Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: LMNA deletion inhibits de novo fat synthesis through AMPKα phosphorylation. ( A ) The expression levels of AMPKα and its phosphorylated form in LMNA -KO 7701 cells are shown with the corresponding plot of the quantitative analysis ( n = 3) ( B ). ( C ) CO-/C IP assays were performed to assess the endogenous interaction between pLamin Aand pAMPKα-T172 or between lamin A/C and pAMPKα-T172 in 7701 cells. IgG was used as a negative control, and the input was used to examine the levels of pAMPKα-T172 and lamin A/C. ( D ) An exogenous reverse pull-down experiment was performed in 7701 cell lines. 7701 cells were transiently transfected with the AMPK α-FLAG plasmid or control vector for 48 h. An anti-FLAG antibody was used to immunoprecipitate the cell lysates. ( E ) HEK293T cells were transiently cotransfected with the plasmid containing the LMNA cDNA and the AMPKα -N, -C and -F plasmid or control vector. An anti-FLAG antibody was used to immunoprecipitate the nuclear lysates. Data are presented as the means ± SD. ns , p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Protein fragments were transferred onto a nitrocellulose (NC) membrane and subjected to immunoblot analyses with the indicated primary antibodies at the following dilutions: anti-lamin A/C (10298-1-AP, Proteintech, Wuhan, China) at 1:1000, anti-phospho-lamin A/C (S22) (2026, CST) at 1:1000, anti-phospho-lamin A/C (S392) (ab58528, Abcam) at 1:1000, anti-AMPK alpha 1 (5831, CST) at 1:1000, anti-phospho-AMPK alpha (T172) (2535S, CST) at 1:1000, anti-acetyl-CoA carboxylase (21923-1-AP, Proteintech) at 1:1000, anti-phospho-acetyl-CoA carboxylase (S79) (3661S, CST) at 1:1000, anti-fatty acid synthase (FASN) rabbit polyclonal antibody (10624-2-AP, Proteintech) at 1:1000, anti– mitochondrial pyruvate carrier (MPC1; 14462, CST) at 1:1000, anti-β-hydroxy-β-methylglutaryl coenzyme A reductase (HMGCR) polyclonal antibody (13533-1-AP, Proteintech) at 1:1000, anti-GAPDH (60004-I-Ig, Proteintech) at 1:5000, anti-β-actin (60008-1-Ig, Proteintech) at 1:5000, and anti-FLAG (80010-1-RR, Proteintech) at 1:5000, which was used as a control.

Techniques: Expressing, Negative Control, Transfection, Plasmid Preparation

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: Abnormalities in Lamin A/C function trigger AMPKα activation. ( A ) Mutations in the LMNA structure and sequence are shown: D203N is located in the rod domain and R482W, G465D, and T528R are located in the LTD. Western blotting was performed to detect changes in lamin A/C, pLamin A/C, AMPKα, and pAMPKα-T172 levels upon the rescue of lamin A/C expression via the transfection of the LMNC cDNA, LMNA-GFP cDNA and LMNA mutants, and the corresponding semiquantitative data obtained after GAPDH normalization are shown. ( B ) TG and lactate synthesis were quantified in the different transfected groups ( n = 3). ( C ) Western blotting was performed to assess concentration-dependent changes in lamin A/C, pLamin A/C, AMPKα, pAMPKα, pACC1 and ACC1 levels in 7701, HepG2 and MHCC97-H cells treated with lonafarnib. The corresponding quantitative data were obtained by standardization with GAPDH. Data are presented as the means of at least three independent experiments ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Protein fragments were transferred onto a nitrocellulose (NC) membrane and subjected to immunoblot analyses with the indicated primary antibodies at the following dilutions: anti-lamin A/C (10298-1-AP, Proteintech, Wuhan, China) at 1:1000, anti-phospho-lamin A/C (S22) (2026, CST) at 1:1000, anti-phospho-lamin A/C (S392) (ab58528, Abcam) at 1:1000, anti-AMPK alpha 1 (5831, CST) at 1:1000, anti-phospho-AMPK alpha (T172) (2535S, CST) at 1:1000, anti-acetyl-CoA carboxylase (21923-1-AP, Proteintech) at 1:1000, anti-phospho-acetyl-CoA carboxylase (S79) (3661S, CST) at 1:1000, anti-fatty acid synthase (FASN) rabbit polyclonal antibody (10624-2-AP, Proteintech) at 1:1000, anti– mitochondrial pyruvate carrier (MPC1; 14462, CST) at 1:1000, anti-β-hydroxy-β-methylglutaryl coenzyme A reductase (HMGCR) polyclonal antibody (13533-1-AP, Proteintech) at 1:1000, anti-GAPDH (60004-I-Ig, Proteintech) at 1:5000, anti-β-actin (60008-1-Ig, Proteintech) at 1:5000, and anti-FLAG (80010-1-RR, Proteintech) at 1:5000, which was used as a control.

Techniques: Activation Assay, Sequencing, Western Blot, Expressing, Transfection, Concentration Assay

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: pLamin A/C is an indicator of increased lipid synthesis. ( A ) Treatment with 25 mM glucose for 12 h promoted lamin A/C phosphorylation and FASN expression in 7701, HepG2 and MHCC97-H cells. Semiquantitative analyses of blots are displayed. ( B ) TG synthesis was quantified ( n = 3). ( C ) Lamin A/C phosphorylation was increased by 25 mM glucose in a time-dependent manner in 7701 and HepG2 cells. Gray values from blots are displayed. ( D ) The inhibition of AMPKα activation was shown in 7701, HepG2 or MHCC97-H cells treated with 25 mM glucose. Semiquantitative analyses of blots are displayed. ( E ) Person’s correlation analysis between pLamin A and FASN or pAMPKα-T172 is shown. Data are shown as the means of at least three independent experiments ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: Protein fragments were transferred onto a nitrocellulose (NC) membrane and subjected to immunoblot analyses with the indicated primary antibodies at the following dilutions: anti-lamin A/C (10298-1-AP, Proteintech, Wuhan, China) at 1:1000, anti-phospho-lamin A/C (S22) (2026, CST) at 1:1000, anti-phospho-lamin A/C (S392) (ab58528, Abcam) at 1:1000, anti-AMPK alpha 1 (5831, CST) at 1:1000, anti-phospho-AMPK alpha (T172) (2535S, CST) at 1:1000, anti-acetyl-CoA carboxylase (21923-1-AP, Proteintech) at 1:1000, anti-phospho-acetyl-CoA carboxylase (S79) (3661S, CST) at 1:1000, anti-fatty acid synthase (FASN) rabbit polyclonal antibody (10624-2-AP, Proteintech) at 1:1000, anti– mitochondrial pyruvate carrier (MPC1; 14462, CST) at 1:1000, anti-β-hydroxy-β-methylglutaryl coenzyme A reductase (HMGCR) polyclonal antibody (13533-1-AP, Proteintech) at 1:1000, anti-GAPDH (60004-I-Ig, Proteintech) at 1:5000, anti-β-actin (60008-1-Ig, Proteintech) at 1:5000, and anti-FLAG (80010-1-RR, Proteintech) at 1:5000, which was used as a control.

Techniques: Expressing, Inhibition, Activation Assay

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: LMNA deletion inhibits de novo fat synthesis through AMPKα phosphorylation. ( A ) The expression levels of AMPKα and its phosphorylated form in LMNA -KO 7701 cells are shown with the corresponding plot of the quantitative analysis ( n = 3) ( B ). ( C ) CO-/C IP assays were performed to assess the endogenous interaction between pLamin Aand pAMPKα-T172 or between lamin A/C and pAMPKα-T172 in 7701 cells. IgG was used as a negative control, and the input was used to examine the levels of pAMPKα-T172 and lamin A/C. ( D ) An exogenous reverse pull-down experiment was performed in 7701 cell lines. 7701 cells were transiently transfected with the AMPK α-FLAG plasmid or control vector for 48 h. An anti-FLAG antibody was used to immunoprecipitate the cell lysates. ( E ) HEK293T cells were transiently cotransfected with the plasmid containing the LMNA cDNA and the AMPKα -N, -C and -F plasmid or control vector. An anti-FLAG antibody was used to immunoprecipitate the nuclear lysates. Data are presented as the means ± SD. ns , p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: One to two micrograms of primary antibodies were added to the remaining lysate and incubated for 6 h at 4 °C with slow shaking; these antibodies included anti-lamin A/C rabbit polyclonal antibody (10298-1-AP, Proteintech, Wuhan, China), anti-phospho-lamin A/C (S22) antibody (2026, CST), anti-phospho-lamin A/C (S392) antibody (ab58528, Abcam), anti-AMPK alpha 1 antibody (5831, CST, Danvers, MA, USA), and anti-phospho-AMPK alpha (T172) antibody (2535S, CST).

Techniques: Expressing, Negative Control, Transfection, Plasmid Preparation

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: Abnormalities in Lamin A/C function trigger AMPKα activation. ( A ) Mutations in the LMNA structure and sequence are shown: D203N is located in the rod domain and R482W, G465D, and T528R are located in the LTD. Western blotting was performed to detect changes in lamin A/C, pLamin A/C, AMPKα, and pAMPKα-T172 levels upon the rescue of lamin A/C expression via the transfection of the LMNC cDNA, LMNA-GFP cDNA and LMNA mutants, and the corresponding semiquantitative data obtained after GAPDH normalization are shown. ( B ) TG and lactate synthesis were quantified in the different transfected groups ( n = 3). ( C ) Western blotting was performed to assess concentration-dependent changes in lamin A/C, pLamin A/C, AMPKα, pAMPKα, pACC1 and ACC1 levels in 7701, HepG2 and MHCC97-H cells treated with lonafarnib. The corresponding quantitative data were obtained by standardization with GAPDH. Data are presented as the means of at least three independent experiments ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: One to two micrograms of primary antibodies were added to the remaining lysate and incubated for 6 h at 4 °C with slow shaking; these antibodies included anti-lamin A/C rabbit polyclonal antibody (10298-1-AP, Proteintech, Wuhan, China), anti-phospho-lamin A/C (S22) antibody (2026, CST), anti-phospho-lamin A/C (S392) antibody (ab58528, Abcam), anti-AMPK alpha 1 antibody (5831, CST, Danvers, MA, USA), and anti-phospho-AMPK alpha (T172) antibody (2535S, CST).

Techniques: Activation Assay, Sequencing, Western Blot, Expressing, Transfection, Concentration Assay

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: pLamin A/C is an indicator of increased lipid synthesis. ( A ) Treatment with 25 mM glucose for 12 h promoted lamin A/C phosphorylation and FASN expression in 7701, HepG2 and MHCC97-H cells. Semiquantitative analyses of blots are displayed. ( B ) TG synthesis was quantified ( n = 3). ( C ) Lamin A/C phosphorylation was increased by 25 mM glucose in a time-dependent manner in 7701 and HepG2 cells. Gray values from blots are displayed. ( D ) The inhibition of AMPKα activation was shown in 7701, HepG2 or MHCC97-H cells treated with 25 mM glucose. Semiquantitative analyses of blots are displayed. ( E ) Person’s correlation analysis between pLamin A and FASN or pAMPKα-T172 is shown. Data are shown as the means of at least three independent experiments ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: One to two micrograms of primary antibodies were added to the remaining lysate and incubated for 6 h at 4 °C with slow shaking; these antibodies included anti-lamin A/C rabbit polyclonal antibody (10298-1-AP, Proteintech, Wuhan, China), anti-phospho-lamin A/C (S22) antibody (2026, CST), anti-phospho-lamin A/C (S392) antibody (ab58528, Abcam), anti-AMPK alpha 1 antibody (5831, CST, Danvers, MA, USA), and anti-phospho-AMPK alpha (T172) antibody (2535S, CST).

Techniques: Expressing, Inhibition, Activation Assay

Journal: iScience

Article Title: Mitochondrial complex I inhibitors suppress tumor growth through concomitant acidification of the intra- and extracellular environment

doi: 10.1016/j.isci.2021.103497

Figure Lengend Snippet: Key resources table

Article Snippet: Anti-phospho-(T172)-AMPK alpha , Cell signaling Technology , Cat#2535; RRID: AB_331250.

Techniques: Recombinant, Synthesized, Staining, Isolation, Activity Assay, Lactate Assay, Software