Supporting Information Materials and Methods
Western blotting Embryonic stem cells (ESCs) derived from E3.5 Grp94+/+ and -/mouse embryos were maintained on feeders in ESC specific media with leukemia
inhibitory factor (LIF) added. Western blots were performed as previously described.1 In
brief, lysates from the untreated or 6 h 300nM ER stress inducer thapsigargin (Tg)
treated cells were extracted in ice-cold RIPA buffer (50 mM Tris-Cl, 150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing cocktails of proteinase
inhibitors and phosphatase inhibitors (Roche), by centrifugation (13000g, 15 min)
following 3 freeze-thaw cycles. Proteins were separated by 6%, 8% or 10% SDS-PAGE,
and transferred to nitrocellulose membrane (Pall). The primary antibodies used include
the following: rat anti-GRP94 (1:5000) from Stressgen; rabbit anti-PERK (H-300)
(1:500), rabbit anti-GADD34 (1:1,000) from Santa Cruz Biotechnology; rabbit antiphosphorylated PERK (Thr980) (1:500), rabbit anti-phosphorylated eIF2 (1:1000) and
rabbit anti-eIF2 (1:1,000) from Cell Signaling; mouse anti- -actin (1:5000) from SigmaAldrich. The experiments were repeated two to three times.
RT-PCR analysis of Xbp-1 mRNA splicing The cells were either untreated or treated
with 300 nM ER stress inducer thapsigargin (Tg) for 6 h. After that, total RNA was
extracted using TRI reagent (Sigma-Aldrich) following the manufacturer’s instructions.
First-strand cDNA was synthesized with SuperScript II (Invitrogen). To detect both
unspliced and spliced Xbp-1 mRNA, PCR was performed as described,2 using primers
5’-GAACCAGGAGTTAAGAACACG-3’ and 5’- AGGCAACAGTGTCAGAGTCC-3’. RT-
PCR of -actin was performed as control at the same condition using primers 5’GACGGCCAGGTCATCACTAT-3’ and 5’-GTACTTGCGCTCAGGAGGAG-3’. These
experiments were repeated three times.
Supporting Information References
1.
Li J, Ni M, Lee B, Barron E, Hinton DR, Lee AS. The unfolded protein response
regulator GRP78/BiP is required for endoplasmic reticulum integrity and stressinduced autophagy in mammalian cells. Cell Death Differ 2008; 15: 1460-1471.
2.
Mao C, Dong D, Little E, Luo S, Lee AS. Transgenic mouse models for
monitoring endoplasmic reticulum stress in vivo. Nat Med 2004; 10: 1013-1014.
Supporting Information Figure Legends
Figure S1 Mice with Grp78 conditional knockout in the Purkinje cells developed
postnatal growth retardation. (a-b) Fasting body weight of Grp78 F/- mice and their
Grp78 F/-; pc-Cre littermates was measured weekly from 3 to 10 wk old. Both male (a)
and female (b) Grp78 F/-; pc-Cre mice exhibited significantly lower body weights than
the sex- and age-matched Grp78 F/- mice. Data are presented as mean±s.e.m. n=10
for each group. *p≤0.05, **p≤0.01, ***p≤0.001 (Student’s t-test).
Figure S2 Purkinje cell-specific knockout of GRP94 caused no morphological or
behavioral defect. (a) Representative immunohistochemical (IHC) staining for GRP94
on the sagittal sections of Grp94 F/F mice and their Grp94 F/-; pc-Cre littermates
cerebella at 5 month of age. (b) Hematoxylin and eosin (H&E) staining on the
cerebellum sections of Grp94 F/F mice and their Grp94 F/-; pc-Cre littermates at 5
month old, demonstrating the Purkinje cell morphology. (c) Graphical representation of
results of rotor rod test (rotating at 30 rpm) given to the Grp94 F/F male mice and their
Grp94 F/-; pc-Cre male littermates for 5 trials at 5 month old, n=5 for each group. The
time of 120 sec denotes that the mice stayed on the rod for the duration of the test and
none fell off the rod. Scale bars represent 50 m (a) and 100 m (b).
Fig S3 Knockout of Grp94 has no effect on UPR signaling pathways. (a) Grp94+/+
and -/- mouse embryonic stem cells (ESCs) were either untreated or treated with 300
nM ER stress inducer thapsigargin (Tg) for 6 h. The cell lysates were subjected to
Western blotting. Lanes that were run on the same gel but noncontiguous are divided by
lines. The size of the proteins (in kDa) is indicated on the left. (b) XBP-1 splicing is not
affected by Grp94 knockout. Grp94+/+ and -/- ESCs were treated the same as in panel
(a). Total RNA was subjected to RT-PCR for detection of the unspliced (u) or spliced (s)
form of XBP-1. The size of the PCR products (in bp) is indicated on the left. For all
panels, the experiments were repeated two to three times.
Figure S4 Reduction of protein ubiquitination status in the prostate epithelial cells of the
Grp78 prostate conditional knockout mice. (a) Representative immunohistochemical
(IHC) (upper panel) and immunofluorescence (IF) (lower panel) staining for GRP78 on
the ventral prostate of the Grp78 F/F mice (control mice) and their Grp78 F/F; PB-Cre
littermates (the prostate epithelial cell-specific knockout mice) at 5 month old. PB-Cre
refers to cre-recombinase driven by the probasin promoter. (b) IF staining for GRP78
(Red) and ubiquitin (Green) on the ventral prostate of Grp78 F/F mice and their Grp78
F/F; PB-Cre littermates at 5 month old. Blue: DAPI. Scale bars represent 200 m (a)
and 50 m (b).
Figure S5 ER expansion and protein aggregation inside the ER of GRP78 null PCs. (a)
Representative Purkinje cell electron micrograph of a 3.5 wk old F/- mouse cerebellum
shows normal Purkinje cell ultrastructural morphology. The white arrowheads indicate
normal ER structure. (b) Electron micrograph of a 3.5 wk old F/-; pc-Cre mouse
cerebellum shows prominent expansion of the ER in the perikaryon and proximal apical
dendrite. (c) Higher magnification of the proximal dendrite area shown in panel B.
Electron-dense aggregates inside the ER are indicated by the yellow arrowheads. (d)
Varying degrees of electron-dense aggregates inside the expanded ER of the GRP78
null PCs as indicated by yellow arrowheads. I to III indicate increasing amount of
electron-dense materials inside the expanded ER. Scale bars represent 5 m (a-b), 3
m (c) and 0.5 m (d).
Supporting Information Video Legend
Representative movement of Grp78 F/- mice (agouti, male) and its F/-; pc-Cre littermate
(black, male) at 5.5 wk old.