Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 5. NaF exposure induces elevated phos phorylation of PRKAA1 expression in vivo and in vitro. (A) The hierarchical cluster heatmap of phosphorylated proteins in rat hippocampus. (B) Statistics of different phosphorylation sites and proteins in rat hippocampus. (C) Volcano plot of differentially phosphorylated proteins in rat hippocampus. The criteria for significance were set at logarithmic fold change (log2 FC) of > 1.2 or < 0.8 (P < 0.05). (D) Phosphorylation of PRKAA1 protein in rat hippocampus. (E-F) Representative western blot and relative quan tifications of PRKAA1 and p-PRKAA1 in rat hippocampus. (G-H) Representative western blot and relative quantifications of PRKAA1 and p-PRKAA1 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Expressing, In Vivo, In Vitro, Phospho-proteomics, Western Blot, Control

Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 6. DM inhibits aberrant mitophagy by pre venting the phosphorylation of PRKAA1 in vitro. (A-B) Representative western blot and relative quantifications of PRKAA1and p-PRKAA1 in SH- SY5Y cells. (C-D) Representative western blot and relative quantifications of PINK1 and Parkin in SH-SY5Y cells. (E-F) Representative images and relative quantifications of immunofluores cence staining of PINK1 and Mito-tracker, after DM intervention. The scale bar represents 50 µm. (G-H) Representative western blot and relative quantifications of TOMM-20 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group, @P < 0.05 compare with the NaF group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Phospho-proteomics, In Vitro, Western Blot, Staining, Control

Journal: Arthritis Research & Therapy

Article Title: Aberrant mechanical loading induces annulus fibrosus cells apoptosis in intervertebral disc degeneration via mechanosensitive ion channel Piezo1

doi: 10.1186/s13075-023-03093-9

Figure Lengend Snippet: Piezo1 promotes AFCs apoptosis via Ca2 + /Calpain2/Caspase3 pathway. a The activity of Calpains in the Ctrl group, CMS group, CMS + Lv-Ctrl group, and CMS + Lv-Piezo1 group as determined by Calpain activity detection kit ( n = 3). b Western blotting analysis showing the Calpain1, Calpain2, Bax, Cleaved-Caspase3 expression in the Ctrl group, CMS group, CMS + Lv-Ctrl group, and CMS + Lv-Piezo1 group ( n = 3). c Representative flow cytometry scatter plots detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group. d Representative Tunel staining fluorescence pictures detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group (scale bar = 50 μm). Apoptotic AFCs appeared red and nuclei were counterstained with Hoechst 33,358 (blue). e Representative MMP staining fluorescence pictures detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group (scale bar = 50 μm). MMP appeared red and nuclei were counterstained with Hoechst 33,358 (blue). f Statistic data of apoptotic AFCs in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by flow cytometry ( n = 3). g Statistic data of Tunel-positive apoptotic AFCs in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by Tunel staining ( n = 3). h Statistic data of mean MMP fluorescent intensity in AFC in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by MMP staining ( n = 3). i Western blotting analysis showing the Bax, Cleaved-Caspase3 expression in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group ( n = 3). ** P < 0. 01, *** P < 0.001, **** P < 0.0001

Article Snippet: Primary antibodies contain calpain1 (10,538–1-AP, Proteintech), calpain2 (11,472–1-AP, Proteintech), Bax (50,599–2-Ig, Proteintech), cleaved-Caspase 3 (9664, Cell Signaling Technology, MA, USA), Piezo1 (5939–1-AP, Proteintech), and Gapdh (60,004–1-Ig, Proteintech).

Techniques: Activity Assay, Western Blot, Expressing, Flow Cytometry, TUNEL Assay, Staining, Fluorescence

Journal: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: miR-214 inhibition and ATF4 overexpression reverse MEG3 knockdown-mediated decreases in FoxO1, G6pc and Pepck protein expression. Cells were transfected with the indicated plasmids, followed by treatment with 0.5 mM palmitate for 4 h. (A–D) Western blot analysis revealed that MEG3 knockdown significantly suppressed the palmitate-mediated increase in (B) FoxO1, (C) Pepck, and (D) G6pc protein expression. (E–H) By contrast, the miR-214 inhibitor additionally induced the palmitate-mediated increase in (F) FoxO1, (G) Pepck and (H) G6pc protein expression. (I–L) ATF4 overexpression also induced the palmitate-mediated increase in (J) FoxO1, (K) Pepck and (L) G6pc protein expression. β-tubulin served as the loading control in the western blot analyses. * P<0.05 vs . group 1; # P<0.05 vs. group 3; $ P<0.05 vs . group 4. si, small interfering RNA; ctrl, control; MEG3, maternally expressed gene 3; miR, microRNA; FoxO1, forkhead box protein O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit; ATF4, activating transcription factor 4.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Inhibition, Over Expression, Expressing, Transfection, Western Blot, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: miR-214 inhibition and ATF4 overexpression reverse the MEG3 knockdown-mediated decrease in FoxO1, G6pc and Pepck mRNA expression. Cells were transfected with the indicated plasmids, followed by treatment with 0.5 mM palmitate for 4 h. (A–C) RT-qPCR results revealed that MEG3 knockdown significantly suppressed the palmitate-mediated increase in (A) FoxO1, (B) Pepck, and (C) G6pc mRNA expression. (D–F) By contrast, the miR-214 inhibitor induced the palmitate-mediated increase in (D) FoxO1, (E) Pepck, and (F) G6pc mRNA expression. (G–I) ATF4 overexpression also induced the palmitate-mediated increase in (G) FoxO1, (H) Pepck, and (I) G6pc mRNA expression. * P<0.05 vs . group 1; # P<0.05 vs. group 3; $ P<0.05 vs . group 4. miR, microRNA; ATF, activating transcription factor 4; MEG3, maternally expressed gene 3; FoxO1, forkhead box protein O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit; si, small interfering RNA; ctrl, control; MEG3.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Inhibition, Over Expression, Expressing, Transfection, Quantitative RT-PCR, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: Proposed associations between target genes. Palmitate time-dependently increased MEG3 and ATF4 expression, but decreased miR-214 expression. MEG3 functioned as a competing endogenous RNA of miR-214 to upregulate ATF4 expression, leading to the promotion of FoxO1 and its downstream gluconeogenic enzymes G6pc and Pepck. This thereby increases gluconeogenesis and promotes insulin resistance. MEG3, maternally expressed gene 3; miR, microRNA; ATF4, activating transcription factor 4; FoxO1, Forkhead box protein O1; O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing