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Yokouchi et al. CDD-06-0627R
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Suppl Figure 1
Induction of ER stress by nephrotoxic heavy metals in renal tubular
cells. (a) LLC-PK1 cells were treated for 6 h with 500 M (CH3COO)2Pb3, 20 M
HgCl2, 1 mM CuSO4, 500 M (CH3COO)2Ni4 or 500 M CoCl2, and expression of
GRPs was evaluated by Northern blot analysis. Expression of GAPDH was used as a
loading control. Densitometric analysis of individual bands normalized by the level of
GAPDH is shown in (b). Cont, untreated control.
Suppl Figure 2
Involvement of ER stress in cadmium-induced apoptosis of LLC-PK1
cells. (a-c) LLC-PK1 cells were stably transfected with a gene coding for ER chaperone
GRP78 or ORP150, and LL/GRP78 cells and LL/ORP150 cells were established. (a,b)
LL/Mock control cells and LL/GRP78 cells were treated with 10 g/ml tunicamycin
(Tun) for 6 h (a) or 20 M CdCl2 for 4 h (b) and subjected to phase-contrast microscopy
(left). Percentages of round cells are shown in the right graph. (c) LL/Mock cells and
LL/ORP150 cells were treated with 20 M CdCl2 for 4 h and subjected to
phase-contrast microscopy and quantitative analysis of round cells. (d) LLC-PK1 cells
were transiently transfected with EGFP together with an empty plasmid (Mock) or
ORP150 expression plasmid (ORP150) and exposed to 20 M CdCl2 for 4 h.
Percentages of EGFP-positive round cells against total EGFP-positive cells were
evaluated by fluorescence microscopy. Data are presented as means  SE. Assays were
performed in quadruplicate. Asterisks indicate statistically significant differences (P
<0.05).
Suppl Figure 3
Roles of the PERK - eIF2 pathway and the ATF6 pathway in
cadmium-induced apoptosis. (a) LLC-PK1 cells were treated with 50 M salubrinal
(Sal), and phosphorylation of eIF2 was evaluated by Western blot analysis. (b) Cells
were exposed to CdCl2 in the presence (+) or absence (-) of salubrinal for 4 h and
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Yokouchi et al. CDD-06-0627R
subjected to phase-contrast microscopy (left, top) and Hoechst staining (left, bottom).
Percentages of round cells are shown in the right graph. (c) Cells were exposed to 20
M CdCl2 in the presence of 0 - 300 M AEBSF for 4 h and subjected to phase-contrast
microscopy (left, top) and Hoechst staining (left, bottom). Percentages of round cells are
shown in the right graph. Asterisks indicate statistically significant differences (P <0.05).
NS, not statistically different.
Suppl Figure 4
Roles of the IRE1 - XBP1 pathway and JNK in cadmium-induced
apoptosis. (a) LLC-PK1 cells were stably transfected with a gene encoding
FLAG-tagged, unspliced form of XBP1 [XBP1(U)], and LL/FLAG-XBP1 cells were
established. Expression of XBP1(U) was confirmed by Northern blot analysis. (b)
LL/Mock cells and LL/XBP1-DN cells were treated with 20 M CdCl2 for 4 h and
subjected to phase-contrast microscopy (left). Percentages of round cells are shown in
the right graph. (c) Cell were exposed to 20 M CdCl2 in the absence (-) or presence (+)
of 300 M AEBSF for 1 h and subjected to Western blot analysis of phosphorylated
JNK. Total protein of JNK is shown at the bottom as a loading control. (d) LLC-PK1
cells were treated with 20 M CdCl2 for 4 h in the absence (-) or presence (+) of 10 M
SP600125 and subjected to phase-contrast microscopy (top) and Hoechst staining
(bottom). Percentages of round cells are shown in the right graph. Asterisks indicate
statistically significant differences (P <0.05).
Suppl Figure 5
Suppression of cadmium-induced GRP78 expression by inhibition of
ATF6. LLC-PK1 cells were exposed to 20 M CdCl2 in the presence of 0 - 300 M
AEBSF for 4 h and subjected to Northern blot analysis of GRP78.
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