Journal: International Journal of Molecular Sciences

Article Title: MAPK Phosphatase-3 Mediates Chronic Endoplasmic Reticulum Stress Promoting Hepatic Gluconeogenesis

doi: 10.3390/ijms27062874

Figure Lengend Snippet: Chronic ER stress promoted gluconeogenesis and Mkp-3 expression. ( A ) Primary mouse hepatocytes were treated with Tg (2 ng/mL) for 10 days or cultured with PA (50 µM) for 4 days, or with their respective controls. ( B ) Expression levels of ER stress marker genes Grp78 , Chop and Xbp1 in Tg-treated hepatocytes ( n = 3). ( C ) Expression levels of gluconeogenic genes Pepck and G6pc , and their regulator Pgc1α in Tg-treated hepatocytes ( n = 3). ( D ) Expression level of Mkp-3 in Tg-treated hepatocytes ( n = 3). ( E ) Expression of ER stress marker genes Grp78 , Chop and Xbp1 in PA-treated hepatocytes ( n = 3). ( F ) Expression level of Mkp-3 in PA-treated hepatocytes ( n = 3). Data are presented as mean ± standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significant difference.

Article Snippet: Briefly, Mkp-3 loxp/loxp mice (S-CKO-13779, Cyagen Biosciences, Guangzhou, China) were mated with albumin-Cre mice (Jackson Laboratory, Bar Harbor, ME, USA) to generate Mkp-3 LKO ( Mkp-3 loxp/loxp , Cre + ) mice.

Techniques: Expressing, Cell Culture, Marker

Journal: International Journal of Molecular Sciences

Article Title: MAPK Phosphatase-3 Mediates Chronic Endoplasmic Reticulum Stress Promoting Hepatic Gluconeogenesis

doi: 10.3390/ijms27062874

Figure Lengend Snippet: Chronic Tg treatment induced ER stress in mouse liver and promoted gluconeogenesis and Mkp-3 expression. ( A ) Mice were intraperitoneally injected with Tg (0.075 mg/kg body weight) or equivalent volume of DMSO/saline solution for 35 days. ( B ) Changes in body weight during the treatment ( n = 6). ( C ) Liver weight ( n = 6). ( D ) Fasting blood glucose concentrations at harvest time ( n = 6). ( E ) Expression levels of ER stress marker genes in mouse liver ( n = 6). ( F ) Expression levels of gluconeogenic genes and their regulator Pgc1α in mouse liver ( n = 6). ( G ) Expression level of the Mkp-3 gene in mouse liver ( n = 6). ( H , I ) Protein levels of p-PERK and MKP-3 in mouse liver ( n = 6). Data are presented as mean ± standard error. * p < 0.05, ** p < 0.01, ns indicates no significant difference.

Article Snippet: Briefly, Mkp-3 loxp/loxp mice (S-CKO-13779, Cyagen Biosciences, Guangzhou, China) were mated with albumin-Cre mice (Jackson Laboratory, Bar Harbor, ME, USA) to generate Mkp-3 LKO ( Mkp-3 loxp/loxp , Cre + ) mice.

Techniques: Expressing, Injection, Saline, Marker

Journal: International Journal of Molecular Sciences

Article Title: MAPK Phosphatase-3 Mediates Chronic Endoplasmic Reticulum Stress Promoting Hepatic Gluconeogenesis

doi: 10.3390/ijms27062874

Figure Lengend Snippet: Prolonged excess energy intake induced hepatic ER stress and promoted gluconeogenesis and Mkp-3 expression. ( A ) Mice were fed with a high-fat diet (HFD) or standard chow diet for 28 weeks. ( B ) Changes in body weight during the treatment ( n = 12). ( C ) Fasting blood glucose concentration in mice ( n = 12). ( D ) Weights of liver, gonadal fat (GF) and subcutaneous fat (SCF) in the mice ( n = 12). ( E ) Serum concentrations of non-esterified fatty acids (NEFA), total cholesterol (TC), and triglyceride (TAG) in the mice ( n = 12). ( F ) Expression levels of Grp78 and Chop in mouse liver ( n = 12). ( G ) Expression levels of Pepck , G6pc and Pgc1α in mouse liver ( n = 12). ( H ) Expression levels of Mkp-3 in mouse liver ( n = 12). ( I , J ) Protein levels of p-PERK, PERK and MKP-3 in mouse liver ( n = 6). Data are presented as mean ± standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significant difference.

Article Snippet: Briefly, Mkp-3 loxp/loxp mice (S-CKO-13779, Cyagen Biosciences, Guangzhou, China) were mated with albumin-Cre mice (Jackson Laboratory, Bar Harbor, ME, USA) to generate Mkp-3 LKO ( Mkp-3 loxp/loxp , Cre + ) mice.

Techniques: Expressing, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: MAPK Phosphatase-3 Mediates Chronic Endoplasmic Reticulum Stress Promoting Hepatic Gluconeogenesis

doi: 10.3390/ijms27062874

Figure Lengend Snippet: Role of MKP-3 in chronic ER stress promoting hepatic gluconeogenesis. ( A ) Wild-type (WT) and liver-specific Mkp-3 knockout ( Mkp-3 LKO) mice were fed with a HFD or standard chow diet for 14 weeks. ( B ) Body weight at harvest time ( n = 5). ( C ) Liver weight (n = 5). ( D ) Gonadal fat weight ( n = 5). ( E ) Fasting blood glucose concentration ( n = 5). ( F ) Expression levels of Grp78 and Chop in mouse liver ( n = 5). ( G ) Expression levels of Pepck , G6pc , and Pgc1α in mouse liver ( n = 5). Data are presented as mean ± standard error. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significant difference.

Article Snippet: Briefly, Mkp-3 loxp/loxp mice (S-CKO-13779, Cyagen Biosciences, Guangzhou, China) were mated with albumin-Cre mice (Jackson Laboratory, Bar Harbor, ME, USA) to generate Mkp-3 LKO ( Mkp-3 loxp/loxp , Cre + ) mice.

Techniques: Knock-Out, Concentration Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: MAPK Phosphatase-3 Mediates Chronic Endoplasmic Reticulum Stress Promoting Hepatic Gluconeogenesis

doi: 10.3390/ijms27062874

Figure Lengend Snippet: Chronic ER stress promoted Mkp-3 expression via the PERK pathway. ( A ) Primary mouse hepatocytes were treated with a PERK agonist CCT020312 at 0.4 μM, gene expression level of Mkp-3 was then measured ( n = 3). ( B ) Primary mouse hepatocytes were co-treated with Tg and a PERK inhibitor GSK2656157 at 0.04 μM, gene expression level of Mkp-3 was then measured ( n = 3). ( C , D ) Primary mouse hepatocytes were treated with an IRE1 inhibitor 4µ8c, protein level of XBP1 ( C ) and gene expression level of Mkp-3 were measured ( n = 3). Data are presented as mean ± standard error. ** p < 0.01, *** p < 0.001, ns indicates no significant difference.

Article Snippet: Briefly, Mkp-3 loxp/loxp mice (S-CKO-13779, Cyagen Biosciences, Guangzhou, China) were mated with albumin-Cre mice (Jackson Laboratory, Bar Harbor, ME, USA) to generate Mkp-3 LKO ( Mkp-3 loxp/loxp , Cre + ) mice.

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Integration of human ERKs and DUSPs into the yeast cell wall integrity pathway

doi: 10.1038/s41598-025-33816-8

Figure Lengend Snippet: Analysis of the dephosphorylation of human ERK1, ERK2 and ERK5 by phosphatases DUSP6 or DUSP3. ( A ) Western blotting analysis from Y00993 ( slt2 Δ) cells co-transformed with the plasmid pEG-KG (vector), pEG-KG-DUSP3 or pEG-KG-DUSP6 and pAG413- TDH3 -EGFP-ERK1 or pAG413- TDH3 -EGFP-ERK2, grown to mid-exponential phase in YPG and then stimulated with 30 µg/mL of CR for 4 h when indicated. Cell extracts were resolved by SDS-PAGE and analysed by immunoblotting with anti-P-p42/44 and anti-GFP to detect the phosphorylation and expression of ERKs, with anti-GST to detect the expression of the different DUSPs, and anti-G6PDH as loading control. The numbers at the left indicate the molecular weight in kDa of the protein ladder and the labels on the right side indicate which band correspond to each protein, according to its molecular weight. ( B ) Western blotting assay was performed as in A, from Y00993 cells co-transformed with the empty vector pEG-KG, pEG-KG-DUSP6 or pEG-KG-DUSP6 PD, encoding the phosphatase dead version of DUSP6, and pAG413- TDH3 -EGFP-ERK1 or pAG413- TDH3 -EGFP-ERK2. ( C ) Western blotting assay was performed as in A, from Y07202 cells co-transformed with pAG414- TDH3 -MEK5 S311D, T315D -mRFP (MEK5*) and pEG-KG (vector), pEG-KG-DUSP3 or pEG-KG-DUSP6 and pAG413- TDH3 -EGFP-ERK5 or pAG413- TDH3 -EGFP-ERK5ΔCt, and cultured in YPG for 6 h. The intensity of immunoreactive bands detected with anti-P-p42/44 was normalized with respect to that of the loading control. The graphs on the right show the fold variation in the intensity of each phosphorylated EGFP-fusion protein from cells co-expressing the corresponding DUSP with respect to that from those not expressing them (vector) under the same conditions. Data is from three independent experiments (mean ± s.d.). * p < 0.05, ** p < 0.01, *** p < 0.001 relative to control (vector) unless otherwise indicated.

Article Snippet: R777-E043 Hs.DUSP6 , SmR , DUSP6 , Donor vector bearing DUSP6, was a gift from Dominic Esposito (Addgene plasmid # 70327; http://n2t.net/addgene:70327 ; RRID: Addgene_70327).

Techniques: De-Phosphorylation Assay, Western Blot, Transformation Assay, Plasmid Preparation, SDS Page, Phospho-proteomics, Expressing, Control, Molecular Weight, Cell Culture

Journal: Scientific Reports

Article Title: Integration of human ERKs and DUSPs into the yeast cell wall integrity pathway

doi: 10.1038/s41598-025-33816-8

Figure Lengend Snippet: Analysis of the dephosphorylation of yeast Slt2, Fus3 and Kss1 by the human phosphatases DUSP6 and DUSP3. Western blotting analysis (left panel) of extracts from wild-type BY4742 (WT) and Y04500 ( itc1 Δ) cells transformed with plasmids pEG-KG (vector), pEG-KG-DUSP3 or pEG-KG-DUSP6. Cells were cultured in SR-G Ura- medium for six hours at 24 °C. Membranes were probed with anti-P-p42/44 to detect phosphorylation of Slt2, Fus3 and Kss1, with anti-GST to detect the expression of GST-DUSP3 and GST-DUSP6, and anti-G6PDH as loading control. The numbers at the left indicate the molecular weight in kDa of the protein ladder and the labels on the right side indicate which band correspond to each protein, according to its molecular weight. The intensity of immunoreactive bands detected with anti-P-p42/44 was normalized with respect to that of the loading control. The graph on the right shows the fold variation in the intensity of phosphorylated Slt2 from cells expressing the corresponding DUSP with respect to that from not expressing them (vector). Data is from three independent experiments (mean ± s.d.). * p < 0.05, ** p < 0.01 relative to control (vector) unless otherwise indicated.

Article Snippet: R777-E043 Hs.DUSP6 , SmR , DUSP6 , Donor vector bearing DUSP6, was a gift from Dominic Esposito (Addgene plasmid # 70327; http://n2t.net/addgene:70327 ; RRID: Addgene_70327).

Techniques: De-Phosphorylation Assay, Western Blot, Transformation Assay, Plasmid Preparation, Cell Culture, Phospho-proteomics, Expressing, Control, Molecular Weight