L. A. Rutkevich et al. Figure 1. Efﬁcacy of PDI family member knockdown and assessment of UPR. (A) PDI family members were depleted from HepG2 cells by RNAi over a period of 6 d. Indicated single or multiple knockdowns were carried out transiently using two independent target- ing siRNAs, designated (1) and (2), or in some instances using cells constitutively expressing shRNAmir targeting ERp57 (ERp57sh). Equiv- alent amounts of cell lysates were immuno- blotted for PDI family members, ER chaper- ones, CHOP, and actin. Images shown are a composite from various experiments, all of which included actin as a loading control. Exposures were adjusted to an equivalent density of the actin control. Data are repre- sentative of a minimum of ﬁve replicate knockdowns. (B) After the indicated knock- downs or overnight treatment with 5 g/ml tunicamycin, total RNA was isolated from HepG2 cells and analyzed for Xbp-1 splicing by reverse-transcription PCR. Xbp-1U, un- spliced Xbp-1 product; Xbp-1S, spliced Xbp-1 product. (C) HepG2 cells were subjected to PDI knockdown for 10 d. On day 9, cells were treated overnight with the indicated concen- trations of tunicamycin (Tm). The following day, cell lysates were immunoblotted to de- tect PDI, BiP, and actin. Lanes shown were assembled from a single gel. Total RNA was also isolated and analyzed for Xbp-1 splicing. ure 1C). These ﬁndings indicate that HepG2 cells depleted of PDI, although not exhibiting an overt UPR themselves, are more sensitive to ER stress imposed through tunicamycin treatment. Because the degree of Xbp-1 splicing and BiP upregulation in control HepG2 cells was remarkably modest after tunicamycin treatment (Figure 1C), it may be that these professional secretory cells have higher basal levels of fold- ing and ER-associated degradation (ERAD) machineries, and so they do not respond as readily as other cell types to stress conditions. Indeed, it has been reported that nearly 75% of endogenous apolipoprotein B-100 in HepG2 cells is constitutively degraded cotranslationally (Adeli et al., 1997), suggesting that misfolded substrates may be disposed of without the need for an additional UPR. The preceding data indicate that the 6-d period chosen for PDI family member depletion is suitable to assess the func- tions of PDI family members without confounding factors such as compensatory upregulation of other family mem- bers or induction of the UPR. Identiﬁcation of PDI Substrates Human HepG2 cells were selected for these experiments because they express many well characterized secretory pro- 3096 teins that possess disulﬁde bonds. Albumin (Alb), -fetopro- tein ( FP), and transferrin (TF) are attractive study subjects since they are monomeric, contain multiple disulﬁdes, and have molecular weights sufﬁciently low for oxidative fold- ing to be monitored by nonreducing SDS-PAGE. Further- more, FP and TF are glycosylated whereas Alb is not, which permits an assessment of glycoprotein preference by PDI family members during catalysis of oxidative folding. To ﬁrst conﬁrm that these secretory proteins are indeed substrates of PDI, HepG2 cells were prepared that constitu- tively express an HA-tagged substrate-trapping mutant of PDI (PDICXXA-HA) (Figure 2A). Mutation of the second Cys in both PDI active sites stabilizes mixed-disulﬁde reaction intermediates with substrates on which it is acting as a reductase or isomerase (Walker and Gilbert, 1997). Although our focus is on oxidative folding, it is likely that in the case of PDI it selects substrates largely through hydrophobic interactions regardless of the reaction catalyzed (Tian et al., 2006). Radiolabeled cells expressing PDICXXA-HA were sub- jected to a ﬁrst round of immunoisolation with anti-HA mAb followed by disruption of immune complexes and a second round of immunoisolation with Ab directed against Molecular Biology of the Cell