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DOI: 10.1038/ncb2593

Figure S1 Analysis of ER microsomes purified from untransfected HeLa cells. MW (kD) at right or left of blots. a, Purified ER microsomes stained with ER-Tracker Red. RER=Rough ER. b, Purified ER microsomes

immunoblotted for indicated proteins. c, Coomassie-stained gel showing amounts of GST-PARP-16 and GST-PARP-16H152Q Y182A utilized for reaction shown in Fig. 2a. Asterisk=full-length proteins. Bar, 1 mm.

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Figure S2 Prolonged overexpression of PARP-16 causes abnormal ER structures. a, Cells expressing GFP-PARP-16, GFP-PARP-16H152Q Y182A, or GFP-PARP-16Cb5 for 16h or 28 h stained for GFP-PARP-16 (green), Calnexin

(red) and DNA (blue). In each condition, expression levels of GFP fusion proteins were monitored via immunoblot (b), and abnormal ER structures quantitated. n=3 (c). Bar, 10 mm.

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Figure S3 Effects of PARP-16 knock-down on ROS generation, Ca2+ leakage from the ER, and cellular responses to DNA damage or cytoplasmic stress. a, ROS generation in control and PARP-16 knock-downs. Shown are values in arbitrary unit (A.U.) for fluorescence intensity of CM-H2DCFDA before and after H2O2 treatment. n=2; p > 0.05 after H2O2 treatment. b, Intracellular Ca2+ concentration in control and PARP-16 knock-downs. Shown are values in arbitrary unit (A.U.) for fluorescence intensity ratio 340 nm/380 under basal conditions (time points 1 – 3),

during Thapsigargin treatment (time points 4 – 7), and after EGTA addition (time points 8 – 11). Each time points are 2 min apart. n=2; p > 0.1 after Thapsigargin treatment. c, Cells treated with or without Cisplatin were immuno-stained for g-H2AX (red) and DNA (blue), and g-H2AX positive cells were counted. n=2; p > 0.1 in PARP-16 knock downs. d, Cells treated with or without Arsenite were immuno-stained for TIA-1 (red) and DNA (blue), and TIA-1 positive cells were counted. n=2; p < 0.05 in PARP-16 knock downs. Bars, 10 mm.

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Figure S4 PARP-16 is required for IRE1a-mediated UPR. Full Images of agarose gels shown in Fig. 4b (a) and Fig. 5a (b). Asterisks= 290 and 183 bp fragments originated from unspliced XBP-1 cDNA, upon digestion with PstI restriction enzyme. Triangle represents hybrid amplicons. (OE)= over-expression; (ctrl)= control; (P-16)= PARP-16; (U)= unspliced; (S)= spliced; (UT)= untreated; (BFA)= Brefeldin A treated; (TUN)= Tunicamycin

treated. c, ER microsome based NAD+ incorporation and kinase assays. ER microsomes containing GFP-PERK were (ADP-ribosyl)ated using either GST-PARP-16 or GST-PARP-16H152Q Y182A in the presence of 32P-NAD+. The duplicate NAD+ incorporation reactions were performed under the same conditions using unlabeled NAD+ instead, and then subjected to kinase assays using 32P-ATP.

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Figure S5 PARP-16 knock-down does not affect ERAD and chaperoning activities. a, Knock-down cells expressing CD3d-YFP were treated with Cycloheximide in the presence or absence of MG132. CD3d-YFP clearance, as a measure of ERAD activity, was monitored via immunoblot of lysates.

Cells co-expressing CD3d-YFP and a mCherry fusion to either PARP-16 or PARP-16H152Q Y182A, were also analyzed. Immunoblot of PARP-16 and mCherry fusions are shown. b, Immunoblots for indicated proteins. (UT)= untreated; (TUN)= Tunicamycin treated; (Ctrl)= control; (P-16)= PARP-16.

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Fig. S6Fig. 3a: Autoradiogram Fig. 3a: IRE1α

Fig. 3b: GFP Fig. 3b: GFP Fig. 3b: GFP Fig. 3b: GFP

Fig. 3b: P-16 Fig. 3b: P-16 Fig. 3b: P-16Fig. 3b: PERK

Fig. 3b: IRE1α

Fig. 3b: ATF6

Fig. 3c: Autoradiogram

Fig. 3d: Autoradiogram

Fig. 3d: GFP Fig. 3d: P-16

F2a: Autoradiogram Fig. 2b: Autoradiogram

Fig. 4f: 32P-UTP

Fig. 4c: IRE1αPFig. 4c: PERKP

Fig. 4c: BiP

Fig. 4c: PERK

Fig. 4c: eIF2αP

Fig. 4c: ATF4

Fig. 4c: eIF2α

Fig. 4e: 32P-ATP Fig. 4e: 32P-NADFig. 4d: 32P-ATP Fig. 4d: 32P-NAD

Fig. 3a: GFP

Fig. 3c: GFP Fig. 3c: P-16

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Fig. 4c: XBP-1

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Fig. 4b: XBP-1 Splicing

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Figure S6 Uncropped data.

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Fig. S6

Fig. S5b: PDI Fig. S5b: ERp57 Fig. S5b: BiP

Fig. S5b: CNXFig. S5b: Tubulin

Fig. S4c: 32P-ATP Fig. 4c: 32P-NAD

Fig. S5a: CD3δ-YFP (O/E)

Fig. S5a: CD3δ-YFP (siRNA) Fig. S5a: Hsp90 (siRNA)

Fig. S5a: Red (O/E)Fig. S5a: Hsp90 (O/E)

Fig. S5a: P-16 (siRNA)

Fig. S2b: GFP

Fig. S2b: Tubulin

Fig. S1c: CoomassieFig. S1b: Tubulin

Fig. S1b: Mannosidase II

Fig. S1b: Hsp90

Fig. S1b: Lamin B

Fig. S1b: Calnexin

Fig. S1b: PERK

Fig. S1b: PARP-16

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Figure S6 continued

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