Journal: Research

Article Title: PDHA1 Hyperactivation Orchestrates Metabolic Reprogramming Promoting Endothelial Senescence

doi: 10.34133/research.1398

Figure Lengend Snippet: Pyruvate dehydrogenase E1 component subunit alpha (PDHA1)-activation-driven endothelial senescence via the cyclic GMP-AMP synthase (cGAS) signaling pathway. Cellular senescence in pulmonary vascular endothelial cells (PVECs) was induced by treatment with AZD7545. (A) The mitochondrial reactive oxygen species (mtROS) in AZD7545-treated PVECs were detected by an ROS kit (scale bar = 20 μm). (B and C) JC-1 staining showing mitochondrial membrane potential in endothelial cells (scale bar = 20 μm). (D) Quantification of cytosolic mitochondrial DNA (mtDNA) for the Nd1 , Cytb , and D-loop in AZD7545-treated PVECs was performed by real-time polymerase chain reaction (PCR). (E and F) Western blot and quantification for cGAS, stimulator of interferon genes (STING), p-TBK1 Ser172 , TBK1, p-IRF3 Ser396 , and IRF3 protein expression. (G) Primary PVECs isolated from Cgas WT and Cgas KO mice were stimulated with AZD7545 (40 μM) for 24 h. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to detect AZD7545-induced senescence in primary PVECs from Cgas WT and Cgas KO mice; scale bar = 20 μm. (H) Ki67 staining was performed in AZD7545-treated primary PVECs from Cgas KO mice; scale bar = 50 μm. (I and J) The expression of senescence-related proteins (cGAS, p53, p21, p16, and phosphorylated histone H2AX [γ-H2AX]) was examined by Western blot in AZD7545-treated primary PVECs isolated from Cgas WT and Cgas KO mice. α-Tubulin was used as the internal control. n = 3. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Anti-p-TBK1 (Ser 172) polyclonal antibody , ABclonal , AP1026 , 1: 2,000.

Techniques: Activation Assay, Staining, Membrane, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Isolation, Control

Journal: Research

Article Title: PDHA1 Hyperactivation Orchestrates Metabolic Reprogramming Promoting Endothelial Senescence

doi: 10.34133/research.1398

Figure Lengend Snippet: Antibody sources and dilutions

Article Snippet: Anti-p-TBK1 (Ser 172) polyclonal antibody , ABclonal , AP1026 , 1: 2,000.

Techniques: Western Blot

Journal: Science Advances

Article Title: Constitutive AMPK activation prevents hepatocellular carcinoma development through inhibition of HNF4α activity

doi: 10.1126/sciadv.aea8017

Figure Lengend Snippet: ( A ) Western blot analysis of female mouse livers fed HFD containing doxycycline (Doxy) to express constitutive active AMPK [a1(1-312); AMPK CA ] for 7 days compared to control livers. ( B ) Immunohistochemical analysis of livers from male control and AMPK CA mice fed HFD+Doxy for 7 months. Scale bars, 100 μm. Right: Quantification of the staining intensity for P-Thr 172 AMPK and P-Ser 79 ACC staining. n = 6 to 10. ±SEM, Welch t test. ( C ) Representative whole-mount (top) and MRI (bottom) image of male control and AMPK CA livers 9 months post–DEN injection. Yellow dashed lines indicate tumors. ( D ) Quantification of the tumor number in male control and AMPK CA livers 6 and 9 months post–DEN injection based on MRI images. 6 months: N = 5 to 7; 9 months: N = 16 to 17; Fisher least significant difference (LSD) test. ( E ) Quantification of the serum AFP levels in control and AMPK CA male mice 6 and 9 months post–DEN injection. n = 9 to 15. Fisher LSD test. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: Phospho-ACC (Ser 79 ) Rabbit mAb , Cell Signaling Technology , 11818.

Techniques: Western Blot, Control, Immunohistochemical staining, Staining, Injection

Journal: Science Advances

Article Title: Constitutive AMPK activation prevents hepatocellular carcinoma development through inhibition of HNF4α activity

doi: 10.1126/sciadv.aea8017

Figure Lengend Snippet: ( A ) Quantification of the tumor size from MRI imaging at 6 and 9 months. n = 10 to 448. Log normal Welch t test. ( B ) Immunohistochemical analysis of BrdU incorporation in tumor tissue from control and AMPK CA livers following a 4-hour BrdU pulse (10 mg/kg). Scale bars, 100 μm. Right: Quantification of the number of positive cells per area. n = 13 to 17. Welch t test. ( C ) Gene expression of Ampka1 normalized to Actin in nontumor and tumor tissue. n = 5 to 6. ±SEM, Fisher LSD test. ( D ) Immunohistochemical analysis of P-Ser 79 ACC in nontumor and tumor tissue from control and AMPK CA livers. Scale bars, 100 μm. ( E ) Quantification of the staining intensity of P-Ser 79 ACC. n = 5 to 14. Fisher LSD test. ( F ) Immunohistochemical analysis of P-Thr 172 AMPK in nontumor and tumor tissue from control and AMPK CA livers. Scale bars, 100 μm. ( G ) Quantification of the staining intensity. n = 6 to 11. Fisher LSD test. ( H ) Western blot analysis of nontumor (NT) and tumor (T) tissue. VINCULIN as loading control. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Phospho-ACC (Ser 79 ) Rabbit mAb , Cell Signaling Technology , 11818.

Techniques: Imaging, Immunohistochemical staining, BrdU Incorporation Assay, Control, Gene Expression, Staining, Western Blot