1. Volume 22, Issue 11, Pages 2809-2817 (March 2018)
A PP6-ASK3 Module Coordinates the Bidirectional Cell Volume Regulation under Osmotic Stress 
Kengo Watanabe, Tsuyoshi Umeda, Kuniyoshi Niwa, Isao Naguro, Hidenori Ichijo 
Cell Reports 
Volume 22, Issue 11, Pages (March 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 2809-2817DOI: (10.1016/j.celrep.2018.02.045)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
A Bidirectional Osmo-Responsive Kinase, ASK3, Is Required for Cell Volume Regulation under Hypoosmotic Stress
(A) Changes in the activities of several osmo-responsive kinases in HeLa and A549 cells. Hypo, 200 mOsm; Iso, 300 mOsm; Hyper, 500 mOsm; 15 min. The right graphs depict the quantification of western blots. Quantified individual values and the mean ± SEM are presented as gray points and black bars, respectively (n = 5). IB, immunoblotting. †Non-specific bands. ‡Remnant bands from prior detection (p-WNK).
(B) Cell volume changes under hypoosmotic stress and RVD efficiency in HeLa cells (hypoosmotic stress: 120 mOsm). The left graph is a representative result with the mean ± SEM from triplicate. In the right graph, the recovery rates of normalized F/Fbefore at 60 min are represented as the mean ± SEM (n = 7 from 2 independent experiments).
(C) Time course of hypoosmosis-induced cell rupture in EGFP-stably expressing HeLa cells (hypoosmotic stress: 20 mOsm). The top panel is a representative image set. In the bottom graphs, the data are presented as the mean ± SEM (n = 7–10 per group).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S1.
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4. Figure 2 PP6 Is an ASK3 Phosphatase under Hyperosmotic Stress
(A) Effects of a pan-protein phosphatase inhibitor on ASK3 inactivation under hyperosmotic stress. Cells were pre-treated with 50 mM sodium fluoride (NaF) for 30 min. NaCl: sodium chloride, a negative pre-treatment control.
(B) Effects of PPP6C depletion on ASK3 activity under hyperosmotic stress. Note that the band for PPP6C appears as both a singlet and a doublet for unknown reasons; this phenomenon was previously discussed (Hwang et al., 2016). PPP5C: an ASK1 phosphatase.
(C) Effects of PPP6C depletion on endogenous ASK and OSR1 activities under hyperosmotic stress. †Non-specific bands.
(D) Effects of PP6 overexpression on ASK3 activity under hyperosmotic stress.
(E) In vitro phosphatase assay of PP6 on ASK3. Prior to purification, PP6-overexpressing cells were exposed to osmotic stress. ‡Remnant bands from prior detection (p-ASK). #IgG light-chain bands.
(A–E) The bottom graph is the quantification of the western blots. Quantified individual values and the mean ± SEM are presented as gray points and black bars, respectively. n = 3 (A, B, and E) and n = 5 (C and D), with the exception of PPP6C WT/D84N (++) n = 4. Hyperosmotic stress (−): 300 mOsm, (+): 400 mOsm (A); 425 mOsm (B–D); 500 mOsm (E), (++): 500 mOsm; 10 min. IB, immunoblotting; WT, wild-type; D84N, inactive PPP6C mutant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S2.
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5. Figure 3
Increased Interaction between PP6 and ASK3 Is Necessary for ASK3 Inactivation under Hyperosmotic Stress
(A) Osmolality dependency of the interaction between the PP6 core enzyme and ASK3. Osmotic stress: 10 min.
(B) Effects of ANKRD52 on the phosphatase activity of the PP6 core enzyme. The top panel is a representative western blot of the purified samples. The bottom graph depicts the result of in vitro phosphatase assays with a phospho-Thr peptide, presented as the mean ± SEM (n = 3). WT, wild-type; D84N, inactive PPP6C mutant.
(C) Effects of ANKRD52 depletion on the interaction between the PP6 core enzyme and ASK3.
(D) Effects of ANKRD52 depletion on endogenous ASK activity under hyperosmotic stress. †Non-specific bands.
(A, C, and D) The right or bottom graph depicts the quantification of western blots. Quantified individual values and the mean ± SEM are presented as gray points and black bars, respectively. n = 3 (A and C) and n = 5 (D). Hyperosmotic stress (−): 300 mOsm, (+): 425 mOsm (D); 500 mOsm (C); 10 min.
Superfluous lanes were digitally eliminated from blot images in (B), (C), and (D) as indicated by black lines. IB, immunoblotting; IP, immunoprecipitation; n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figures S3 and S5.
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6. Figure 4
ASK3 Inactivation Failure Inhibits RVI and Leads to Cell Death under Hyperosmotic Stress
(A) Time course of endogenous ASK activity change under hyperosmotic stress. The right graph depicts the quantification of western blots. Quantified individual values and the mean ± SEM are presented as gray points and black bars, respectively (n = 3). Hyperosmotic stress (−): 300 mOsm, (+): 600 mOsm. IB, immunoblotting. †Non-specific bands.
(B) Cell volume changes under hyperosmotic stress and RVI efficiency in HeLa cells.
(C) Hyperosmotic stress-induced cell death in HeLa cells. The left panel is a representative image set. The white scale bar represents 100 μm. PI, propidium iodide.
(B and C) The data are presented as the mean ± SEM; n = 5 (B) and n = 9 (C). Hyperosmotic stress: 600 mOsm.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figures S1, S4, and S5.
Cell Reports  , DOI: ( /j.celrep )
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