rat aortic smooth muscle a10 cells  (ATCC)

Journal: bioRxiv

Article Title: Plasma membrane mediated GLUT10 mitochondrial targeting regulates intracellular ascorbic acid homeostasis

doi: 10.1101/2025.01.09.632083

Figure Lengend Snippet: ( A ) GLUT10/GFP and Mito/DsRed fusion proteins. ( B ) Confocal images show colocalization of GLUT10/GFP and mitochondria in live cells. A10 cells expressing both GLUT10/GFP and Mito/DsRed were treated with 100 μM H 2 O 2 ; imaging was performed every 1 h for 6 h. Scale bar, 10 μm. ( C ) Quantification of the percentage of GLUT10/GFP colocalized with Mito/DsRed, as in B . ( D ) Merged confocal images of a live A10 cells expressing GLUT10/GFP and Mito/DsRed. Scale bar, 25 μm. ( E ) Magnified time-lapse confocal images from ( D ); time points from 0-4 s. Scale bar, 1μm. ( F ) Intensity plots along the line from E (t = 0-4 s). B , D and E , Green, GLUT10/GFP; magenta , Mito/DsRed; white, merged.

Article Snippet: Mouse aortic smooth muscle (MOVAS) cells (CRL-2797), rat aortic smooth muscle (A10) cells (CRL-1476), and HEK293T cells (CRL-11268) were obtained from ATCC (American Type Culture Collection, ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin and streptomycin (Gibco).

Techniques: Expressing, Imaging

Journal: bioRxiv

Article Title: Plasma membrane mediated GLUT10 mitochondrial targeting regulates intracellular ascorbic acid homeostasis

doi: 10.1101/2025.01.09.632083

Figure Lengend Snippet: ( A ) The N-glycosylation site (N-X-S, underlined triplet) in mouse GLUT10 was predicted with NetNglyc1.0. The asparagine (N) predicted to be N-glycosylated is highlighted in red. Immunoblot analysis showed a molecular weight shift of GLUT10/GFP in mitochondrial-enriched fractions from GLUT10/GFP-expressing A10 cells treated with PNGaseF (indicated by black and red arrows). HSP60, Heat shock protein 60, a mitochondrial marker. ( B ) Confocal images of GLUT10/GFP colocalized with ER in A10 cells expressing GLUT10/GFP treated with or without 5 μM BFA for 1 h; ER was stained with ER Tracker Red. Green, GFP; magenta , ER Tracker Red; white, merged. Scale bars, 10 µm. ( C ) Immunoblots to detect molecular weight changes of GLUT10/V5 in subcellular fractions. GLUT10/V5 T-REx-293 cells were induced with TET for 12 h. After 4 h of induction, cells were treated with or without 3.56 μM BFA for 4 h. BFA was then removed, and induction continued for another 4 h. IB of V5 for GLUT10/V5; TIM50, mitochondrial marker; CRT, calreticulin, ER marker. ( D ) Diagram shows GLUT10/GFP, GLUT10 N334Q mutant fused to GFP (GLUT10 N334Q/GFP), and Mito/DsRed fusion protein. ( E ) Confocal images of A10 cells expressing GLUT10/GFP or GLUT10 N334Q/GFP and Mito/DsRed treated without (Mock) of with 100 μM H 2 O 2 for 12 h. Green, GFP; magenta , Mito/DsRed; white, merged. Scale bar, 25 µm. ( F ) Quantification of the percentage of GLUT10/GFP (WT) or GLUT10 N334Q/GFP (N334Q) colocalized with mitochondria, as in B . Data represent the mean ± SEM, n = total of 13-22 cells from three independent experiments. Unpaired two-tailed Student’s t-test. **** P < 0.0001.

Article Snippet: Mouse aortic smooth muscle (MOVAS) cells (CRL-2797), rat aortic smooth muscle (A10) cells (CRL-1476), and HEK293T cells (CRL-11268) were obtained from ATCC (American Type Culture Collection, ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin and streptomycin (Gibco).

Techniques: Glycoproteomics, Western Blot, Molecular Weight, Expressing, Marker, Staining, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Plasma membrane mediated GLUT10 mitochondrial targeting regulates intracellular ascorbic acid homeostasis

doi: 10.1101/2025.01.09.632083

Figure Lengend Snippet: ( A ) Amino acid alignments of GLUT10 C-terminus from several species analyzed by CLUSTAL W program. The YXXΦ motif is underlined. Residues in black shading are conserved, while gray shading indicates amino acids belonging to the same conservation group. ( B ) GLUT10/GFP and YXXΦ motif-deleted GLUT10/GFP (GLUT10d/GFP) fusion proteins. ( C ) Confocal images of MOVAS cells expressing GLUT10/GFP or GLUT10d/GFP. Scale bar 10 µm. ( D ) Quantification of the relative intensity of plasma membrane-localized GFP, as in C . A custom ImageJ macro was used as described in Supplementary materials and Supplementary figure 8. Data are shown as mean ± SEM, n = total 16-19 cells from 3 independent experiments. ( E ) Quantification of the DHA uptake. A10 cells expressing GFP, GLUT10/GFP or GLUT10d/GFP were incubated 5 mM DHA for 30 mins, and intracellular AA levels were measured by HPLC and presented as relative levels compared to cells expressing GFP control. Data are shown as mean ± SEM from 5 independent experiments. ( F ) Confocal images of MOVAS cells expressing GLUT10/GFP or GLUT10d/GFP stained with MitoTracker Red. Green, GFP; magenta , MitoTracker Red; white, merged. Scale bars, 5 µm. ( G ) Quantification of the percentages of GLUT10/GFP and GLUT10d/GFP colocalized with MitoTracker, as in F . Data represent the mean ± SEM, n = 14-15 cells from 3 independent experiments. ( H ) GLUT1/GFP and GLUT1-YXXΦ/GFP fusion proteins. ( I ) Confocal images of MOVAS cells expressing GLUT1/GFP or GLUT1-YXXΦ/GFP. Scale bar 10 µm. ( J ) Quantification of the relative intensity of plasma membrane GFP, as in I . Images were analyzed using a custom ImageJ macro described in Supplementary materials and Supplementary figure 8. Data are shown as mean ± SEM, n = total 12-16 cells from 3 independent experiments. ( K ) Confocal images of GLUT10/GFP-expressing A10 cells treated with or without 20 μM CPZ for 6 h. Arrows indicate plasma membrane-localized GLUT10/GFP. Scale bar, 10 µm. ( L ) Immunoblots of GLUT10/V5 levels in mitochondria-enriched fractions from T-REx-293 cells with induced GLUT10/V5 expression at indicated time points. IB, V5 for GLUT10/V5, TIM50, mitochondrial marker. ( M and N ) Immunoblots of GLUT10/V5 levels in ( M ) mitochondria-enriched fractions and in ( N ) total protein lysates of T-REx-293 cells pretreated with 20 μM CPZ for 1 h before induction of GLUT10/V5 expression for 6 h. IB, V5 for GLUT10/V5, TIM50, mitochondrial marker, beta-actin served as total protein loading control. Statistical comparisons were made with two-tailed Student’s t-test in D , E and H . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mouse aortic smooth muscle (MOVAS) cells (CRL-2797), rat aortic smooth muscle (A10) cells (CRL-1476), and HEK293T cells (CRL-11268) were obtained from ATCC (American Type Culture Collection, ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin and streptomycin (Gibco).

Techniques: Expressing, Clinical Proteomics, Membrane, Incubation, Control, Staining, Western Blot, Marker, Two Tailed Test

Journal: Circulation Research

Article Title: Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney Disease

doi: 10.1161/circresaha.122.321939

Figure Lengend Snippet: Figure 1. Circulating sEVs from CKD rodent models facilitate vascular calcification in aortic VSMCs. A, Establishment of a CKD rodent model by giving wild-type Wister rats a diet containing 0.75% adenine for 4 weeks, and an outline of calcification assay using A10 clonal embryonic rat aortic smooth muscle cells. Calcification of VSMCs was assayed with Alizarin red staining 48 hours after culture in CM containing 5% rodent serum and 4 mM-inorganic phosphate. B, Masson Trichrome staining showed tubulointerstitial fibrosis and extensive tubular dilation in the kidneys of adenine-treated rats. C, Serum urea nitrogen and creatinine indicating uremic solutes were elevated in CKD serum (n=8 per group). D, Calcium staining with Alizarin red was performed 48 hours, after cell treatment with CM containing 4.0 mM Pi and serum derived from control (Ctrl) or CKD serum. E, Calcium contents after the lysis of calcified deposits (Continued )

Article Snippet: Facilitate VC in Aortic VSMCs To examine the effect of CKD serum on VC, we evaluated the formation of calcium phosphate crystals in the A10 clonal embryonic rat aortic smooth muscle cell line (A10 cells; ATCC)16 under treatment with the serum of CKD model rats.

Techniques: Staining, Derivative Assay, Control, Lysis

Journal: The Kaohsiung journal of medical sciences

Article Title: Effect of profilin-1 on the asymmetric dimethylarginine-induced vascular lesion-associated hypertension.

doi: 10.1002/kjm2.12468

Figure Lengend Snippet: FIGURE 1 The effect of exogenous ADMA on the expression of profilin-1 and proliferation ability in cultured RASCMs. (A) The effect of ADMA (1, 3, 10, and 30 μM) for 24 h on the protein expression of profilin-1 in RASMCs, **p < 0.01 versus control. (B) The effect of ADMA (30 μM) for different times (6, 12, 18, and 24 h) on the protein expression of profilin-1 in RASMCs, *p < 0.05 versus control and **p < 0.01 versus control. Data are from three independent experiments (n = 3). (C) The effect of ADMA (1, 3, 10, and 30 μM) for 24 h on the proliferation of RASMCs by MTT, **p < 0.01 versus control. (D) The effect of ADMA (30 μM) for different times (6, 12, 18, and 24 h) on the proliferation of RASMCs by MTT, **p < 0.01 versus control. Data were obtained from six independent experiments (n = 6)

Article Snippet: Rat aortic smooth muscle cells (RASMCs) A10 were purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Cell Culture, Control

Journal: Journal of materials chemistry. B

Article Title: Noncovalent functionalization of mesoporous silica nanoparticles with amphiphilic peptides.

doi: 10.1039/c4tb00037d

Figure Lengend Snippet: Fig. 4 Cytotoxicity results of bare and peptide functionalized MSNs. (a) A10 cells incubated for 4 h with particles and 20 h in particle free media and (b) HUVEC cells incubated for 4 h with particles and 20 h in particle free media.

Article Snippet: Viability and uptake experiments were performed by using human umbilical vein endothelial cells (HUVECs) and A10 rat aortic smooth muscle cells (ATCC® Cat# CRL-1476TM).

Techniques: Incubation

Journal: Journal of materials chemistry. B

Article Title: Noncovalent functionalization of mesoporous silica nanoparticles with amphiphilic peptides.

doi: 10.1039/c4tb00037d

Figure Lengend Snippet: Fig. 5 Uptake results of bare and peptide functionalized MSNs. Confocal results showing that peptide functionalized particles were internalized more both A10 and HUVEC cell lines. Upper images at left show the fluorescence of particles, lower images at left show the fluorescence of actin filaments stained by Phalloidin-TRITC and panels on right show the merged images.

Article Snippet: Viability and uptake experiments were performed by using human umbilical vein endothelial cells (HUVECs) and A10 rat aortic smooth muscle cells (ATCC® Cat# CRL-1476TM).

Techniques: Staining

Journal: Journal of materials chemistry. B

Article Title: Noncovalent functionalization of mesoporous silica nanoparticles with amphiphilic peptides.

doi: 10.1039/c4tb00037d

Figure Lengend Snippet: Fig. 6 Flow cytometry analysis of A10 cells and HUVECs treated with bare and peptide functionalized MSNs. (a) Flow cytometry histograms. (b) Graph demonstrates the improved uptake of peptide functionalized MSNs. Data were generated from at least three independent experi- ments. According to Student's t-test, **p < 0.001 and ***p < 0.0001.

Article Snippet: Viability and uptake experiments were performed by using human umbilical vein endothelial cells (HUVECs) and A10 rat aortic smooth muscle cells (ATCC® Cat# CRL-1476TM).

Techniques: Flow Cytometry, Generated