Journal: Cell Death Discovery

Article Title: NR4A1 enhances MKP7 expression to diminish JNK activation induced by ROS or ER-stress in pancreatic β cells for surviving

doi: 10.1038/s41420-021-00521-0

Figure Lengend Snippet: A The relative mRNA levels of MKP7 in the islets from WT and KO mice fed with normal diet were assayed with qPCR. B The relative protein level of MKP7 in the islets from WT or KO mice fed with a normal diet was obtained with Western Blotting. After 4 months of high-fat diet feeding, WT and KO mice were tested for the basal blood glucose level ( C ) and GTT ( D ). The relative p-JNK levels and the relative MKP7 levels in the islets from WT or KO mice fed with a high-fat diet for four months were analyzed ( E – F ). These data represented the means of three independent experiments, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. ns. Error bars were shown as SD values.

Article Snippet: The antibodies used in the experiment were as follows: cleaved caspase-3 Antibody (9661) and rabbit anti-pJNK antibody (4668) were purchased from CST; mouse anti-GAPDH (10494-1-AP), rabbit anti-MKP2 antibody (10739-1-AP), and rabbit anti-MKP7 antibody (14237-1-AP) were derived from Proteintech Group (Wuhan, China); rabbit anti-NR4A1 antibody (ab13851 and DF7850) was purchased from Abcam and Affinity; mouse anti-β-Actin (BS6007M) and rabbit anti-tubulin α (BS1699) were purchased from Bioworld Technology.

Techniques: Western Blot

Journal: Cell Death Discovery

Article Title: NR4A1 enhances MKP7 expression to diminish JNK activation induced by ROS or ER-stress in pancreatic β cells for surviving

doi: 10.1038/s41420-021-00521-0

Figure Lengend Snippet: A The relative mRNA levels of MKP2 and MKP7 in NC and OV cells were determined with qPCR. B and C The relative MKP2 and MKP7 protein levels in both OV and NC cells were determined with Western Blotting. D The protein levels of MKP7 and NR4A1 in response to 100 μM H 2 O 2 in NC cells. E – H The protein levels of MKP7 in response to 100 μM H 2 O 2 and 0.5 μM TG at various time points in both OV and NC cells. The data represented the means of three independent experiments, * P < 0.05, ** P < 0.01 vs. ns. Error bars were shown as SD values.

Article Snippet: The antibodies used in the experiment were as follows: cleaved caspase-3 Antibody (9661) and rabbit anti-pJNK antibody (4668) were purchased from CST; mouse anti-GAPDH (10494-1-AP), rabbit anti-MKP2 antibody (10739-1-AP), and rabbit anti-MKP7 antibody (14237-1-AP) were derived from Proteintech Group (Wuhan, China); rabbit anti-NR4A1 antibody (ab13851 and DF7850) was purchased from Abcam and Affinity; mouse anti-β-Actin (BS6007M) and rabbit anti-tubulin α (BS1699) were purchased from Bioworld Technology.

Techniques: Western Blot

Journal: Cell Death Discovery

Article Title: NR4A1 enhances MKP7 expression to diminish JNK activation induced by ROS or ER-stress in pancreatic β cells for surviving

doi: 10.1038/s41420-021-00521-0

Figure Lengend Snippet: A – B β-TC6 cells were infected with Lentivirus encoding shRNA targeting to MKP7 or with Lentivirus encoding a scrambled shRNA as control. The protein levels of MKP7 in KD-MKP7 (MKP7 knockdown) or CON-MKP7 cells were determined with Western Blotting. C – F The phosphorylated JNK (p-JNK) level in response to 100 μM H 2 O 2 and 0.5 μM TG in KD-MKP7 cells and CON-MKP7 cells were analyzed with western Blotting. These data represented the means of three independent experiments, * P < 0.05, *** P < 0.001 vs. ns. Error bars were shown as SD values.

Article Snippet: The antibodies used in the experiment were as follows: cleaved caspase-3 Antibody (9661) and rabbit anti-pJNK antibody (4668) were purchased from CST; mouse anti-GAPDH (10494-1-AP), rabbit anti-MKP2 antibody (10739-1-AP), and rabbit anti-MKP7 antibody (14237-1-AP) were derived from Proteintech Group (Wuhan, China); rabbit anti-NR4A1 antibody (ab13851 and DF7850) was purchased from Abcam and Affinity; mouse anti-β-Actin (BS6007M) and rabbit anti-tubulin α (BS1699) were purchased from Bioworld Technology.

Techniques: Infection, shRNA, Control, Knockdown, Western Blot

Journal: Cell Death Discovery

Article Title: NR4A1 enhances MKP7 expression to diminish JNK activation induced by ROS or ER-stress in pancreatic β cells for surviving

doi: 10.1038/s41420-021-00521-0

Figure Lengend Snippet: A Sketch for NR4A1 putative binding site in MKP7 promoter. B A diagram of MKP7 promoters with different lengths was designed for luciferase reporter construction. C The relative luciferase activity of MKP7 promoters with different lengths exhibited in both OV and NC cells. D and E ChIP analysis were exploited to detect the physical association between NR4A1 and the promoter region of MKP7; the exogenous NR4A1-HA expression with adenoviral infection in MIN6 cells was associated with chromatin at some specific DNA sequences, after chromatin immunoprecipitation with anti-HA antibodies, the pulled-down DNA fragments were subjected to PCR analysis with specific pairs of primers. The two putative NR4A1 binding sites (−55 to −50, −1637 to −1632) in the MKP7 promoter regulatory sequence were confirmed with specific PCR amplification. These data represented the means of three independent experiments, ** P < 0.01, *** P < 0.001 vs. ns. Error bars were shown as SD values.

Article Snippet: The antibodies used in the experiment were as follows: cleaved caspase-3 Antibody (9661) and rabbit anti-pJNK antibody (4668) were purchased from CST; mouse anti-GAPDH (10494-1-AP), rabbit anti-MKP2 antibody (10739-1-AP), and rabbit anti-MKP7 antibody (14237-1-AP) were derived from Proteintech Group (Wuhan, China); rabbit anti-NR4A1 antibody (ab13851 and DF7850) was purchased from Abcam and Affinity; mouse anti-β-Actin (BS6007M) and rabbit anti-tubulin α (BS1699) were purchased from Bioworld Technology.

Techniques: Binding Assay, Luciferase, Activity Assay, Expressing, Infection, Chromatin Immunoprecipitation, Sequencing, Amplification

Journal: Cell Death Discovery

Article Title: NR4A1 enhances MKP7 expression to diminish JNK activation induced by ROS or ER-stress in pancreatic β cells for surviving

doi: 10.1038/s41420-021-00521-0

Figure Lengend Snippet: A ROS may result in ER-stress. ROS or/and ER-stress incurs JNK activation. The activated JNK leads to pancreatic β cell apoptosis via mitochondria-dependent or independent pathways. B Acute ROS or ER-stress results in the expression of NR4A1, which in turn enhances the expression of MKP7, while MKP7 is able to abolish partial JNK activation by exerting its phosphatase activity, therefore, largely reduces pancreatic β cell apoptosis. The fate of pancreatic β cells depends on the balance between the availability of NR4A1 or MKP7 and the severity of ROS or ER-stress.

Article Snippet: The antibodies used in the experiment were as follows: cleaved caspase-3 Antibody (9661) and rabbit anti-pJNK antibody (4668) were purchased from CST; mouse anti-GAPDH (10494-1-AP), rabbit anti-MKP2 antibody (10739-1-AP), and rabbit anti-MKP7 antibody (14237-1-AP) were derived from Proteintech Group (Wuhan, China); rabbit anti-NR4A1 antibody (ab13851 and DF7850) was purchased from Abcam and Affinity; mouse anti-β-Actin (BS6007M) and rabbit anti-tubulin α (BS1699) were purchased from Bioworld Technology.

Techniques: Activation Assay, Expressing, Activity Assay

Journal: Cell Death Discovery

Article Title: NR4A1 enhances MKP7 expression to diminish JNK activation induced by ROS or ER-stress in pancreatic β cells for surviving

doi: 10.1038/s41420-021-00521-0

Figure Lengend Snippet: For real-time quantitative PCR.

Article Snippet: The antibodies used in the experiment were as follows: cleaved caspase-3 Antibody (9661) and rabbit anti-pJNK antibody (4668) were purchased from CST; mouse anti-GAPDH (10494-1-AP), rabbit anti-MKP2 antibody (10739-1-AP), and rabbit anti-MKP7 antibody (14237-1-AP) were derived from Proteintech Group (Wuhan, China); rabbit anti-NR4A1 antibody (ab13851 and DF7850) was purchased from Abcam and Affinity; mouse anti-β-Actin (BS6007M) and rabbit anti-tubulin α (BS1699) were purchased from Bioworld Technology.

Techniques: Sequencing