Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A) Immunohistochemistry staining of DKK1 in the livers of patients with NAFLD (n = 5) and normal individuals (n = 3); scale bar = 100 µ m, (B) in liver samples from WT mice fed with chow and HFD for 24 wk (n = 5). Scale bar = 100 µ m. (C) qPCR analysis of DKK1 mRNA expression in livers of chow- and HFD-fed 24-wk mice (n = 4 mice for each group). (D, E) Representative Western blot of DKK1 in liver samples of chow- and HFD-fed mice (n = 4). Each lane represents liver lysates from individual mouse. (F) Serum DKK1 protein levels in chow- or HFD-fed mice for 24 wk (n = 15–17). (G) Western blot of DKK1 in different primary cells isolated from HFD-fed mice liver. Hep, hepatocytes; HSC, hepatic stellate cells; LSEC, liver sinusoidal endothelial cells; KC, Kupffer cells. (H) Western blot analysis of DKK1 in AML12 cells under different concentrations of FFA exposures (N = 2). ** P < 0.01 as compared with the indicated controls by two-tailed t tests (two groups). All data are shown as the means ± SD. Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Expressing, Western Blot, Isolation, Two Tailed Test

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A) Represented images of DKK1 immunohistochemistry staining on liver sections from NAFLD patients (n = 5) and normal controls (n = 3); scale bar = 100 µ m. (B) LW and LW/BW index of chow- and HFD-fed 6-mo mice (n = 5). (C) Lipid accumulations in the livers were analyzed by H&E staining and ORO staining (n = 3); scale bar = 100 µ m. (D, E) Serum ALT, AST; (E) serum lipids measurement (n = 14–17). ** P < 0.01, *** P < 0.001, **** P < 0.0001 as compared with the indicated controls by two-tailed t tests. All data are shown as the means ± SD. Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Two Tailed Test

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A) Schematic illustration of experiment procedure. AAV tail intravenous injection with AAV-GFP-NC (n = 4), AAV-OE-DKK1 (n = 5), or AAV-sh-DKK1 (n = 6) and then fed HFD for 20 wk before euthanasia. (B) Dynamic body weight tracking of chow- and HFD-fed mice with DKK1 manipulations. (C) Representative Western blot of DKK1 in liver samples of HFD-fed mice after 20 wk (n = 3). Each lane represents liver lysates from individual mouse. (D) H&E and ORO staining. Scale bar = 200 µ m; (E, F) lipid contents in liver of mice with different DKK1 gene manipulations under chow or HFD fed (n = 3). (G, H) Serum lipid contents with different DKK1 gene manipulations under chow or HFD fed. (I) The expression confirmations of lipid metabolism–related genes in liver of mice with different DKK1 gene manipulations under chow or HFD fed (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 as compared with the indicated controls by two-tailed t tests. All data are shown as the means ± SD. ns, not significant. Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Injection, Western Blot, Staining, Expressing, Two Tailed Test

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A) H&E staining of livers from controls and different DKK1 gene manipulations with 20 wk chow or HFD fed; scale bar = 100 µ m. (B) mRNA levels of Wnt-related genes in WT and DKK1-KO HepG2 cells treated with FFA or BSA for 24 h (n = 3). (C) Representative Western blot of ROCK1, total and phosphorylated JNK in WT, and DKK1-KO HepG2 cells treated with FFA or BSA for 24 h (N = 2). * P < 0.05, ** P < 0.01 as compared with the indicated controls by two-tailed t tests. All data are shown as the means ± SD. Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Two Tailed Test

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A, B, C, D, E, F, G, H) The targeted locus of DKK1 and sequencing analysis of homozygous knockout HepG2 and AML12 (A, B) cells that were confirmed with Western blot (C, D), and the paralleled experiments performed on CD36 knockout cells (E, F, G, H). (I, J, K) The DKK1-overexpressed (OE-DKK1) or DKK1-knock-down cell lines (sh-DKK1) were constructed by transfecting overexpression lentivirus or shRNA lentivirus, respectively. Scale bar = 200 µ m (I), and the DKK1 levels were confirmed by Western blot analysis (J, K). Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Sequencing, Knock-Out, Western Blot, Knockdown, Construct, Over Expression, shRNA

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A, B, C) Under DKK1 knockout conditions, cell steatosis was induced by FFA (A, B, C), n = 3 in each group. (A, B, C) Oil red O staining (A) scale bar = 50 µ m; TG measurements in DKK1 −/− AML12 (B) and DKK1 −/− HepG2 (C) cells. n = 3 in each group. (D, E) The changed TG was further confirmed with administration of a DKK1 inhibitor, WAY262611 in AML12 (D) and HepG2 (E) cells. n = 3 in each group. (F, G, H) On other hand, under DKK1 overexpression condition, the induced steatosis status was parallel analyzed, oil red O staining (F), and TG measurements in DKK1 −/− AML12 (G) and DKK1 −/− HepG2 (H) cells. n = 3 in each group. (I, J) Furthermore, the changed trend of TG was further confirmed with administration of recombinant DKK1 protein in AML12 (I) and HepG2 (J) cells. n = 3 in each group. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared with the indicated controls by two-tailed t tests. All data are shown as the means ± SD. Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Knock-Out, Staining, Over Expression, Recombinant, Two Tailed Test

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A, B) Volcano map (A) and KEGG analysis (B) of up- and down-regulated genes in LV-OE-DKK1 AML12 cells compared with LV-GFP-NC AML12 cells under FFA exposure for 24 h. (C, D, E, F) The expression confirmations of lipid metabolism–related genes in cell lines with different DKK1 gene manipulations under FFA treatment; DKK1 overexpression in AML12 (C) and HepG2 (D); DKK1 knockout AML12 (E) and HepG2 (F) cells. n = 3 in each group. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared with the indicated controls by two-tailed t tests. All data are shown as the means ± SD. ns, not significant. Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Expressing, Over Expression, Knock-Out, Two Tailed Test

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A, B) Representative Western blot analyses of lipid metabolism–related proteins in DKK1 overexpression- or knock-down mice (A) and DKK1 overexpression in AML12 cell line (B), in which the numbers marked above the controls were the ratio between CD36 compared with the β-Tubulin. (C, D, E, F) The ORO staining of WT and CD36 −/− cells cultured with recombinant DKK1 protein (r-hDKK1 or r-mDKK1) with or without FFA induction (C, D) scale bar = 100 µ m, and measurement of TG contents in CD36 −/− HepG2 (E) and CD36 −/− AML12 (F) cells. n = 3 in each group. (G) The CD36-promoter-driven luciferase reporter assay under DKK1 overexpression condition. n = 3 in each group. (H) Western blot analysis shows the overexpression of DKK1 increased pERK and nucleus PPARγ in AML12 cells with independent duplicates. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared with the indicated controls by two-tailed t tests. All data are shown as the means ± SD. Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Western Blot, Over Expression, Knockdown, Staining, Cell Culture, Recombinant, Luciferase, Reporter Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: (A, B) The GTT (A) and ITT (B) analyses of mice with treatments of AAV-GFP-NC, AAV-OE-DKK1, or AAV-sh-DKK1 and HFD-fed 20 wk (n = 3). (C) Western blot analyses of phosphorylated JNK in AML12 cells under rDKK1 stimulation (N = 2). (D) Representative Western blot analyses of phosphorylated AKT and FOXO1 in AML12 cells under insulin and rDKK1 stimulation (N = 2). * P < 0.05, ** P < 0.01, *** P < 0.001 as compared with the indicated controls by two-tailed t tests. All data are shown as the means ± SD. Source data are available for this figure.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Western Blot, Two Tailed Test

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: In response to continuous challenge with HFD, (1) the DKK1 expression is mainly induced in hepatocytes and the increased serum DKK1 could have served as a diagnostic bio-marker for steatohepatitis progression. (2) DKK1 enhances hepatic CD36 expression by activating ERK-PPARγ signaling and consequently leads to increased hepatic fatty acid uptake and hepatocyte steatosis. (3) DKK1 activates JNK to decrease the phosphorylation of AKT and FOXO1, leading to insulin resistance. Hepatic fatty acid uptake and insulin resistance synergistically exacerbate fatty acid accumulation and the resultant hepatic steatosis.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Expressing, Diagnostic Assay, Marker, Phospho-proteomics

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: List of primers used for DKK1 and CD36 knock out and identify.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Knock-Out, Sequencing

Journal: Life Science Alliance

Article Title: Hepatic DKK1-driven steatosis is CD36 dependent

doi: 10.26508/lsa.202201665

Figure Lengend Snippet: Antibodies used for Western blot and IHC.

Article Snippet: ALT, AST, TC, TG levels were measured using an automatic biochemical analyzer (7020; Hitachi) in Fengrui Biotechnology Co. Serum levels of DKK1 were measured using a commercially available mouse ELISA kit (EK0925; Boster Biological) according to the manufacturer’s instructions.

Techniques: Western Blot

Journal: Molecular Biology of the Cell

Article Title: Response regulator–mediated MAPKKK heteromer promotes stress signaling to the Spc1 MAPK in fission yeast

doi: 10.1091/mbc.E12-10-0727

Figure Lengend Snippet: (A) The Spc1 signaling pathway in the fission yeast S. pombe . See Introduction for details. It remains to be determined how osmolarity stress signals are transmitted to the Spc1 MAPK cascade. HK, histidine kinase; HPt, histidine phosphotransferase; RR, response regulator. (B) Both Wis4 and Win1 MAPKKKs are required for activation of the Spc1 MAPK cascade in response to osmostress. Wild-type, ∆wis4 , win1-1 , and ∆wis4 win1-1 strains were exposed to high-osmolarity stress of 0.6 M KCl, and their cell lysate was analyzed by immunoblotting to detect the phosphorylated, active Spc1 (pSpc1) and the total Spc1 protein level. (C) Overexpressed Win1 MAPKKK is unable to complement the ∆wis4 defect in osmostress-induced activation of the Spc1 MAPK. ∆wis4 strains carrying a multicopy plasmid to express either Win1 or Wis4 were exposed to osmostress of 0.6 M KCl, and their cell lysate was analyzed by immunoblotting with anti-Spc1 and anti–phospho-Spc1 (pSpc1) antibodies.

Article Snippet: The Spc1 (also known as Sty1) MAPK cascade in the fission yeast Schizosaccharomyces pombe ( ) is highly homologous to the HOG pathway ( Millar et al. , 1995 ; ), but, like mammalian SAPKs, stress stimuli that activate the Spc1 MAPK are not limited to osmostress ( Degols et al. , 1996 ).

Techniques: Activation Assay, Western Blot, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Response regulator–mediated MAPKKK heteromer promotes stress signaling to the Spc1 MAPK in fission yeast

doi: 10.1091/mbc.E12-10-0727

Figure Lengend Snippet: The Wis4 and Win1 MAPKKKs form a heteromer complex. (A) win1:myc and wis4:myc strains were used to monitor the expression levels of the MAPKKKs before and after osmostress of 0.6 M KCl by immunoblotting with anti- myc antibodies (α- myc ). The Spc1 protein in the samples was detected with anti-Spc1 antibodies (α-Spc1) as an internal control. (B, C) Association between the Wis4 and Win1 MAPKKKs before and after stress. Strains that express one MAPKKK tagged with the myc epitope and the other tagged with HATAP were exposed to osmostress of 0.6 M KCl (B) or oxidative stress of 0.73 mM H 2 O 2 (C), and their cell lysate was subjected to affinity purification with IgG–Sepharose, followed by immunoblotting with anti- myc and anti-HA antibodies. The strains expressing no HATAP-tagged MAPKKK served as negative controls ( wis4 + and win1 + ). Bottom, levels of myc -tagged Win1 (B) and Wis4 (C) in the cell lysate used for the affinity purification.

Article Snippet: The Spc1 (also known as Sty1) MAPK cascade in the fission yeast Schizosaccharomyces pombe ( ) is highly homologous to the HOG pathway ( Millar et al. , 1995 ; ), but, like mammalian SAPKs, stress stimuli that activate the Spc1 MAPK are not limited to osmostress ( Degols et al. , 1996 ).

Techniques: Expressing, Western Blot, Control, Affinity Purification

Journal: Molecular Biology of the Cell

Article Title: Response regulator–mediated MAPKKK heteromer promotes stress signaling to the Spc1 MAPK in fission yeast

doi: 10.1091/mbc.E12-10-0727

Figure Lengend Snippet: A Wis4–Win1 heteromer with only one active MAPKKK can induce activation of the Spc1 cascade. (A) The ∆wis4 mutant, as well as strains expressing myc -tagged wild-type or KM mutants of the Wis4 MAPKKK, were exposed to osmostress of 0.6 M KCl and activation of the Spc1 MAPK was monitored by immunoblotting. The expression levels of Wis4- myc and Wis4KM- myc were determined by anti- myc antibodies (bottom). (B) A strain expressing the myc -tagged wild-type Wis4, as well as wis4KM:myc , win1KM:myc , and wis4KM:myc win1KM:myc strains, was exposed to osmostress of 0.6 M KCl and analyzed as in A. (C, D) Strains that express myc -tagged Wis4 (C) or Wis4KM (D), together with Win1 or Win1KM with the HATAP tag, were exposed to osmostress of 0.6 M KCl. The cell lysate was subjected to affinity purification with IgG–Sepharose, followed by immunoblotting using anti- myc and anti-HA antibodies. The strains expressing no HATAP-tagged MAPKKK served as negative controls (–). Bottom, levels of Wis4- myc (C) and Wis4KM- myc (D) in the cell lysate used for the affinity purification.

Article Snippet: The Spc1 (also known as Sty1) MAPK cascade in the fission yeast Schizosaccharomyces pombe ( ) is highly homologous to the HOG pathway ( Millar et al. , 1995 ; ), but, like mammalian SAPKs, stress stimuli that activate the Spc1 MAPK are not limited to osmostress ( Degols et al. , 1996 ).

Techniques: Activation Assay, Mutagenesis, Expressing, Western Blot, Affinity Purification

Journal: Molecular Biology of the Cell

Article Title: Response regulator–mediated MAPKKK heteromer promotes stress signaling to the Spc1 MAPK in fission yeast

doi: 10.1091/mbc.E12-10-0727

Figure Lengend Snippet: The N-terminal segment of Wis4 and the central region of Win1 are important for physical interaction between the MAPKKKs and for activation of the Spc1 cascade. (A) Yeast two-hybrid assays to detect interaction of amino acid residues 282–1123 of Win1 (bait) with a series of truncated Wis4 fragments of indicated amino acid residues (prey). ND, not determined. (B) Yeast two-hybrid assays to detect interactions of the Wis4 fragment of amino acid residues 1–300 (prey) with a series of truncated Win1 fragments as indicated (bait). (C) Designs of chimera MAPKKKs. Wis4 and Win1 MAPKKKs were divided into three segments: the N-terminal region with the CMiN, the central, noncatalytic domain, and the kinase domain. Chimeras were constructed by different combinations of the three segments as schematically shown. (D, E) In a ∆wis4 ∆win1 strain, chimera MAPKKKs shown in C were expressed at low levels from plasmids using the thiamine-repressible nmt1 promoter in the presence of 2 μM thiamine. Activation of the Spc1 MAPK was monitored by immunoblotting along the time course after exposure to osmostress of 0.6 M KCl. Wild-type and ∆wis4 ∆win1 strains carrying the empty vector plasmid were included as controls.

Article Snippet: The Spc1 (also known as Sty1) MAPK cascade in the fission yeast Schizosaccharomyces pombe ( ) is highly homologous to the HOG pathway ( Millar et al. , 1995 ; ), but, like mammalian SAPKs, stress stimuli that activate the Spc1 MAPK are not limited to osmostress ( Degols et al. , 1996 ).

Techniques: Activation Assay, Construct, Western Blot, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Response regulator–mediated MAPKKK heteromer promotes stress signaling to the Spc1 MAPK in fission yeast

doi: 10.1091/mbc.E12-10-0727

Figure Lengend Snippet: The conserved motif in N-terminus (CMiN) of the Wis4 and Win1 MAPKKKs is required for interaction with the Mcs4 response regulator. (A) Alignment of the CMiN sequences of fungal stress MAPKKKs using ClustalW2 ( Goujon et al. , 2010 ). A.f., Aspergillus fumigatus ; A.n., Aspergillus niger ; N.c., Neurospora crassa ; S.j., Schizosaccharomyces japonicus ; S.p., Schizosaccharomyces pombe ; Y.l., Yarrowia lipolytica . Asterisks indicate amino acid residues identical to those in S. pombe Wis4. The consensus sequence is shown at the bottom. –, acidic residues; +, basic residues; B, branched-chain residues. (B) Mcs4 binds to the N-terminal region of Wis4 MAPKKK. GST and GST fused to amino acid residues 120–320 of Wis4 were expressed in a mcs4:myc strain and affinity purified using glutathione–Sepharose. Proteins bound to the beads and Mcs4- myc in the crude lysate were detected with anti- myc and anti-GST antibodies. (C, D) Deletion of the CMiN (∆CMiN) in the MAPKKKs impairs their association with the Mcs4 response regulator. Cell lysate from mcs4:myc strains expressing Wis4-HATAP (C) or Win1-HATAP (D) with or without CMiN were subjected to IgG–Sepharose affinity purification, and proteins bound to the beads and Mcs4- myc in the lysate were detected by immunoblotting. N, negative controls with strains expressing no HATAP-tagged MAPKKK. (E, F) Osmostress-induced activation of the Spc1 MAPK in cells expressing Wis4∆CMiN-HATAP (E) or Win1∆CMiN-HATAP (F) was compared with that in wild-type cells (WT). Spc1 phosphorylation was determined as in .

Article Snippet: The Spc1 (also known as Sty1) MAPK cascade in the fission yeast Schizosaccharomyces pombe ( ) is highly homologous to the HOG pathway ( Millar et al. , 1995 ; ), but, like mammalian SAPKs, stress stimuli that activate the Spc1 MAPK are not limited to osmostress ( Degols et al. , 1996 ).

Techniques: Sequencing, Affinity Purification, Expressing, Western Blot, Activation Assay, Phospho-proteomics