EMBO/soda-nel huggingface dataset:
Cysteamine-bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis (2021)
Figure 2

Figure 2. Metabolomic and proteomic profiling reveal αKG accumulation in cystinosis.

(A) Principal component analysis (PCA) of control (CTNSWT), CRISPR-generated cystinotic (CTNS-/-), and patient-derived cystinotic (CTNSPatient) cells based on the metabolites measured. Each dot represents one sample, and the dots of the same colour are biological replicates (n = 3).

(B) Heatmap analysis of top 25 metabolites distinctively expressed in control and CTNS-/- cells. Metabolites significantly decreased (P < 0.05) were displayed in green, while metabolites significantly increased (P < 0.05) were displayed in red (n = 3).

(C) Global test pathway enrichment analysis of the intracellular metabolic interactions distinctively affected in CTNS-/- cells compared to healthy control cells (n = 3). Larger circles further from the y-axis and orange-red colour show higher impact of pathway.

(D) List of metabolites that were shared and significantly altered in both the CTNS-/- and CTNSPatient cells compared to control cells (n = 6).

(E) Plasma levels of αKG (µM) in healthy individuals (n=4) and cystinotic patients (n=6). All cystinotic patients were on cysteamine treatment at the time of blood sampling. For this experiment, non-parametric Mann-Whitney t-test was used to demonstrate the significance.

(F) Principal component analysis (PCA) of the measured proteins in CTNSWT, CTNS-/-, and CTNSPatient (n = 3).

(G) Volcano plot illustrates differentially abundant proteins (n = 3). The -log10 (Benjamini-Hochberg corrected P-value) is plotted against the log2 (fold change: CTNSWT/CTNS-/-).

(H) Gene ontology (GO) analysis illustrates classes of proteins differing between CTNSWT and CTNS-/- cells (n = 3).

(I) List of proteins that were significantly upregulated and downregulated in CTNS-/- cells compared to control cells (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, LIPA; Lysosomal acid lipase, ACP2; Lysosomal acid phosphatase, CTSS; Cathepsin S, CTSC; Dipeptidyl peptidase, IGF2R; Cation-independent mannose-6-phosphate receptor, SORT1; Sortilin, CAT; Catalase, CTSA; Lysosomal protective protein, and SOD; Superoxide dismutase.

Data information: Data are expressed as mean ± SEM. *P-values < 0.05 were considered to be significant. One‐way ANOVA with Dunnett's correction (A, B, C, D, E, F, G, H and I). Exact P-values and statistical tests are listed in Appendix Table S1.
Cysteamine-bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis (2021)
Figure 3

Figure 3. αKG is a key metabolite responsible for impaired autophagy and proximal tubule dysfunction in cystinotic proximal tubule cells.

(A) Relative reactive oxygen species (ROS/mg protein) production in control (CTNSWT), CRISPR-generated cystinotic cells (CTNS-/-), and patient-derived cystinotic (CTNSPatient) cells under fed condition (n = 3).

(B, C, D) ROS production (ROS/mg protein) in CTNSWT, CTNS‑/-, and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG (4 hrs), respectively (n = 3).

E, Viability test in starved (-AA) CTNSWT, CTNS‑/-, and CTNSPatient cells in the presence or absence of DMKG (2 mM) for 24 hrs. Results are shown relative to the fed condition (n = 3).

(F, G) Representative confocal micrographs (scale bars are 20 µm) and immunofluorescence analysis of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3).

(H, I) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in CTNSWT, CTNS‑/-, and CTNSPatient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 hrs, respectively (n = 3).

Data information: Data are expressed as mean ± SEM. P-values < 0.05 were considered to be significant. One‐way ANOVA with Dunnett's correction (A, B, C, D, E, G, and I) or Unpaired t‐test (B, C, D, E, and G). Exact P-values and statistical tests are listed in Appendix Table S1.
Cysteamine-bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis (2021)
Figure 4

Figure 4. Bicalutamide-cysteamine combination treatment corrects the metabolome and proteome profile of cystinotic proximal tubule cells.

(A) Metabolomic analysis of CRISPR-generated cystinotic cells (CTNS-/-) treated with cysteamine (100 µM), bicalutamide (35 µM), and a combination of cysteamine and bicalutamide (100 µM and 35 µM, respectively) (n = 3).

(B) Heatmap analysis of the measured metabolites in CTNS-/- cells upon treatment with cysteamine or cysteamine-bicalutamide combination treatment (n = 3). Metabolites significantly decreased were displayed in green, while metabolites significantly increased were displayed in red.

(C, D) Heatmap and REACTOME analysis of the altered proteins in CTNS-/- cells upon treatment with cysteamine, bicalutamide, and cysteamine-bicalutamide combination treatment (n = 3). Proteins significantly decreased were displayed in green, while metabolites significantly increased were displayed in red.

(E), Proteomic analysis of CTNS-/- cells treated with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide, respectively (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, GLUD1; GLUD2; Mitochondrial glutamate dehydrogenase 1/2, IGF2R; Cation-independent mannose-6-phosphate receptor, GSTK1; Glutathione S-transferase kappa 1, COX6B1; Cytochrome c oxidase subunit 6B1, ACACA; Acetyl-CoA carboxylase 1-Biotin carboxylase, GLS; Glutaminase kidney isoform, mitochondrial, CASP3; Caspase-3, NPC1; Niemann-Pick C1 protein.

Data information: Data are expressed as mean ± SEM. P-values < 0.05 were considered to be significant. One‐way ANOVA with Dunnett's correction (A ,B, C, D, and E) * Significantly different from CTNSWT cells (p < 0.05). # significantly different from CTNS-/- cells (p < 0.05). Exact P-values and statistical tests are listed in Appendix Table S1.
Cysteamine-bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis (2021)
Figure 5

Figure 5. Cysteamine-bicalutamide combination treatment efficiently lowers lysosomal cystine, abolishes αKG-mediated autophagy distortion and cell death in cystinotic proximal tubule cells.

(A) Cell viability test of DMKG (2 mM)-treated CRISPR-generated cystinotic cells (CTNS‑/-) upon pre-treatment with cysteamine (100 µM), bicalutamide (35 µM), and a combination of cysteamine and bicalutamide (100 µM and 35 µM, respectively). Results are shown relative to the starved condition (n = 6).

(B) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in DMKG (2 mM)-treated CTNSWT and CTNS-/- cells upon pre-treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 3).

(C) Relative reactive oxygen species (ROS/mg protein) production in DMKG-treated CTNSWT and CTNS‑/- cells upon pre-treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n = 3).

(D, E) Representative confocal micrographs and quantification of DQ-BSA in CTNSWT, and CTNS-/- cells upon treatment with bicalutamide. Scale bars are 20 µm (n = 8-14 quantified images).

(F) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNS-/- in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 6), bicalutamide (n = 6), and a combination of cysteamine and bicalutamide (n = 6).

(G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNSPatient in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 4), bicalutamide (n = 4), and a combination of cysteamine and bicalutamide (n = 6).

Data information: Data are expressed as mean ± SEM. *P-values < 0.05 were considered to be significant. One‐way ANOVA with Dunnett's correction (A, B, C, D, E, F, and G) or Unpaired t‐test (B, C, E, F and G). Exact P-values and statistical tests are listed in Appendix Table S1.
Cysteamine-bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis (2021)
Figure 6

Figure 6. Cysteamine-bicalutamide combination treatment is effective in patient-derived cystinotic tubuloids and cystinotic zebrafish.

(A) Immunocytochemistry of patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) and healthy kidney tissue-derived control tubuloids (CNTSWT-1 and CTNSWT-2) for PAX8, TP63 and F-actin. Scale bar = 100 µm.

(B) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in control and cystinotic tubuloids (n = 3).

(C, D) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in two different patient-derived cystinotic tubuloids in the absence of the drugs (NT) or upon treatment with cysteamine (100 μM), bicalutamide (35 μM) or cysteamine (100 μM)-bicalutamide (35 μM) combination treatment (n = 3).

(E) αKG levels measured in patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) in the absence of the drugs (NT) or upon treatment with cysteamine, bicalutamide or cysteamine-bicalutamide combination treatment using metabolomics (n = 3).

(F) Representative images of control and cystinotic zebrafish.

(G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in control and cystinotic zebrafish (n = 3).

(H) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in cystinotic zebrafish after treatment with cysteamine (1000 µM), bicalutamide (10 µM), and a combination of cysteamine and bicalutamide (1000 µM and 10 µM, respectively) (n=40 embryos per group).

(I) Survival rates in ctns-/- zebrafish upon treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).

(J) Deformity rates in ctns-/- zebrafish after treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).

(K) Hatching rates in surviving ctns-/- zebrafish evaluated at 72 and 96 hrs post fertilization (hpf) with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).

Data information: Data are expressed as mean ± SEM. *P-values < 0.05 were considered to be significant. One‐way ANOVA with Dunnett's correction (B, C, D, E, G, H, and J) or Unpaired t‐test (C, D, and H). Exact P-values and statistical tests are listed in Appendix Table S1.
A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction (2020)
Figure 1

Figure 1 - LUZP1 is a microtubule (MT)-binding protein localized at tight junction (TJ)-associated circumferential rings (CRs).

Membrane overlay assay of Taxol-stabilized MTs using the apical junctional complex (AJC)-enriched fraction. The AJC-enriched fraction was further fractionated using ultra-centrifugation and ion-exchange chromatography with an SP-Sepharose and a Q-Sepharose column (see also Fig EV1C). The bands (marked by an arrowhead and red dots) correspond to LUZP1. 1: Ho, homogenate; 2: Sup, supernatant; 3: Pel, pellet; 4: SP-Pass, passing material through an SP-Sepharose column; 5: SP-E3(2), 2nd elution fraction through an SP-Sepharose column; 6: Q-Pass, passing material through a Q-Sepharose column; 7 to 9: Q-E3(1) to Q-E3(3), 1st to 3rd elution fractions through a Q-Sepharose column.

MT co-sedimentation assay with purified Flag-LUZP1. Flag-LUZP1 directly bound MTs with a dissociation constant (Kd) of 0.78 ± 0.13 μM. A representative immunoblot and a plot in which all results are plotted with the Michaelis-Menten fitted curve are shown. n = 3. S, supernatant; P, pellet.

Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical and sub-apical planes. LUZP1 was associated with TJs which are positive for ZO-1. Scale bar, 10 μm.

A representative super-resolution micrograph of immunostained Eph4 epithelial cell sheets with the graph showing fluorescent intensities along the line. LUZP1 co-localized better with myosin IIB than with ZO-1. a.u., arbitrary units. Scale bar, 10 μm.

Magnified images of Fig 1D. Scale bar, 1 μm.

Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane. The apical MTs were associated with both LUZP1 and TJs. Scale bar, 10 μm (low magnification) and 1 μm (high magnification).

Representative immunoelectron micrographs of Eph4 cells. Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin. MV, microvilli; AJ, adherens junction, DS, desmosome. Scale bars, 200 nm.

A schematic drawing of LUZP1 localization in association with TJs, apical MTs, and actomyosin-based CRs.

Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ZO-1/-2 DKO cells were marked by asterisks (*). The LUZP1 junctional localization was apparently disrupted in TJ-deficient ZO-1/-2 DKO cells. Scale bar, 10 μm.

Representative confocal micrographs of transient E-cadherin knockout (KO) in WT Eph4 cells. E-cadherin KO cells were marked by asterisks (*). The LUZP1 junctional localization seemed not to change between AJ-deficient E-cadherin KO and WT cells. Scale bar, 10 μm.
A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction (2020)
Figure 2

Figure 2 - LUZP1 knockout (LUZP1 KO) cells display apical constriction defects with a significant reduction in di-phosphorylated myosin light chain (ppMLC) levels within circumferential rings (CRs).

Representative confocal micrographs of co-cultures of Venus-LUZP1-expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells in the apical and basal plane. Scale bar, 10 μm.

Box plots with dot density plots showing that the apical area/basal area ratio was significantly smaller in REV cells than in LUZP1 KO cells [0.65 ± 0.16 (REV) vs. 1.30 ± 0.17 (LUZP1 KO)]. n = 10. **p < 0.01 (unpaired t-test). The value < 1.0 indicates apical constriction. The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range.

Representative micrographs of co-cultures of wild-type (WT) and LUZP1 KO Eph4 cells. ppMLC levels within CRs were clearly reduced in LUZP1 KO cells compared to those in WT cells. Scale bar, 10 μm.

Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of LUZP1 KO cells [118.34 ± 57.84 μm2 (WT) vs. 185.32 ± 76.69 μm2 (LUZP1 KO)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± standard deviation (SD).

Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [19.19 ± 10.02 arbitrary units (a.u.) (WT) vs. 2.92 ± 1.81 a.u. (LUZP1 KO)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD.

A scatter plot showing that mean fluorescence intensities of ppMLC and those of LUZP1 significantly correlated within CRs [Pearson's correlation coefficients (r) = 0.75, p < 0.01].

Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ppMLC levels within CRs were clearly reduced in ZO-1/-2 DKO cells compared to those in WT cells. Scale bar, 10 μm.

Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO-1/-2 DKO cells [119.62 ± 31.62 μm2 (WT) vs. 199.96 ± 94.22 μm2 (LUZP1 KO)] (See also Fig 1I). n = 3. **p < 0.01 (unpaired t test). Bars and error bars represent the mean ± SD.

Representative confocal micrographs of co-cultures of WT and LUZP1 KO MTD-1A cells. ppMLC levels within CRs were clearly reduced in LUZP1 KO MTD-1A cells compared to those in WT MTD-1A cells. Scale bar, 10 μm.

Representative confocal micrographs of co-cultures of WT and LUZP1 KO CSG120/7 cells. ppMLC levels within CRs were clearly reduced in LUZP1 KO CSG120/7 cells compared to those in WT CSG120/7 cells. Scale bar, 10 μm.
A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction (2020)
Figure 3

Figure 3 - LUZP1 binding affinity for myosin light chain (MLC) depends on the phosphorylation status of MLC.

Representative confocal micrographs of wild-type (WT) Eph4 cells treated with 100 μM Y27632 for 30 min. Artificial reductions in di-phosphorylated myosin light chain (ppMLC) levels with Y27632 inhibited LUZP1 junctional localization. Scale bar, 10 μm.

Bar plots with dot density plots showing that LUZP1 mean intensities within circumferential rings (CRs) were significantly reduced after Y27632 treatment [39.75 ± 6.74 arbitrary units (a.u.) (control) vs. 27.7 ± 4.21 a.u. (Y27632)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± standard deviation (SD).

Representative confocal micrographs of WT Eph4 cells treated with 100 nM Calyculin A for 30 min. Artificial upregulations in ppMLC levels with calyculin A promoted LUZP1 junctional localization. Scale bar, 10 μm.

Bar plots with dot density plots showing that LUZP1 mean intensities within CRs were significantly upregulated after calyculin A treatment [39.19 ± 8.32 a.u. (control) vs. 42.24 ± 5.40 a.u. (calyculin A)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD.

A schematic drawing of the positive feedback loop between ppMLC and LUZP1 at tight junction (TJ)-associated CRs to promote apical constriction.

Representative confocal micrographs of Venus-LUZP1-expressing LUZP1 KO (REV) Eph4 cells in the apical plane. Scale bar, 10 μm.

Magnified micrographs of Fig 3F. Binary images on the right show the colocalization of ppMLC and LUZP1. Scale bar, 1 μm.

Schematics of wild-type MLC (WT-MLC), di-dephosphomimetic MLC (AA-MLC, with T18A and S19A mutations), and di-phosphomimetic MLC (DD-MLC, with T18D and S19D mutations).

In vitro direct binding assay between FLAG-LUZP1 and GST-MLC. Representative immunoblots are shown. S, supernatant; P, pellet.

A representative plot in which the amount of bound GST-WT-, AA-, or DD-MLC/FLAG-LUZP1 was plotted with the Michaelis-Menten fitted curve.

Bar plots with dot density plots showing that the dissociation constant (Kd) of LUZP1 from DD-MLC was significantly lower than that from WT-MLC and AA-MLC [4.04 ± 1.05 μM (WT-MLC) vs. 4.88 ± 1.79 μM (AA-MLC) vs. 0.85 ± 0.39 μM (DD-MLC)]. n = 5 (WT-MLC and AA-MLC) and n = 4 (DD-MLC). *p < 0.05 [Kruskal-Wallis test followed by Steel-Dwass test]. Bars and error bars represent the mean ± SD.
A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction (2020)
Figure 4

Figure 4 - Calyculin A treatment reverses the effect of LUZP1 on di-phosphorylated myosin light chain (ppMLC) levels.

Representative confocal micrographs of co-cultures of wild-type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm.

Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells [40.87 ± 9.95 arbitrary units (a.u.) (WT) vs. 39.48 ± 6.04 a.u. (LUZP1 KO)]. n = 3. p = 0.54 (Mann-Whitney U test). Bars and error bars represent the mean ± standard deviation (SD).

In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0-5 μg GST-LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio [1.00 (1st lane, control) vs. 1.13 ± 0.24 (2nd lane) vs. 1.01 ± 0.44 (3rd lane) vs. 1.08 ± 0.73 (4th lane)]. n = 4. p = 0.49 (Kruskal-Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting.

Representative confocal micrographs of co-cultures of Venus-LUZP1-expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm.

Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells [control, 21.14 ± 16.80 a.u. (WT) vs. 3.10 ± 1.72 a.u. (LUZP1 KO); calyculin A, 25.24 ± 10.54 a.u. (WT) vs. 20.65 ± 5.62 a.u. (LUZP1 KO); washout, 22.09 ± 7.90 a.u. (WT) vs. 7.92 ± 4.01 a.u. (LUZP1 KO)]. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD. n = 3.

Representative immunoblot of WT, LUZP1 KO, and Venus-LUZP1-expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min.

Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [WT, 1.00 (control) vs. 1.40 ± 0.06 (calyculin A) vs. 1.14 ± 0.33 (washout); KO, 0.09 ± 0.04 (control) vs. 1.49 ± 0.06 (calyculin A) vs. 0.81 ± 0.99 (washout); REV, 2.06 ± 1.78 (control) vs. 1.82 ± 1.50 (calyculin A) vs. 1.80 ± 1.14 (washout)]. n = 3. Bars and error bars represent the mean ± SD.
A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction (2020)
Figure 5

Figure 5 - LUZP1 inhibits the activity of protein phosphatase 1c β/δ (PP1c β/δ), the catalytic subunit of myosin phosphatase.

A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20-kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC.

Representative confocal micrographs of co-cultures of wild-type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm.

Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells [28.68 ± 9.60 arbitrary units (a.u.) (WT) vs. 25.04 ± 9.47 a.u. (LUZP1 KO)]. p = 0.09 (Mann-Whitney U test). n = 3. Bars and error bars represent the mean ± standard deviation (SD).

Co-immunoprecipitation of HA-PP1c β/δ and GFP-LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting.

In vitro MLC phosphorylation assay using 1 μg GST-PP1c β/δ in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0-5 μg GST-LUZP1. Quantification of the di-phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 up-regulated ppMLC/MLC levels in a dose-dependent manner [1.00 (1st lane, control) vs. 1.27 ± 0.33 (2nd lane) vs. 1.76 ± 0.68 (3rd lane) vs. 2.53 ± 1.65 (4th lane) vs. 2.93 ± 2.45 (5th lane)]. n = 3 or 6. **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.

In vitro Merlin phosphorylation assay using 1 μg GST-PP1c β/δ, 100 ng GST-Merlin, 2 pg p21-activated kinase 1 (PAK1), and 5 μg GST-LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 up-regulated pMerlin/Merlin levels [0.23 ± 0.15 (1st lane) vs. 1.00 (2nd lane, control) vs. 0.32 ± 0.17 (3rd lane) vs. 0.97 ± 0.42 (4th lane) vs. 1.25 ± 0.39 (5th lane)]. n = 4 or 9. *p < 0.05, **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 3rd lane)]. Bars and error bars represent the mean ± SD.

A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)-associated CRs to promote apical constriction.