Article Figures & Data
Figures
- Fig. 1.
Generation and RNA-seq analysis of ZIP7−/− cells. (A) Representative images of ZIP7+/+ and ZIP7−/− cells stained with ZIP7 (green), PDI (red), and 4′,6′-diamidino-2-phenylindole (blue). ZIP7+/+ cells show colocalization of ZIP7 with PDI. ZIP7−/− cells exhibit minimal background staining with the ZIP7 antibody. (B) Western blot of ZIP7+/+ and ZIP7−/− cell lysates for ZIP7 and GAPDH expression. There is no detectable ZIP7 protein in ZIP7−/− clones. (C) Histographs of differentially expressed genes in ZIP7−/− cells. (D) Quantification of ZIP8, ZIP14, ZNT1, and ZNT5 in ZIP7+/+ and ZIP7−/− cells. Error bars represent S.E.M. (n = 4). Significant differences between ZIP7+/+ and ZIP7−/− were determined by Student’s t test (**P < 0.01). (E) GO analysis of ZIP7+/+ and ZIP7−/− cells. Protein processing in ER and cell cycle were the two significantly different pathways in ZIP7+/+ compared with ZIP7−/−.
- Fig. 2.
ZIP7 KO causes decreased proliferation and induction of ER stress. (A) Quantification of cell number in ZIP7+/+ and ZIP7−/− cells after 24, 48, and 72 hours. There are significantly less ZIP7−/− cells compared with ZIP7+/+ cells, 48 and 72 hours after plating. (B) Quantification of PDI intensity in ZIP7+/+ and ZIP7−/− cells. There is a significant increase in PDI intensity in ZIP7−/− relative to ZIP7+/+ cells. Values are expressed as a fold change in intensity relative to ZIP7+/+ cells. (C) Representative images of ZIP7+/+ and ZIP7−/− cells. PDI (green) and CellMask (red). (D) Quantification of ER area in ZIP7+/+ and ZIP7−/− cells. There is a significant increase in the ER area in ZIP7−/− cells compared with ZIP7+/+ cells. (E) Quantification of CHOP in the nucleus versus the cytoplasm in ZIP7+/+ and ZIP7−/− cells. There is a significant increase in CHOP in the nucleus versus the cytoplasm in ZIP7−/− cells compared with ZIP7+/+ cells. (F) Representative images of ZIP7+/+ and ZIP7−/− cells stained with CHOP (green). Error bars represent S.E.M. from three independent experiments (n = 3). Significant differences between ZIP7+/+ and ZIP7−/− cells were determined by Student’s t test (**P < 0.01).
- Fig. 3.
Overexpression of WT ZIP7 rescues cell proliferation and ER stress phenotypes. (A) Representative images of ZIP7−/− cells either untransduced or transduced with GFP, WT ZIP7, or ZIP7G178D for 72 hours and stained with PDI (orange). (B) Quantification of cell number after 72 hours of transduction. WT ZIP7 significantly increased cell number compared with untransduced cells. There were no significant differences in ZIP7 G178D-transduced cells. (C) Quantification of nuclear/cytoplasmic CHOP after 72 hours of transduction. Translocation of CHOP was significantly decreased in cells transduced with WT ZIP7 compared with untransduced cells. (D) Quantification of PDI intensity 72 hours after virus transduction. PDI intensity was significantly decreased in WT ZIP7-transduced cells. (E) Quantification of ER area. ER area was significantly decreased in WT ZIP7-transduced cells. Error bars represent S.E.M. from three independent experiments (n = 3). Significant differences were determined by one-way analysis of variance, followed by Dunnett’s test (**P < 0.01).
- Fig. 4.
ZIP7 KO causes reduced zinc in the cytoplasm and increased zinc in the ER. (A) ER concentrations of zinc from ZIP7+/+ and ZIP7−/− cells with vehicle and 500 nM pyrithione treatment. ZIP7−/− cells exhibit significantly increased ER zinc levels compared with ZIP7+/+ cells with vehicle treatment. ER zinc levels are not significantly changed with pyrithione treatment. (B) Cytosol concentrations of zinc from ZIP7+/+ and ZIP7−/− cells with vehicle and 500 nM pyrithione treatment. ZIP7−/− cells have significantly decreased cytosol zinc levels compared with ZIP7+/+ cells. Error bars represent S.E.M. (n = 6). Significant differences between vehicle- and pyrithione-treated cells were determined by Student’s t test (**P < 0.01).
- Fig. 5.
Pyrithione treatment rescues ER stress and proliferation in ZIP7−/− cells. (A) Representative images of ZIP7−/− cells treated with vehicle or pyrithione for 72 hours. Cells were stained with CHOP (green) and PDI (orange). (B) Quantification of cell number in vehicle- and pyrithione-treated cells. (C) Quantification of CHOP in the nucleus versus the cytoplasm in vehicle- and pyrithione-treated cells. (D) Quantification of PDI intensity in vehicle- and pyrithione-treated cells. Values are expressed as fold change relative to vehicle. (E) Quantification of ER area in vehicle- and pyrithione-treated cells. Values are expressed as fold change relative to vehicle. In all graphs, error bars represent S.E.M. from three independent experiments (n = 3). Significant differences between vehicle- and pyrithione-treated cells were determined by Student’s t test (**P < 0.01).
- Fig. 6.
Phenotypic screen for small molecules that rescue ER stress and proliferation in ZIP7−/− cells. (A) Schematic of screening results. Two thousand eight hundred and sixteen small molecules were screened at a concentration of 1 μM. There were 33 primary hits that inhibit CHOP translocation to the nucleus, and 30 of those were confirmed in the secondary screen. One compound was identified to rescue all ZIP7−/− phenotypes, including cell number, PDI intensity, and ER area. (B) Representative images of vehicle and compounds that rescue CHOP nuclear translocation, PDI intensity, and PDI area. Cells are stained with CHOP (green) and PDI (orange). (C) Structures of compound A. (D) Quantification of cell number in vehicle- and compound-treated cells. (E) Quantification of CHOP in the nucleus versus the cytosol. Values are expressed as relative to vehicle. (F) Quantification of PDI intensity in vehicle- and compound-treated cells. Values are expressed as relative to vehicle. (G) Quantification of ER area in vehicle- and compound-treated cells. Values are expressed as relative to vehicle. Error bars in all graphs represent S.E.M. from three independent experiments (n = 3). Significant differences between vehicle- and compound-treated cells were determined by Student’s t test (**P < 0.01).
- Fig. 7.
Compound A rescues ZIP7−/− phenotypes independent of zinc levels. (A) Quantification of cytosolic zinc concentration by ICP-MS. Compound A does not significantly alter cytosolic zinc levels. (B) Quantification of ER zinc levels by ICP-MS. Compound A does not significantly alter ER zinc levels. Error bars represent S.E.M. from three biologic replicates. (C) Quantification of CHOP in the nucleus versus the cytosol in cells treated with inducers of ER stress. Tunicamycin, brefeldin A, and thapsigargin induce CHOP translocation to the nucleus, and compound A does not block the CHOP translocation. Significant differences between vehicle and ER stress inducer were determined by one-way analysis of variance, followed by Dunnett’s test (**P < 0.01). There were no significant differences between ER stress inducer–treated cells versus ER stress inducer and compound A–treated cells. Error bars represent S.E.M. from three independent experiments (n = 3). ns, not significant.
Additional Files
Data Supplement
- Supplemental Data -
Supplemental Table 1 - Compound A Inhibits Multiple Kinases
Supplemental Figure 1 - Compound A dose response
Supplemental Figure 2 - Compound A has no effect on cell number, CHOP translocation, PDI
intensity or ER area in ZIP7+/+ cellsSupplemental Methods
- Supplemental Data -
In this issue
ZIP7 Maintains Cytosolic Zinc Levels
