1. Volume 27, Issue 6, Pages 1886-1896.e6 (May 2019)
The Ion Transporter NKCC1 Links Cell Volume to Cell Mass Regulation by Suppressing mTORC1 
Wael L. Demian, Avinash Persaud, Chong Jiang, Étienne Coyaud, Shixuan Liu, Andras Kapus, Ran Kafri, Brian Raught, Daniela Rotin 
Cell Reports 
Volume 27, Issue 6, Pages e6 (May 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 1886-1896.e6DOI: (10.1016/j.celrep.2019.04.034)
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3. Figure 1
NKCC1 Binds the Leucine Transporter LAT1 and Inhibits LAT1 Function
(A) Co-immunoprecipitation (co-IP) of NKCC1 with LAT1. HeLa cells were transfected with HA-NKCC1 and FLAG-LAT1 or FLAG-FGFR1 (control). Following FLAG IP, the presence of co-immunoprecipitated NKCC1 was verified by immunoblotting with HA antibodies. Quantification of binding is depicted below the blot (mean ± SEM, N = 3 independent experiments).
(B) Co-IP of endogenous NKCC1 with endogenous LAT1. Endogenous NKCC1 was immunoprecipitated from HeLa cells using NKCC1 antibodies, and binding to endogenous LAT1 was verified by immunoblotting for LAT1. Lower panels in (A) and (B) depict controls for the IPs and for protein loading and represent three independent experiments.
(C) NKCC1 knockdown enhances LAT1-mediated [3H]-Leu uptake. HeLa cells stably expressing scrambled or NKCC1 shRNA (NKCC1 knockdown [KD] cells) were serum and nutrient starved and pre-treated with 1 mM Gln for 30 min followed by stimulation for 10 min with [3H]-Leu in the absence or presence of 10 mM of the LAT1 inhibitor BCH, and cellular content of 3H-Leu was analyzed using scintillation counting.
(D) Inhibition of ASCT2 in NKCC1-depleted cells reduces [3H]-Leu uptake. Scrambled or NKCC1-KD HeLa cells were serum and nutrient starved and pre-treated with 1 mM Gln for 30 min followed by stimulation for 10 min with 14 nM [3H]-Leu (in 0.4 mM Leu) in the absence or presence of 10 mM of the ASCT2 inhibitor GPNA.
(E) Enhanced Leu-dependent [3H]-Gln efflux upon NKCC1 KD. Scrambled or NKCC1-KD HeLa cells were serum and nutrient starved and preloaded with [3H]-Gln for 30 min, washed, and treated with either 0.4 mM Leu or 10 mM BCH for 10 min.
In (C)–(E), histogram data are mean ± SEM of average rate of Leu uptake or Gln efflux per min. N = 3 independent experiments, each performed in triplicate; p values were calculated using Student’s t test. See also Figures S1–S4 and Table S1.
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4. Figure 2 Depletion of NKCC1 Enhances mTORC1 Activation
(A–C) NKCC1 KD in cells enhances mTORC1 activation by essential amino acids (EAAs) and Gln/Leu. Scrambled or NKCC1-KD HeLa (A and C) or Hek293T (B) cells were serum and nutrient starved and stimulated with EAA, Gln, Leu, Gln+Leu (Gln/Leu), or Arg for 15 min, as indicated, and mTORC1 activation (activated S6K1 [p-p70]) was analyzed. Lower panels are quantification of S6K1 (mTORC1) activation (p-p70/p70 ratio) in N = 3 separate experiments. Data are mean ± SEM; p values were calculated using Student’s t test.
(D) Knockout of NKCC1 activates mTORC1 in vivo. Colonic epithelia were harvested from wild-type (WT) or NKCC1−/− mice. Lower panels in (D) represent activation (phosphorylation) of Akt (pAkt) and loading controls. Data are mean ± SEM, and p values were calculated using Student’s t test from N = 5 mice per genotype. Of the two WT controls shown, WT(1) is a male and WT(2) is a female.
In all panels, data summaries are depicted beneath their respective immunoblots. See also Figure S3.
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5. Figure 3
Inhibition of NKCC1 Activity with Bumetanide Activates mTORC1, and NKCC1 Depletion Enhances Lysosomal mTOR Translocation
(A–C) NKCC1 inhibition enhances mTORC1 activation.
(A) Scrambled or NKCC1-KD HeLa cells were serum and nutrient starved and treated with 100 μM bumetanide for 30 min followed by stimulation with either Gln/Leu or EAA for 15 min, as indicated.
(B and C) Mouse colonic organoids (B) or epithelial cells harvested from the mouse distal colon (C) were treated (or not) with 100 μM bumetanide for 30 min. mTORC1 activation was determined using immunoblotting for activated S6K1 (p-p70). Lower panels depict loading controls. Histograms beneath immunoblots represent their respective quantifications (p-p70/p70 ratio) and are mean ± SEM of N = 3 independent experiments; p values were calculated using Student’s t test.
(D and E) NKCC1 depletion leads to enhanced mTOR translocation to the lysosomal membrane.
(D) Scrambled and NKCC1-KD HeLa cells were serum and nutrient starved for 2 h and then pre-treated with 2 mM Gln before stimulation with 0.4 mM Leu for 15 min. The cells were fixed and stained for the nucleus (DAPI, cyan), lysosomes (LAMP1, green), and mTOR (anti-mTOR, red).
(E) Quantification of mTOR that co-localized with the lysosome. N = 95–100 cells per treatment. Data are mean ± SEM. FI, fluorescence intensity.
See also Figure S4.
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6. Figure 4
Depletion Of NKCC1 Promotes Akt Activation And TSC2 Phosphorylation
(A and B) NKCC1 KD enhances Akt (S473) and Akt-dependent TSC2 phosphorylation (A) and Akt (T308) phosphorylation (B). Scrambled or NKCC1-KD HeLa cells were serum and nutrient starved before stimulation with EAA for 15 min and analysis of Akt or TSC2 phosphorylation.
(C and D) Inhibition of Akt and Erk1/2 (C) and PI3K (D) block mTORC1 activation in NKCC1-depleted cells. Scrambled and NKCC1-KD HeLa cells were serum and nutrient starved and treated with either 5 μM of the Akt inhibitor mk2206 (Akti), 50 μM of Erk1/2 inhibitor PD98059 (Erk1/2i), or 10 μM of the PI3K inhibitor LY mTORC1 activation was assessed using immunoblotting for S6K1 phosphorylation; phosphorylation of Akt, the Akt dependent phosphorylation site on TSC2 (S939), and Erk1/2 was assessed using anti p-Akt (S473 and S308), p-TSC2 (S939), and p-Erk1/2 (T202/Y204) antibodies, respectively, as indicated. Lower panels depict controls. Histograms beneath the immunoblots depict quantification of mTORC1 activation (p-p70/p-70 ratio), Akt activation (p-Akt/Akt ratio ratio), and TSC2 phosphorylation (p-TSC2/actin).
Values are mean ± SEM (N = 3, except p-Akt(S473)/Akt in A, where N = 16); p values were calculated using Student’s t tests. See also Figure S5.
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7. Figure 5
Enhanced Insulin Receptor Expression and Activity Promote Akt and mTORC1 Activation in NKCC1-Depleted Cells
(A) Increased expression of and activity of the insulin receptor (IR) in NKCC1-KD cells. Scrambled and NKCC1-KD HeLa cells were serum starved overnight, stimulated with 0.5 nM insulin for 15 min, and analyzed for IR levels and activity, measured by Akt activation (pAkt(S473)).
(B) Increased activity but not expression of the IGF-1R in NKCC1-KD cells. Scrambled and NKCC1-KD HeLa cells were serum starved overnight, stimulated with 0.25 nM IGF-1 for 15 min and analyzed as in (A).
(C) Increased cell surface expression of the IR (but not the IGF-1R) in NKCC1-KD cells. Scrambled or NKCC1-KD HeLa cells were treated with sulfo-NHS-LC biotin to label cell surface proteins, and IR and IGF-1R plasma membrane amounts were determined by precipitating biotinylated proteins with streptavidin-agarose and immunoblotting with either IR or IGF-1R antibodies.
(D) KD of IR, but not IGF-1R, blocks Akt and mTORC1 activation in NKCC1-depleted cells. NKCC1-KD HeLa cells were transfected with either control siRNA or siRNA against the IR, IGF-1R, or both, serum and nutrient starved overnight and stimulated with EAA for 15 min, as indicated. mTORC1 and Akt activation was assessed using immunoblotting with S6K1 phosphorylation (p-p70) and p-Akt (S473) antibodies, respectively. Lower panels depict controls for IR and IGF-1R expression and loading controls.
Histograms beneath the immunoblots in (D) depict quantification of mTORC1 activation (p-p70/p-70 ratio) and Akt activation (p-Akt/Akt ratio). All data are mean ± SEM (N = 3); p values were calculated using Student’s t tests. See also Figure S5.
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8. Figure 6
NKCC1 Independently Regulates LAT1 or the Akt (or Erk) Pathways to Control mTORC1 Activation
(A) Increased LAT1 activity in NKCC1-depleted cells enhances mTORC1 activation independently of Akt or Erk. Scrambled or NKCC1-KD HeLa cells were transfected with constitutively active Flag-Rheb(N153T), treated with Akt and Erk1/2 inhibitor (Akti and Erk1/2i) under starvation conditions, and stimulated with EAA for 15 min, where indicated. mTORC1 and Erk1/2 activation was assessed using immunoblotting for S6K1 phosphorylation (p-p70), p-Akt (S473), and p-Erk1/2 (T202/Y204) antibodies, respectively. Lower panels depict controls for NKCC1, Flag-Rheb(N153T), Akt, and Erk expression, as indicated.
(B) Constitutively active Rag complex enhances mTORC1 activation in NKCC1-KD cells in the absence of amino acids. Scrambled or NKCC1-KD HeLa cells were transfected with activated Rag complex (RagA(Q66L)/RagC(S75L)), and mTORC1 activation (p-p70/p-70 ratio) was analyzed in serum-starved cells in the absence of amino acids (i.e., in the absence of LAT1 contribution). For both (A) and (B), quantifications of activated mTORC1 (p-p70/p-70), or Akt (p-Akt(S473)/Akt), are shown beneath or beside the immunoblots and depict mean ± SEM (N = 3); p values were calculated using Student’s t tests.
(C) Model: NKCC1-mediated regulation of mTORC1 activation. NKCC1 suppresses mTORC1 activation by inhibiting the Leu transporter LAT1, the IR/PI3K/Akt pathway, and the Erk pathway. Several components of the mTORC1 regulatory machinery at the lysosomal membrane are not included here for simplicity.
See also Figure S6.
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9. Figure 7 NKCC1 KD Decreases Cell Size and Promotes Cell Proliferation
(A and B) NKCC1 depletion increases cell proliferation and decreases the relative duration of G1 phase of the cell cycle.
(A) HeLa cell proliferation (cell count) of either scrambled or two NKCC1-KD clones was measured over the course of 4 days using Countess Coulter counter and normalized to day 0. Data are mean ± SEM (N = 3 independent experiments).
(B) NKCC1-KD cells display a decrease in the fraction of cells in G1 phase and an increase in the fraction of cells in S/G2 phase.
(C–F) NKCC1 depletion results in smaller cell volume and mass. NKCC1-KD cells display reduced (C) cell volume and (D) cell mass (upper panel shows the distribution of single cell mass, and lower panel compares average cell mass from replicate samples).
(E) Cell mass for NKCC1-KD cells is also smaller for cells in the G1 phase, indicating that the smaller size phenotype in NKCC1-KD is not due to a redistribution of cell cycle (the upper and lower panels are similar to those in D).
In (D) and (E), upper graphs represent the raw data of cell mass measurement by the protein dye fluorescent of succinimidyl ester dye (see STAR Methods). PDF, probability density function.
(F) NKCC1-KD cells display smaller nuclear size compared with the scrambled control.
Data are shown as mean ± SEM (N = 3); p values indicated in (E) and (F) were calculated using Student’s t tests.
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