ﬁve residues upstream of the C-terminal KDEL sequence, was ampliﬁed using primer pair PDI-forward: 5 -ATATATAAGCTTGCGGCCGCGCCAC- CatgCTGCGCCGC-GCTCTGCTGTGCC-3 and PDI-reverse-1: 5 -AGCGT- AATCTGGAACATCGTATGG-GTAATCGTCTTCCTCCATGTCTGGCTC-3 . These primers encode 5 NotI and HindIII sites (underlined), a Kozak se- quence (italics), start codon (lower case) and the HA-tag (bold). This PCR product was used as template in a second PCR with primer pair PDI-forward and PDI-reverse-2: 5 -ATATATGGATCCttaCAGTTCATCTTTCACAG- CTTTCTGATCAGCGT-AATCTGGAACATCGTATGGGTA-3 to amplify the complete PDI cDNA and encode the remainder of PDI following the HA-tag (bold) including the stop codon (lower case) and a BamHI site (underlined). The product was digested with HindIII and BamHI and ligated into pBluescript (Stratagene, La Jolla, CA), producing pBS-PDIWT. To generate the double mutant PDI-C56A, C400A (PDICXXA-HA), a modiﬁed QuikChange mutagenesis protocol was performed (Zheng et al., 2004) using plasmid pBS-PDIWT as template and the following primer pairs: C56A-forward: 5 -GTGTGGCCACgcCAAaGCTtTGGCCCCTGAGTATGCCAAAG-3 and C56A- reverse: 5 -CAGGGGCCAaAGCtTTGgcGTGGCCACACCAAGGGGCATAG-3 ; and C400A-forward: 5 -GTGTGGTCACgcCAAACAGTTGGCTCCCATTTG-3 and C400A-reverse: 5 -CACTGTTT-GgcGTGACCACACCATGGGGCATAG-3 (mutagenic nucleotides in lower case, mutated codons underlined, silent restric- tion site in italics). The constructs encoding PDIWT-HA and PDICXXA-HA were subcloned into plasmid pQCXIH (Clontech, Mountain View, CA) using NotI/ BamHI sites. Recombinant Moloney virus packaged with the respective pQCXIH plasmids was used to infect HepG2 cells (Ireland et al., 2008), with stable trans- formants selected in 200 g/ml hygromycin B. RNA Interference Stealth Select siRNAs (40 nm ﬁnal; Invitrogen), either speciﬁc for PDI, ERp57, ERp72, or P5, or a negative nontargeting control, were mixed with 6 L of Oligofectamine (Invitrogen) in a ﬁnal volume of 500 L of Opti-MEM me- dium in 6-well plates. To each well, 2.5 105 HepG2 cells in 1.5 ml of DMEM were added and, after 4 h at 37°C, the medium was supplemented with 1 ml DMEM and FBS to a ﬁnal concentration of 10%. After knockdown for 3 d, the cells were trypsinized, pooled, and resuspended for a second transfection with fresh complexes using the same targeting siRNA. After an additional 3 d, cells were prepared for analysis. For transfections with two different siRNAs, the above protocol was carried out but treatment with each siRNA was staggered by one day, extending the total time to 7 d. For constitutive knockdown of ERp57, recombinant Moloney virus pack- aged with pSM2c-ERp57shRNAmir (OpenBiosystems, Huntsville, AL) was used to infect HepG2 cells as described above. Transformants were subjected to a second infection with virus as well as clonal selection to maximize knockdown efﬁciency. Double and triple knockdowns were carried out in this cell line using siRNAs as described above. Unfolded Protein Response Induction and RT-PCR Positive controls for unfolded protein response (UPR) induction were carried out by treating HepG2 cells with either 0.5 or 5 g/ml tunicamycin (Sigma) dissolved in DMSO or an equivalent volume of DMSO only (control) over- night at 37°C. Cells were split for immunoblot analysis and for RNA isolation. Total RNA was subjected to one-step reverse transcription PCR (Qiagen, Chatsworth, CA) using human Xbp-1 ampliﬁcation primers: 5 -GGAGTTAA- GACAGCGCTTGG-3 and 5 -GAGATGTTCTGGAGGGGTGA-3 . Metabolic Radiolabeling and Immunoisolation After RNAi, HepG2 cells (1 106) were starved for 30 min with Met-free RPMI 1640 and radiolabeled for 3 min in 1 ml of medium containing 0.1 mCi of [35S]Met ( 1000 Ci/mmol; Perkin Elmer, Boston, MA). The cells were chased for various times in RPMI 1640 supplemented with 1 mM Met and then incubated on ice for 3 min in 2 ml of PBS supplemented with 20 mM N-ethylmaleimide (NEM; Sigma). Cells were then lysed at 4°C in 1 ml RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate (DOC), 0.1% SDS, and protease inhibitors) containing 20 mM NEM and subjected to serial immunoisolations with 30 g anti-albumin, anti-transferrin, anti– -fetoprotein, anti– -2-HS-glycoprotein, and anti– -1- antitrypsin antibodies. In each case, following 2 h at 4°C, immune complexes were recovered by incubation for 1 h with 30 L of protein-A agarose beads (GE Healthcare, Little Chalront, Buckinghamshire, United Kingdom). Agar- ose-bound immune complexes were washed four times with 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 0.025% DOC, 0.01% SDS and split for elution in SDS-PAGE sample buffer either lacking or containing 40 mM DTT. Samples were analyzed by SDS-PAGE (10% gels), detected using a Storm phosphorimager (Molecular Dynamics; GE Healthcare), and quantiﬁed with ImageQuant software (GE Healthcare). For sequential immunoisolation of mixed disulﬁde complexes between PDICXXA-HA and substrates, HepG2 cells expressing mutant PDI or the viral vector were pulsed for 15 min with 50 Ci/plate of [35S]Met, incubated with PBS containing 20 mM NEM, and lysed as outlined above. Immunoisolation was carried out using anti-HA antibody and protein-A agarose with immune complexes eluted and disrupted by incubation at 40°C for 1 h in 0.2% SDS in PBS (pH 6.8). The eluates were adjusted to 2% NP-40, 5 mg/ml gelatin, and Vol. 21, September 15, 2010 Oxidative Folding by PDI Family Members 50 M HA peptide (Genscript, Piscataway, NJ) in PBS (pH 6.8) before a second round of immunoisolation with anti-substrate antibody. Immune complexes were analyzed by reducing SDS-PAGE (10% gel) and visualized by ﬂuorography. RESULTS Effective Knockdown of PDI Family Members without Observable UPR Induction Because attempts have failed to obtain mammalian cell lines that constitutively express very low levels of PDI (Park et al., 2006), a transient RNAi knockdown approach was used to assess the functions of PDI family members in human hep- atoma cells. For high efﬁciency knockdown, it was necessary to carry out the knockdown over a period of 6 d with two transfections of the targeting siRNA. Cell growth was equiv- alent in both control and knockdown conditions after the ﬁrst transfection, but following the second application of siRNA a slowing of growth rate was observed in cells treated with targeting siRNA compared with cells treated with control siRNA ( 75% of control rate for PDI knockdown and 85% for knockdown of other PDI family members). However, no accompanying loss of cell viability or impairment of protein translation was observed (data not shown). Using the two-step transfection method, 95% knock- down efﬁciency was attained for PDI, ERp57, ERp72, and P5 using either of two independent siRNAs (Figure 1A). HepG2 cells constitutively expressing shRNA targeting ERp57 (ERp57sh) also exhibited knockdown of ERp57 comparable to that observed with siRNA. These cells facilitated experi- ments in which both ERp57 and a second or third PDI family member were depleted in combination. Knockdown efﬁ- ciency also exceeded 95% in the various combination knock- downs (Figure 1A). No off-target knockdown was observed for other PDI family members, and in no case was compen- satory upregulation of remaining family members observed. We anticipated that depletion of PDI family members might induce the UPR. Surprisingly, there was no increase in the expression of BiP, PDI or CHOP, representing middle and late stages of the UPR, in any of the single or multiple knockdowns (Figure 1A) (Wu and Kaufman, 2006). Addi- tionally, there was no alteration in the levels of Cnx or Crt. To monitor the IRE-1 pathway directly, we tested for Xbp-1 splicing (Yoshida, 2007), and the spliced product was not observed in any of the knockdown conditions compared with cells treated with tunicamycin as a positive control (Figure 1B). Similarly, there was no evidence of PERK path- way activation as measured by phosphorylation of eIF2 (data not shown). We further tested whether PDI-depleted cells might be more susceptible to externally imposed ER stress. HepG2 cells were subjected to knockdown for an extended period of ten days including an additional siRNA transfection on day 6 to maintain cellular siRNA concentration. As shown in Figure 1C, greater than 95% knockdown of PDI was main- tained during this period, yet remarkably there was no evidence of UPR induction as assessed either by Xbp-1 splic- ing or by increased BiP expression (Figure 1C, compare lanes 1 and 4). These cells were subsequently tested for their sensitivity to ER stress induced by tunicamycin. Control cells exhibited Xbp-1 splicing and a 1.4-fold increase in BiP level only at 5 g/ml tunicamycin, but in 10-d PDI-depleted cells Xbp-1 splicing and increased BiP expression (1.5-fold) were apparent at a 10-fold lower concentration of tunicamy- cin. Furthermore, at 5 g/ml tunicamycin, the increase in BiP level (1.9-fold) and extent of Xbp-1 splicing in PDI- depleted cells exceeded those observed in control cells (Fig- 3095