Journal: Frontiers in Pharmacology

Article Title: GLP-1 Receptor Activation Abrogates β-Cell Dysfunction by PKA Cα-Mediated Degradation of Thioredoxin Interacting Protein

doi: 10.3389/fphar.2019.01230

Figure Lengend Snippet: Deletion of PKA Cα enhances TXNIP level in β-cells. (A) Three designed sgRNAs were inserted into LentiV2 plasmid. These plasmids were transfected into INS-1 cells, and the TXNIP protein level was detected using WB (n = 3). (B) Single PKA Cα-KO β-cell was screened out using 96-well plate and the PKA Cα gene was sequenced in WT and PKA Cα-KO cells. Red sequence indicates GGG in the CRISPR -Cas9 sgRNA design and the green sequence indicates the whole sgRNA sequence. (C) EYFP vector or EYFP- PKA Cα plasmids were transfected into WT or PKA Cα-KO INS-1 cells separately for 24 h. THAP was added to the indicated cells. TXNIP and PKA Cα were analyzed using WB (n = 3). (D) WT or PKA Cα-KO INS-1 cells were treated with THAP, FSK or H89 for 2 h. TXNIP and PKA Cα were analyzed using WB (n = 3). (E) WT or PKA Cα-KO INS-1 cells were treated with THAP, and GSIS assay was performed as described in materials and methods (n = 3). (F) The mRNA level of IL-1β and IL-6 were analyzed in WT or PKA Cα-KO INS-1 cells by qRT-PCR (n = 3). Bars represent the mean ± SEM of independent samples. Significant difference in expression between groups as labeled was analyzed by one-way ANOVA, corrected for multiple comparisons with the Bonferroni test. (ns indicates no statistically significant, ** indicates P value < 0.01, *** indicates P value < 0.001).

Article Snippet: INS-1cells were transfected with the lentiCRISPRv2 [lentiCRISPRv2 puro was a gift from Brett Stringer (Addgene plasmid # 98290)] with the guide RNA sequence 5’-CCTCCCAATCCGCCGTAAGT-3’ against PKA Cα (Gene ID: 25636).

Techniques: Plasmid Preparation, Transfection, Sequencing, CRISPR, Quantitative RT-PCR, Expressing, Labeling

Journal: Frontiers in Pharmacology

Article Title: GLP-1 Receptor Activation Abrogates β-Cell Dysfunction by PKA Cα-Mediated Degradation of Thioredoxin Interacting Protein

doi: 10.3389/fphar.2019.01230

Figure Lengend Snippet: FSK treatment reduces TXNIP level. (A) INS-1 cells were incubated with THAP (0.5 µM), and TXNIP protein was detected using WB (n = 3). (B) INS-1 cells were incubated with THAP (0.5 µM) and FSK (10 µM) at the same time, and then TXNIP protein was detected using WB (n = 3). (C) INS-1 cells were incubated with THAP (0.5 µM) and FSK (10 µM) together, then mRNA level of TXNIP was detected by qRT-PCR (n = 3). (D) INS-1 cells were incubated with MG132 (1 µM) or Bortezomib (0.5 µM) for 1 h, and then THAP (0.5 µM) and FSK (10 µM) was added for 2 h, then TXNIP was detected using WB (n = 3). (E) INS-1 cells were incubated with indicated inhibitors for 1 h, and then THAP (0.5 µM) and FSK (10 µM) was added for 8 h, then TXNIP and PKA Cα were detected using WB (n = 3). (F) INS-1 cells were incubated with indicated inhibitors for 8 h, and then TXNIP and PKA Cα were detected using WB (n = 3). Bars represent the mean ± SEM of independent samples. Significant difference in expression between un-treated group and the drug treatment group as labeled was analyzed by one-way ANOVA, corrected for multiple comparisons with the Bonferroni test. (* indicates P value < 0.05, ** indicates P value < 0.01, *** indicates P value < 0.001).

Article Snippet: INS-1cells were transfected with the lentiCRISPRv2 [lentiCRISPRv2 puro was a gift from Brett Stringer (Addgene plasmid # 98290)] with the guide RNA sequence 5’-CCTCCCAATCCGCCGTAAGT-3’ against PKA Cα (Gene ID: 25636).

Techniques: Incubation, Quantitative RT-PCR, Expressing, Labeling

Journal: Frontiers in Pharmacology

Article Title: GLP-1 Receptor Activation Abrogates β-Cell Dysfunction by PKA Cα-Mediated Degradation of Thioredoxin Interacting Protein

doi: 10.3389/fphar.2019.01230

Figure Lengend Snippet: PKA Cα directly interacts with TXNIP. (A) TXNIP-VC155 (0.5 µg/ml) and PKA Cα-VN173 (0.5 µg/ml) plasmids were transfected into INS-1 cells, with or without MG132. The FRET signal (Ex/Em: 515/528 nm) was observed using confocal microscope after 24 h. Nucleus was stained with DAPI (n = 3). (B) ECFP-TXNIP or EYFP- PKA Cα plasmids were transfected into INS-1 cells separately or together for 24 h. EYFP (red) or ECFP (green) signal was observed using confocal microscope (n = 3). INS-1 cells were incubated with THAP (0.5 µM) or FSK (10 µM) for 2 h, followed by lysis, anti- PKA Cα (C) or anti-TXNIP (D) antibody was used for Co-IP assay (n = 3). HEK-293 cells were transfected with PKA Cα or TXNIP (3.1a) plasmid and then cells were lysed. PKA Cα (E) or TXNIP (F) antibody was used for Co-IP assay (n = 3).

Article Snippet: INS-1cells were transfected with the lentiCRISPRv2 [lentiCRISPRv2 puro was a gift from Brett Stringer (Addgene plasmid # 98290)] with the guide RNA sequence 5’-CCTCCCAATCCGCCGTAAGT-3’ against PKA Cα (Gene ID: 25636).

Techniques: Transfection, Microscopy, Staining, Incubation, Lysis, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Frontiers in Pharmacology

Article Title: GLP-1 Receptor Activation Abrogates β-Cell Dysfunction by PKA Cα-Mediated Degradation of Thioredoxin Interacting Protein

doi: 10.3389/fphar.2019.01230

Figure Lengend Snippet: PKA Cα overexpression alleviates TXNIP-induced β-cell dysfunctions. TXNIP and PKA Cα plasmids were transfected into INS-1 cells (A) or PKA Cα-KO INS-1 cells (B) , and GSIS assay was performed as described in materials and methods (n = 3). (C) TXNIP plasmid was transfected into WT or PKA Cα-KO INS-1 cells, with or without EYFP- PKA Cα plasmids. Then the mRNA level of IL-1β and IL-6 were analyzed using qRT-PCR (n = 3). (D) The mRNA level of IL-1β and IL-6 were analyzed by qRT-PCR in PKA Cα-KO INS-1 cell, with or without PKA Cα transfection (n = 3). Bars represent the mean ± SEM of independent samples. Significant difference in expression between groups as labeled was analyzed by one-way ANOVA, corrected for multiple comparisons with the Bonferroni test. (* indicates P value < 0.05, ** indicates P value < 0.01, *** indicates P value < 0.001).

Article Snippet: INS-1cells were transfected with the lentiCRISPRv2 [lentiCRISPRv2 puro was a gift from Brett Stringer (Addgene plasmid # 98290)] with the guide RNA sequence 5’-CCTCCCAATCCGCCGTAAGT-3’ against PKA Cα (Gene ID: 25636).

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Labeling

Journal: Frontiers in Pharmacology

Article Title: GLP-1 Receptor Activation Abrogates β-Cell Dysfunction by PKA Cα-Mediated Degradation of Thioredoxin Interacting Protein

doi: 10.3389/fphar.2019.01230

Figure Lengend Snippet: TXNIP (S308A) mutant resists the degradation effects of PKA Cα. (A) TXNIP phosphorylation site prediction by pkaPS software (n = 3). (B) TXNIP, TXNIP (S308A) and TXNIP (S307/308A) were transfected into INS-1 cells separately, then the TXNIP protein level was detected using WB after FSK treatment for 2 h (n = 3). (C) TXNIP, TXNIP (S308A), TXNIP (S307/308A) and EYFP- PKA Cα plasmids were transfected into INS-1 cells for 24 h and TXNIP level was detected using WB (n = 3). (D) TXNIP-VC155 (0.5 µg/ml), TXNIP (S307/308A)-VC155 (0.5 µg/ml), TXNIP (S308A)-VC155 (0.5 µg/ml) and PKA Cα-VN173 (0.5 µg/ml) plasmids were transfected separately into INS-1 cells, and the FRET signal (Ex/Em: 515/528 nm) was observed using confocal microscope after 24 h. Nucleus was stained with DAPI (n = 3). Bars represent the mean ± SEM of independent samples. Significant difference in expression between groups as labeled was analyzed by one-way ANOVA, corrected for multiple comparisons with the Bonferroni test. (ns indicates no statistically significant, * indicates P value < 0.05, *** indicates P value < 0.001).

Article Snippet: INS-1cells were transfected with the lentiCRISPRv2 [lentiCRISPRv2 puro was a gift from Brett Stringer (Addgene plasmid # 98290)] with the guide RNA sequence 5’-CCTCCCAATCCGCCGTAAGT-3’ against PKA Cα (Gene ID: 25636).

Techniques: Mutagenesis, Phospho-proteomics, Software, Transfection, Microscopy, Staining, Expressing, Labeling

Journal: Frontiers in Pharmacology

Article Title: GLP-1 Receptor Activation Abrogates β-Cell Dysfunction by PKA Cα-Mediated Degradation of Thioredoxin Interacting Protein

doi: 10.3389/fphar.2019.01230

Figure Lengend Snippet: Exendin-4 mediated β-cell protective effects are dependent on PKA Cα/TXNIP pathway. (B) (A) WT and PKA Cα-KO pancreatic β-cells were treated with exendin-4 for 2 h and TXNIP level was detected using WB (n = 3). (B) TXNIP, TXNIP (S308A) and TXNIP (S307/308A) plasmids were transfected separately into INS-1 cells for 24 h, followed by treatment with exendin-4 for 2h. TXNIP level was detected using WB (n = 3). (C) The quantification of (B) . (D) WT and PKA Cα-KO pancreatic β-cells were treated with THAP or exendin-4 for 2 h and TXNIP level was detected using WB (n = 3). (E) TXNIP and TXNIP (S307/308A) mutant plasmids were transfected separately into INS-1 cells for 24 h. The mRNA level of IL-1β and IL-6 were detected using qRT-PCR (n = 3). (F) The graphic model of PKA Cα/TXNIP pathway in pancreatic β-cells. Red arrow indicated the negative effect, Green arrow indicated the positive effect. Bars represent the mean ± SEM of independent samples. Significant difference in expression between groups as labeled was analyzed by one-way ANOVA, corrected for multiple comparisons with the Bonferroni test. (ns indicates no statistically significant, * indicates P value < 0.05, ** indicates P value < 0.01, *** indicates P value < 0.001).

Article Snippet: INS-1cells were transfected with the lentiCRISPRv2 [lentiCRISPRv2 puro was a gift from Brett Stringer (Addgene plasmid # 98290)] with the guide RNA sequence 5’-CCTCCCAATCCGCCGTAAGT-3’ against PKA Cα (Gene ID: 25636).

Techniques: Transfection, Mutagenesis, Quantitative RT-PCR, Expressing, Labeling