Fig 1: RBP4 (retinol‐binding protein 4), regulates ischemia‐hypoxia‐induced oxidative stress and cell injury in cardiomyocytes. A, The indexes of oxidative stress including glutathione peroxidase, superoxide dismutase, malondialdehyde, and reactive oxygen species were measured at day‐3 post‐acute myocardial infarction and sham controls. B, Lactate dehydrogenase release in the myocardium at day‐3 post‐acute myocardial infarction and sham controls; n=6; *P<0.05 vs sham. C and D, Primary cardiomyocytes were treated with ischemia‐hypoxia for 6 hours. The indexes of oxidative stress including glutathione peroxidase, superoxide dismutase, malondialdehyde, and reactive oxygen species (C) as well as the cell viability (D) were measured; n=6; *P<0.05 vs control. E and F, Primary cardiomyocytes were transfected with adenovirus carrying shRBP4 or scramble RNA for 48 hours and then treated with ischemia‐hypoxia for 6 hours. The indexes of oxidative stress (E) and cell viability (F) were measured; n=6; *P<0.05 vs ischemia‐hypoxia+Ad scramble. G and H, Primary cardiomyocytes were transfected with adenovirus carrying the expression sequence of RBP4 or GFP for 48 hours. The indexes of oxidative stress (G) and cell viability (H) were measured; n=6; *P<0.05 vs Ad‐GFP. AMI indicates acute myocardial infarction; CCK‐8, Cell Counting Kit‐8; GFP, green fluorescent protein; GSH‐PX, glutathione peroxidase; I/H, ischemia/hypoxia; RBP4, retinol‐binding protein 4; ROS, reactive oxygen species; and SOD, superoxide dismutase.
Fig 2: RBP4 (retinol‐binding protein 4) regulates cardiomyocyte pyroptosis via interacting with NLRP3 (nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3). A, Co‐immunoprecipitation identified a protein interaction between RBP4 and NLRP3 in primary cardiomyocytes. B through D, Primary cardiomyocytes were transfected with adenovirus carrying the expression sequence of RBP4 for 24 hours, and then transfected with NLRP3 siRNA or scramble for 24 hours. Protein levels of RBP4, NLRP3 inflammasome and pyroptosis‐related genes were detected by western blot (B). The percentage of propidium iodide (red) positive cells (C) and lactate dehydrogenase release (D) were reduced by silencing of NLRP3; n=6; *P<0.05 vs scramble; **P<0.01 vs scramble. E through G, Primary cardiomyocytes were transfected with adenovirus carrying the expression sequence of RBP4 for 24 hours, and then treated with NLRP3 inhibitor MCC950 (10 μmol/L) for 24 hours. Protein levels of RBP4, NLRP3 inflammasome and pyroptosis‐related genes were detected by western blot (E). The percentage of propidium iodide (red) positive cells (F) and lactate dehydrogenase release (G) were reduced by inhibition of NLRP3; n=6; *P<0.05 vs DMSO; **P<0.01 vs DMSO. AMI indicates acute myocardial infarction; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; GSDMD, gasdermin‐D; IB, immunoblotting; I/H, ischemia/hypoxia; IgG, immunoglobulin G; IL‐1β, interleukin‐1β; IL‐18, interleukin‐18; LDH, lactate dehydrogenase; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3; PI, propidium iodide; and RBP4, retinol‐binding protein 4 .
Fig 3: Early and late dedifferentiation biomarkers predict human chondrocyte plasticity. a Confirmation of the immunostained scRNA-seq-defined markers RBP4, SOD3, IFITM3, and F-actin in human P0-8 chondrocytes, as a human chondrocyte dedifferentiation model; scale bars: 50 μm. b Quantitative data of immunostained scRNA-seq-defined markers, cell/nucleus size and cell relative viability in human P0-8 chondrocytes. c Immunostained RBP4, SOD3, IFITM3, and F-actin in human articular chondrocytes from 2 other donors (Donor 1 and 2) for phenotype evaluation; scale bars: 50 μm. d Heatmap of captured parameters in the human chondrocyte dedifferentiation model and articular chondrocyte samples from 2 donors, including immunostained RBP4, SOD3, IFITM3 and F-actin intensity, as well as cell size and nucleus size. Each cell represents the detection of one biomarker in one cell. Each row represents a round of detection. Twenty cells were detected for one biomarker in each batch. e, f Sample-to-sample distance heatmap and principal component analysis (PCA) plot showing the similarity of Donor-1 and Donor-2 chondrocytes with the human chondrocyte dedifferentiation model (P0-8). g Alcian blue–stained micromasses formed by the human chondrocyte dedifferentiation model (P0-8) and Donor-1 and 2 chondrocytes; scale bars: 3 mm. All data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: atRA suppressed RBP4 mRNA levels and altered the mRNA levels of p27 and cyclin D1 in human embryonic palatal mesenchymal cells. (A) mRNA expression of RBP4 was decreased in the atRA-treated group compared with that in the Con group. (B) mRNA expression of p27 was increased in the atRA-treated group compared with the Con group. (C) mRNA expression of cyclin D1 was reduced in the atRA-treated group compared with the Con group. There were 3 duplicate cultures in each group, and experiments were performed in triplicate. *P<0.05, **P<0.01 and ***P<0.001. atRA, all-trans retinoic acid; RBP4, retinol binding protein 4; Con, control.
Fig 5: Knockdown of RBP4 induced upregulation of p27 and downregulation of cyclin D1 at the mRNA level. (A) When compared with the Con group, RBP4 mRNA levels were downregulated in HEPM cells treated with RBP4 siRNA. (B) P27 mRNA levels were upregulated in HEPM cells treated with RBP4 siRNA via Con. (C) Cyclin D1 mRNA levels were downregulated in HEPM cells treated with RBP4 siRNA compared with Con. A total of 3 duplicate samples were used in each group, and experiments were performed in triplicate. *P<0.05, **P<0.01 and ***P<0.001. RBP4, retinol binding protein 4; HEPM, human embryonic palatal mesenchymal; siRNA, small interfering RNA; Con, control siRNA.
Supplier Page from Abcam for Anti-RBP4 antibody [EP3657]