection, except a subset of experiments described in Fig. 2c that were performed at 28 h post-transfection. GFP-PERK, GFP-IRE1a, GFP-ATF6, and GFP-SEC61b were expressed for 24 h. To induce ER stress, Brefeldin A (Sigma-Aldrich), Tunicamycin (Sigma-Aldrich) and Thapsigargin (Sigma-Aldrich) were added to cell culture at 5 mg/ml, 3 mg/ml, and 0.2 mM, respectively.

Cytological, Protein and Immunological Techniques

Immunofluorescence analysis was performed as described6. ER-Tracker Red and a lypophilic dye DiI (Molecular Probes) were used at 1 mM. Trypan blue (Sigma-Aldrich) was used at 0.2%. Immunoprecipitation and immunoblotting were carried out as described6, with the exception that in some cases proteins were immunoprecipitated from ER microsomes. Fluorescence, biochemical protease protection assays and membrane extraction assays were performed as described223. PARP-16H152Q Y182A, PARP-16ΔC, and PARP-16AA were generated via PCR-mediated site-directed mutagenesis using pfu polymerase24. To construct the PARP-16Cb5 chimera, DNA sequence of the C-tail of Cytochrome b525 was added to the reverse primer for PCR. All mutations were confirmed via DNA sequencing. XBP-1 splicing assays were performed as described26. Antibodies used: PARP-16 (Aviva ARP33751; Cocalico Biologicals, custom made, HM933), Calnexin (BD, 610523), Lamin B1 (Abcam, ab20396), Lamin B2 (Abcam, ab8983), Lamin A/C (Abcam, ab8984), Tubulin (Abcam, ab6161), GFP (Invitrogen, A11120; Rockland, 600-401-215), Red (Chromotek, 5F8), PERK (Sigma-Aldrich, HPA015737, P0074; Santa Cruz, sc-9481), phospho-PERK (Santa Cruz, sc-32577), eIF2a (Abcam, ab50733), phospho-eIF2a (Sigma-Aldrich, SAB4300221), IRE1a (Cell Signaling, 3294), phospho-IRE1a (Thermo Scientific, PA1-16927), ATF4 (Abcam, ab1371), XBP-1 (Abcam, ab37152), PDI (Abcam, ab2792), ERp57 (Abcam, ab10287), BiP (Cell Signaling, 3177), ATF6 (Abcam, ab11909), Mannosidase II (Abcam, ab12277), Hsp90 (Stressgen, SPA-830), p230 (gift from F. Gertler), EEA1 (Abcam, ab15846), MTCO2 (Abcam, ab3298), phospho-gH2AX (Millipore, 05-636), TIA-1 (Santa Cruz, sc-1751). Antibodies were used at 1:1,000 for immunoblotting (except for anti-eIF2a used at 1:50); 1:500 for immunoprecipitation; and 1:100 for immunofluorescence.

NAD+ Incorporation Assays

For ER microsome-based assays including NAD+ incorporation and immunoprecipitation, ER microsomes were fractionated from HeLa cells via isopycnic flotation method as described12, and incubated with 100 mM b-NAD+ (MP Biomedicals) and 2.5 mCi of 32P-NAD+ (Perkin Elmer) in a PARP reaction buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 1 mM DTT, 0.1 mM Na3VO4, 50 mM NaF, 5 mM b-glycerophosphate, 1 mM ADP-HPD, and protease inhibitor cocktail) at 25°C for 30 min. The ratio between unlabeled and labeled NAD+ was kept constant upon titration of NAD+ concentration. The 32P-labeled ER microsomes were then lysed by addition of Triton X-100 at 1%. Proteins were immunoprecipitated, eluted in a 1X Laemmli sample buffer by boiling at 65°C for 10 min, and then analyzed using autoradiogram and immunoblotting. For assays using recombinant proteins, GST-PARP-16 wild-type and mutant isoforms were purified from BL 21 RIPL cells, following manufacturer’s protocol (Stratagene). NAD+ incorporation reactions were performed under the same conditions described above.

Kinase Assays

PERK and IRE1a kinase assays were performed as described27,28, with an exception that the kinase activities were measured post NAD+ incorporation by PARP-16 in the context of ER microsomes. In brief, ER microsomes were subject to NAD+ incorporation assays in a PARP buffer as described in the NAD+ Incorporation Assays section using 100 mM unlabeled b-NAD+ (MP Biomedicals) and GST-PARP-16 wild-type or catalytically inactive mutant proteins purified from bacteria. The (ADP-ribosyl)ated ER microsomes were incubated with 100 mM ATP (New England BioLabs) and 2.5 mCi of [g-32P]ATP (Perkin Elmer) in a kinase buffer (for GFP-PERK activity, 20 mM HEPES pH 7.4, 50 mM KCl, 1.5 mM DTT, 2 mM Mg(OAc)2, 2 mM MnCl2, 0.1 mM Na3VO4, 50 mM NaF, 5 mM b-glycerophosphate, and protease inhibitor cocktail; for GFP- IRE1a activity, 20 mM HEPES pH 7.4, 1 mM DTT, 10 mM Mg(OAc)2, 50 mM K(OAc)2, 0.1 mM Na3VO4, 50 mM NaF, 5 mM b-glycerophosphate, and protease inhibitor cocktail) at 25°C for 30 min. The 32P-labeled ER microsomes were then lysed by addition of Triton X-100 at 1%. Proteins were immunoprecipitated, eluted in 1X Laemmli sample buffer by heating at 65°C for 10 min, and then analyzed using autoradiogram and immunoblotting.

IRE1a Endonuclease Assasys

A 479 bp mouse XBP-1 DNA fragment containing the intron and flanking exons on both sides (263 bp on the 5’ end and 191 bp on the 3’ end) was amplified via PCR using a reverse primer containing T7 RNA polymerase promoter sequence. In vitro transcription of XBP-1 mRNA and XBP-1 cleavage assays were performed essentially as described28. Gel-purified transcript equivalent to ~ 20,000 cpm was incubated with (ADP-ribosyl)ated GFP-IRE1a immunoprecipitates in an IRE1a kinase buffer plus 2 mM unlabeled ATP at 25°C for 30 min. The cleavage products were analyzed on 10% TBE-UREA polyacrylamide gels.

ROS Generation and Ca2+ Measurement

ROS was evaluated using a cell permeant carboxymethyl derivative of fluorescein (CM-H2DCFDA, Invitrogen), following the manufacturer’s protocol (Invitrogen). Cells were loaded with CM-H2DCFDA at 5 M for 30 min at 37°C, then treated with H2O2 at 100 mM for 10 min at 37°C. The fluorescence intensity of oxidized ROS probe was measured using a microplate reader (Tecan). To measure intracellular Ca2+ concentration, cells were loaded with a cell permeant Ca2+ probe, Fura-4F (Invitrogen) at 2 M in the Krebs-Ringer solution containing HEPES and 2 mM CaCl2 for 30 min at 37°C. The ratiometric fluorescence at 340 nm/380 nm was measured every 2 min for 20 min, using a microplate reader (Tecan). Thapsigargin was added at 1 M, and EGTA used at 3 mM to chelate the released Ca2+. To induce cytoplasmic stress and DNA damage, Arsenite (Sigma-Aldrich) and Cisplatin (Sigma-Aldrich) were used at 100 µM and 10 M, for 30 min and 8 h, respectively. To inhibit translation and the proteasome, Cycloheximide (Sigma-Aldrich) and MG132 (Sigma-Aldrich) were used at 100 µg/ml and 10 µM, respectively. Total RNA was extracted using RNeasy kits and QIAshredder (Qiagen), and cDNA was amplified using Quantitect reverse transcription kit (Qiagen). RT-qPCR was performed with Quantitect SYBR Green PCR kit (Qiagen), using a LightCycler 480 (Roche). mRNA levels were normalized against GAPDH mRNA.

Statistics

All experiments were repeated a minimum of two times and the unpaired Student’s t-test was used for statistical analysis.

22.  Chang, P., Coughlin, M., and Mitchison, T..J. (2005). Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nat Cell Biol 7, 1133-1139.

23.  Lorenz, H., Hailey, D.W., and Lippincott-Schwartz, J. (2005). Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization. Nat Methods 3, 205-210.

24.  Zheng, L., Baumann, U., and Reymond, J.L. (2004). An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucl Acids Res 32, e115.

25.  Pedrazzini, E., Villa, A., and Borgese, N. (1996). A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane. PNAS 93, 94207-94212.

26.  Calfon M., Zeng H., Urano F., Till J.H., Hubbard S.R., Harding H.P., Clark S.G. and Ron D. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415, 92-96 (2002).

27.  Harding, H., Zhang, Y, and Ron, D. (1999). Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271-274.

28.  Sidrauski, C. and Walter, P (1997). The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell 90, 1031-1039.

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