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

Journal: Oncology Research

Article Title: MINDY1 Induces PD-L1 Deubiquitination to Promote Immune Escape in Hepatocellular Carcinoma by the Wnt/β-Catenin Pathway

doi: 10.32604/or.2025.067638

Figure Lengend Snippet: MINDY1 promotes PD-L1 deubiquitination through Wnt/β-catenin pathway. ( A–C ) MINDY1/Vector and sh-MINDY1/sh-NC were transfected, and Western blot analysis assessed that overexpression of MINDY1 elevated β-catenin and p-GSK3β(Tyr216)/GSK3β levels in HCC cells, and knockdown of MINDY1 did the opposite. ( D – F ) Western blot measured that SKL2001 (Wnt pathway activator) elevated the activating effect of overexpression of MINDY1 on the Wnt/β-catenin pathway, while pathway inhibitor LiCl decreased this phenomenon. (G) Western blot measured that SKL2001 increased PD-L1 protein expression, whereas LiCl led to PD-L1 downregulation. (H) Western blot examined that SKL2001 promoted the deubiquitination of PD-L1 in HCC cells, while LiCl suppressed its deubiquitination. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Vector/sh-NC; & p < 0.05, && p < 0.01, &&& p < 0.001 vs. MINDY1

Article Snippet: Additionally, HCC cells (Hep3B and Huh-7 cells) transfected with MINDY1 were treated with the Wnt signaling pathway activator SKL2001 (20 nM, S793781, Macklin, Shanghai, China) for 24 h, and this group was denoted as the MINDY1+SKL2001 group [ ].

Techniques: Plasmid Preparation, Transfection, Western Blot, Over Expression, Knockdown, Expressing

Journal: Oncology Research

Article Title: MINDY1 Induces PD-L1 Deubiquitination to Promote Immune Escape in Hepatocellular Carcinoma by the Wnt/β-Catenin Pathway

doi: 10.32604/or.2025.067638

Figure Lengend Snippet: MINDY1 hinders CD8 + T cell activation through the Wnt/β-catenin pathway. ( A ) CFSE staining confirmed that co-culture with HCC cells transfected with MINDY1 resulted in diminished proliferation of CD8 + T cells, which was further diminished by SKL2001 treatment, whereas LiCl treatment did the opposite. ( B ) Flow cytometry indicated a rise in CD8 + T cell apoptosis after they were co-cultured with HCC cells overexpressing MINDY1, with an additional rise observed by SKL2001 treatment, while LiCl treatment reduced the apoptosis rate. ( C ) The cytotoxicity of CD8 + T cells was assessed through the LDH kit. ( D , E ) Flow cytometry confirmed that co-culturing with HCC cells overexpressing MINDY1 led to a decrease in Perforin + and TNF-α + T cells. After treatment with SKL2001, this decrease was further exacerbated, while LiCl increased activated CD8 + T cells. ( F , G ) ELISA was used to detect IFN-γ and IL-2 levels. ( H ) CCK-8 assay was employed to examine the survival rates of HCC cells. n = 3. * p < 0.05, *** p < 0.001 vs. Vector; & p < 0.05, && p < 0.01, &&& p < 0.001 vs. MINDY1

Article Snippet: Additionally, HCC cells (Hep3B and Huh-7 cells) transfected with MINDY1 were treated with the Wnt signaling pathway activator SKL2001 (20 nM, S793781, Macklin, Shanghai, China) for 24 h, and this group was denoted as the MINDY1+SKL2001 group [ ].

Techniques: Activation Assay, Staining, Co-Culture Assay, Transfection, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Plasmid Preparation