1. Volume 65, Issue 2, Pages 296-309 (January 2017)
Lysyl Oxidase 3 Is a Dual-Specificity Enzyme Involved in STAT3 Deacetylation and Deacetylimination Modulation 
Li Ma, Chao Huang, Xiong-Jun Wang, Dazhuan Eric Xin, Li-shun Wang, Quanli C. Zou, Ya-nan S. Zhang, Min-dian Tan, Yu-mei Wang, Ting C. Zhao, Devasis Chatterjee, Rachel A. Altura, Chuangui Wang, Yan S. Xu, Jing-hua Yang, Yong-sheng Fan, Bao-hui Han, Jianmin Si, Xiaoren Zhang, Jinke Cheng, Zhijie Chang, Y. Eugene Chin 
Molecular Cell 
Volume 65, Issue 2, Pages (January 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 65, 296-309DOI: (10.1016/j.molcel.2016.12.002)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Loxl3 Interacts with Stat3 in Nuclei upon Cytokine Stimulation
(A) Stat3 proteins purified from HeLa cells were analyzed with mass spectrometry.
(B) HeLa cells treated with or without OSM were fractionated and blotted with the indicated antibodies.
(C) Mouse spleen was immunostained with Loxl3 antibody. The hematoxylin staining is shown in the lower panel.
(D) Various Loxl3 isoforms were detected in different mouse organs.
(E) The C- and N-terminal domains (CD and ND, respectively) of Loxl3 are illustrated. CAT, catalytic domain; CRL, cytokine receptor-like domain; NES, nuclear export signal; SP, signal peptide; SRCR, scavenger receptor cysteine-rich; SRCR-L1/L2, SRCR-like repeat.
(F) GFP-Loxl3 with N-terminal or C-terminal domain deletion was expressed in HEK293T cells stained with DAPI.
(G) Loxl3 and Stat3 interaction in the nuclear fractions prepared from OSM-treated HeLa cells. IgG, immunoglobulin G.
(H) Interaction between various Myc-Loxl3 constructs and full-length HA-Stat3 in HEK293T cells.
(I) Interaction between various Myc-Stat3 constructs and full-length FLAG-Loxl3 in HEK293T cells.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Loxl3 Is Responsible for Stat3 Deacetylation In Vivo
(A) Immunoprecipitated Stat3 from HEK293T cells with Stat3 and CBP cotransfections was trypsinized for mass spectrometric analysis.
(B) Stat3 from BAPN-treated HeLa cells was analyzed for acetylation with the indicated antibodies.
(C) OSM-induced Stat3 acetylation was abolished by Loxl3 overexpression and was partially recovered by BAPN treatment in HeLa cells.
(D) HEK293T cells were transfected with the indicated plasmids and Stat3 acetylation was analyzed with pan acetyl-lysine antibody.
(E) MCF-7 cells were transfected with Loxl3 siRNA and treated with or without OSM. Subcellular fractions were subjected to western blot with the indicated antibodies.
See also Figure S2.
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5. Figure 3
The N-Terminal SRCR Repeats of Loxl3 Contain Major Deacetylation Activity toward Stat3
(A) Different forms of Loxl3 were analyzed for Stat3 deacetylation in HEK293T cells. Acetyl-Stat3 was blotted with the pan acetyl-lysine antibody.
(B) Loxl3 is highly symmetric between the N-terminal and the C-terminal β sheets linked by SRCR repeat 4 (left). Purified Loxl3 FL, C-terminal domain (CD), and N-terminal domain (ND) all yielded typical ββ sheet spectra by circular dichroism analysis (right).
(C) HEK293T cells were transfected with the indicated plasmids and acetyl-Stat3 was detected with the pan acetyl-lysine antibody. The representative western blot result is shown and densitometry is shown as the means and SEM of three independent experiments.
(D) Stat3 deacetylation by Loxl3 wild-type (WT), Loxl3-HQ (H607, H609Q), Loxl3-CA-1 (C83, C214, C345, C459A), or Loxl3-CA-2 (C376, C446, C492A) in HEK293T cells. The representative western blot result is shown and densitometry is shown as the means and SEM of three independent experiments.
(E) Myc-Loxl1-ND was expressed along with FLAG-Stat5 and CBP in HEK293T cells. (Upper panel) Acetyl-Stat5 was detected with anti-Stat5-K694ac. (Lower panel) HEK293T cells were transfected with the indicated plasmids and acetyl-Snail was detected with pan anti-acetyl lysine.
(F) Purified full-length Loxl3 and acetyl-Stat3 proteins were used for in vitro Loxl3 deacetylase activity assay. Purified Sirt1 plus NAD was also included as a control.
(G) Acetyl-STAT3 proteins were incubated with various forms of Loxl3 and slot blotting was performed with the indicated antibodies.
(H) Indicated acetyl peptides of the Stat3 or H3K4me2 peptide were incubated with the purified Loxl3 proteins. The reaction products were blotted with pan anti-acetyl lysine or anti-H3K4me2.
See also Figure S3 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Loxl3 Catalyzes Stat3 Deacetylation and Deacetylimination In Vitro
(A) Biotin-hydrazide conjugated Stat3 with oxidized lysine residues was immunoprecipitated from HEK293T cells with avidin beads and blotted with HA antibody.
(B) Acetyl-Stat3 and wild-type or mutant Loxl3 were purified from transfected HEK293T cells for in vitro detection of H2O2 production. Data represent the means and SEM of three independent experiments.
(C and D) In vitro K685ac-peptide (C) and K601ac-peptide (D) deacetylation and deaminacetylation by purified Loxl3 proteins of different forms (i.e., FL, ND [SRCR repeats 1–3], and CD [ ]) in vitro were analyzed with mass spectrometry.
(E) Kinetics of Loxl3 in deacetylation and deacetylimination. Data were obtained by following the methods described by Du et al. (2011).
See also Figure S4.
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7. Figure 5
Loxl3 Inhibits Stat3 Transcriptional Activity and Slows the Cell Cycle
(A) Loxl3 effect on Stat3 dimerization in PC3 cells. Anti-HA immunoprecipitates were blotted with anti-Myc or anti-HA.
(B) Various forms of Stat3 as indicated and Stat3 2xSIE-luciferase reporter were transfected with or without CBP into Stat3−/− MEF cells. Data represent the means and SEM of three independent experiments.
(C) Splenocytes from Loxl3+/+ and Loxl3−/− mice were used for Stat3 acetylation analysis with the indicated antibodies.
(D) Various forms of Loxl3 were tested for their effects on the transcriptional activity of Stat3 (left). The SIE-luciferase assay was performed in the presence of various forms of Loxl3 with or without OSM treatment (right). Both experiments were performed in Loxl3−/− spleen fibroblasts. Data represent the means and SEM of three independent experiments.
(E) Indicated genes expressions were analyzed with real-time PCR in HeLa cells introduced with Loxl3 and control (CTL) shRNA. Data represent the means and SEM of three independent experiments.
(F) Spleens from Loxl3+/+ and Loxl3−/− mice were used for the gene expression microarray. Agilent whole genome oligo microarrays were used for analysis.
(G) Splenocytes from Loxl3+/+ and Loxl3−/− mice were treated with or without OSM. Real-time PCR for indicated gene expressions was performed. The data were normalized to ββ-actin and shown as fold changes relative to Loxl3+/+ without OSM. Data represent the means and SEM of three independent experiments.
(H) Kidney cells and splenocytes were prepared from Loxl3+/+ and Loxl3−/− mice for cell cycle analysis by FACS.
See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Loxl3-Stat3 Signaling Pathway in Inflammation Responses
(A) Naive CD4+ T cells were purified from spleen of Loxl3+/+ and Loxl3−/− mice and analyzed with FACS under the conditions for Th17 cell differentiation.
(B) Expression levels of indicated cytokines from Th17 cells from (A) were analyzed by real-time PCR. The data were normalized to ββ-actin and shown as folds of Th0 of Loxl3+/+. Data represent the means and SEM of three independent experiments.
(C) Naive CD4+ T cells were purified from spleen of Loxl3+/+ and Loxl3−/− mice and analyzed with FACS under the conditions for Th1 cell differentiation.
(D) Naive CD4+ T cells were purified from spleen of Loxl3+/+ and Loxl3−/− mice and analyzed with FACS under the conditions for Treg cell differentiation.
(E) Loxl3 protein expression was analyzed by western blot during differentiation of Th0 cells into Th1, Th17, or Treg cells.
(F) Splenocytes from Loxl3+/+, Loxl3+/−, and Loxl3−/− mice were treated with or without IL-6. Whole cell lysates were subjected to western blot with the indicated antibodies.
(G) Splenocytes from Loxl3+/+ and Loxl3−/− mice were treated with or without IL-6 for 12 hr. Real-time PCR for indicated gene expressions was performed. The data were normalized to ββ-actin and shown as fold changes relative to Loxl3+/+ without IL-6. Data represent the means and SEM of three independent experiments.
(H) Examples of colon thickening in response to DSS administration in Loxl3+/+ and Loxl3−/− mice.
(I) H&E staining of colonic sections from Loxl3+/+ and Loxl3−/− mice on day 9 of 2.5% DSS treatment.
(J) Spleen tissue from Loxl3+/+ mice administered DSS was subjected to western blotting with the indicated antibodies.
See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 Illustration of LOX-Mediated Reactions
Whereas HDAC and SIRT are responsible for catalyzing protein deacetylation reactions, LOX family members are dual-specificity enzymes that catalyze both deacetylation and deaminacetylation reactions.
Molecular Cell  , DOI: ( /j.molcel )
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