Journal: EMBO Molecular Medicine

Article Title: Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation

doi: 10.15252/emmm.202013074

Figure Lengend Snippet: A, B Western immunoblots for phosphorylated pyruvate dehydrogenase at pS293 (PDH; n = 6, each replicate consists of a pool of 3–4 EHTs from 2 different separate batches), AMP‐dependent protein kinase at Thr172 (AMPK; n = 6, each replicate consists of a pool of 3–4 EHTs from 2 different separate batches), galectin‐3 ( n = 3; each replicate consists of a pool of 3–4 EHTs from 2 different separate batches), SAFB‐like transcription modulator (SLTM; n = 3; each replicate consists of a pool of 3–4 EHTs from 2 different separate batches), LIM and cysteine‐rich domains 1 (LMCD1; n = 3; each replicate consists of a pool of 3–4 EHTs from 2 different separate batches), calnexin ( n = 6; each replicate consists of a pool of 3–4 EHTs from 2 different separate batches), and reticulocalbin‐3 (RCN‐3; n = 3; each replicate consists of a pool of 3–4 EHTs from 2 different separate batches); Coomassie staining was used as a loading control, mean ± SEM, unpaired two‐sided Student's t ‐test, * P < 0.05. C Immunofluorescence of 2D hiPSC‐CM from PLNic and PLN p.Arg14del with antibodies recognizing calnexin (green), α‐actinin (red), and DAPI staining for nuclei (blue); scale bar 20 µm. Source data are available online for this figure.

Article Snippet: Primary antibodies used were as follows: alpha‐actinin (1:1,000, Sigma‐Aldrich, A7811), phospholamban (1:1,000, Novus, NBP2‐19807), phospholamban pSer16 (1:5,000, Badrilla, A010‐12AP), pyruvate dehydrogenase (phospho S293) (Abcam, ab177461), pyruvate dehydrogenase (Abcam, ab168379), AMPK (phospho threonine 172) (Cell Signaling Technology, CST 2535), AMPKα (Cell Signaling Technology, CST D5A2), galectin‐3 (Thermo Fisher, MA1‐940), reticulocalbin‐3 (Abcam, ab204178), LMCD1 (Thermo Fisher, MA5‐25603), SAFB‐like transcription modulator (Novus Biologicals, NBP2‐38464), and calnexin (Novus Biologicals, AF18).

Techniques: Western Blot, Staining, Control, Immunofluorescence

Journal: Journal of translational medicine

Article Title: Small-molecule agonist AdipoRon alleviates diabetic retinopathy through the AdipoR1/AMPK/EGR4 pathway.

doi: 10.1186/s12967-023-04783-3

Figure Lengend Snippet: Fig. 8 a. Altered AdipoR1 pathway-related proteins in the mouse retina. An increase in AMPK and ACC phosphorylation was noticed after AdipoRon treated.ACC and AMPKα remain unchanged, and the expression of p-AMPK and p-ACC is represented by p-AMPK/AMPKα and p-ACC/ACC. b: Strips quantified using Image J software are shown. The results are shown as mean ± standard deviation, **p < 0.01, ***p < 0.001. ACC, acetyl CoA carboxylase; AMPK, 5’ adenosine monophosphate-activated protein kinase; HFD, high-fat diet; DM, high-fat diet + diabetes mellitus control; DMSO, high-fat diet + diabetes mellitus + dimethyl sulfoxide negative control; AdipoRon, high-fat diet + diabetes mellitus + AdipoRon

Article Snippet: The following antibodies were used: Rabbit monoclonal to AdipoR1 (EPR6626,Abcam, Cambridge, UK); rabbit polyclonal to RNA-binding protein with multiple splicing (RBPMS; Abcam); rabbit monoclonal to glutamine synthetase (EPR13022(B),Abcam); chicken polyclonal to glial fibrillary acidic protein (GFAP; Abcam); rabbit polyclonal to EGR4 (Abcam); phospho-AMPKα rabbit monoclonal antibody (mAb) and AMPKα rabbit mAb (40H9/D5A2,Cell signaling, Boston, USA); phospho-acetyl CoA carboxylase (ACC) rabbit mAb and ACC rabbit mAb (D7D11/C83B10,Cell signaling); cleaved caspase-3 rabbit mAb (Cell signaling), β-actin (8H10D10,Cell signaling); donkey antirabbit AlexaFluor-594 and donkey anti-chicken Alexa 488 (Jackson ImmunoResearch, Philadelphia, USA).

Techniques: Phospho-proteomics, Expressing, Software, Standard Deviation, Control, Negative Control

Journal: Journal of translational medicine

Article Title: Small-molecule agonist AdipoRon alleviates diabetic retinopathy through the AdipoR1/AMPK/EGR4 pathway.

doi: 10.1186/s12967-023-04783-3

Figure Lengend Snippet: Fig. 10 a. Quantitative analysis of mRNA expression of AdipoR1 with different treatments. b. Changes in the expression of AdipoR1 pathway in Müller glia. Upregulation of AdipoR1 promoted the phosphorylation of the downstream AMPK/ACC and this effect was reversed by the AdipoR1-specific siRNA. ACC and AMPKα are the total proteins, and p-AMPK and p-ACC expression levels are represented by p-AMPK/AMPKα and p-ACC/ACC. c. Strips quantified using Image J software. The results are shown as mean ± standard deviation, *p < 0.05, **p < 0.01, *** p < 0.001. c. The different expressions of cleaved-caspase 3 among groups. Strips quantified using Image J software. The results were shown as mean ± standard deviation, **p < 0.01. ACC, acetyl CoA carboxylase; AMPK, 5ʹ adenosine monophosphate-activated protein kinase; AdipoR1, adioponectin receptor 1

Article Snippet: The following antibodies were used: Rabbit monoclonal to AdipoR1 (EPR6626,Abcam, Cambridge, UK); rabbit polyclonal to RNA-binding protein with multiple splicing (RBPMS; Abcam); rabbit monoclonal to glutamine synthetase (EPR13022(B),Abcam); chicken polyclonal to glial fibrillary acidic protein (GFAP; Abcam); rabbit polyclonal to EGR4 (Abcam); phospho-AMPKα rabbit monoclonal antibody (mAb) and AMPKα rabbit mAb (40H9/D5A2,Cell signaling, Boston, USA); phospho-acetyl CoA carboxylase (ACC) rabbit mAb and ACC rabbit mAb (D7D11/C83B10,Cell signaling); cleaved caspase-3 rabbit mAb (Cell signaling), β-actin (8H10D10,Cell signaling); donkey antirabbit AlexaFluor-594 and donkey anti-chicken Alexa 488 (Jackson ImmunoResearch, Philadelphia, USA).

Techniques: Expressing, Phospho-proteomics, Software, Standard Deviation

Journal: Journal of translational medicine

Article Title: Small-molecule agonist AdipoRon alleviates diabetic retinopathy through the AdipoR1/AMPK/EGR4 pathway.

doi: 10.1186/s12967-023-04783-3

Figure Lengend Snippet: Fig. 14 a, c, e. Differences in EGR4 and p-AMPK expression in Müller glia after different treatments.Compound C inhibited p-AMPK and down-regulated EGR4, on the contrary,downregulation of EGR4 did not have an effect on p-AMPK expression. b, d, f. Strips quantified using Image J software. Results are shown as mean ± standard deviation, *p < 0.05, **p < 0.01. g, h. Total intracellular ROS and mtROS were measured using DCFH-DA probes and MitoSOX probes in Müller by flow cytometry. EGR4 may act both in the nucleus and mitochondria, effectively reduced oxidative stress and protected cellular functions. Results are shown as mean ± standard deviation, *p < 0.05, ** p < 0.01. EGR4, early growth response factor 4 AMPK, 5ʹ-adenosine monophosphate-activated protein kinase

Article Snippet: The following antibodies were used: Rabbit monoclonal to AdipoR1 (EPR6626,Abcam, Cambridge, UK); rabbit polyclonal to RNA-binding protein with multiple splicing (RBPMS; Abcam); rabbit monoclonal to glutamine synthetase (EPR13022(B),Abcam); chicken polyclonal to glial fibrillary acidic protein (GFAP; Abcam); rabbit polyclonal to EGR4 (Abcam); phospho-AMPKα rabbit monoclonal antibody (mAb) and AMPKα rabbit mAb (40H9/D5A2,Cell signaling, Boston, USA); phospho-acetyl CoA carboxylase (ACC) rabbit mAb and ACC rabbit mAb (D7D11/C83B10,Cell signaling); cleaved caspase-3 rabbit mAb (Cell signaling), β-actin (8H10D10,Cell signaling); donkey antirabbit AlexaFluor-594 and donkey anti-chicken Alexa 488 (Jackson ImmunoResearch, Philadelphia, USA).

Techniques: Expressing, Software, Standard Deviation, Flow Cytometry