Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 2 Analysis of the responses of human VSMCs to cholesterol stimulation. Human VSMCs were treated with cholesterol- supplemented medium (Panserin, chol:MbCD 10 µg/mL) for three days. The expression of genes encoding determinants of cholesterol and phospholipid processing was analyzed by semi-quantitative PCR (A, **P < .01, *P < .05, n = 6, the expression of the housekeeping gene RPL32 served as internal standard). NFAT5 was detected by immunostaining. NFAT5-positive nuclei were detected by immunofluorescence-based methods and quantified by automated image analysis (B, ***P < .001, n = 10, scale bar: 50 µm). NFAT5 protein abundance was determined in nuclear and cytosolic fractions of cell lysates by immunoblot techniques (C and D, *P < .05, n = 6, histone H3 and α-tubulin served as loading controls for the nuclear and cytosolic fractions, respectively. Ctr., control; Chol., cholesterol)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Immunofluorescence, Quantitative Proteomics, Western Blot, Control

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 3 Analysis of the responses of mouse aortic VSMCs to cholesterol stimulation. VSMCs isolated from aortae of Nfat5fl/fl mice were exposed to cholesterol-supplemented medium (Panserin, chol:MbCD 10 µg/mL) or the respective solvent control for three days. Abca1, Acat1, Hmgcr, Lgals3, and Cd68 expression was analyzed by real-time quantitative PCR (A). The expression of the housekeeping gene Rps12 served as internal control (A, *P < .05, ***P < .001 vs control, n = 6-7). NFAT5 protein was detected by capillary electrophoresis in cytosolic and nuclear fractions of cell lysates B and C. HDAC1 and α-tubulin served as loading controls for the nuclear and cytosolic fractions respectively (C, Cytosol: n.s. vs control; Nucleus: *P < .05 vs control, n = 3, Ctr., control; Chol., cholesterol; n.s., not significant)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Isolation, Solvent, Control, Expressing, Real-time Polymerase Chain Reaction, Electrophoresis

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 4 Genetic ablation of Nfat5 and its effect on cholesterol-exposed VSMCs. Genetic ablation of Nfat5 was achieved by expressing Cre-recombinase in murine Nfat5fl/fl VSMCs via adenoviral transduction (AdCre, an empty vector adenovirus (AdPl) served as control). The knockout of Nfat5 was evidenced by real-time quantitative PCR analysis (A, ***P < .001 vs control, n = 6, Rps12 expression level served as reference) and confirmed on protein level (B, results from capillary electrophoresis and automated signal detection, Valosin-containing protein (VCP) served as loading reference). VSMCs were exposed to cholesterol- supplemented medium for three days (Panserin, chol:MbCD 10 µg/ mL) 24 h after viral transduction which did not alter the expression of genes associated with a foam-cell like phenotype (C, n.s. vs control, n = 6, Chol., cholesterol; n.s., not significant)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Knock-Out, Real-time Polymerase Chain Reaction, Electrophoresis

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 5 Identification of Nfat5-controlled arteriosclerosis-associated transcriptional targets. The Nfat5-regulated expression of selected transcripts associated with the development of arteriosclerotic plaques was analyzed by applying a macroarray-based PCR screening protocol comparing cholesterol-exposed murine Nfat5fl/fl and Nfat5−/− VSMCs. Transcripts which were at least four-fold (rounded up) down- regulated in Nfat5-deficient VSMCs were summarized (A, significantly regulated transcripts were marked in yellow and their individual results were shown in B, *P < .05 vs control, n = 3, Ctr., control; Chol., cholesterol; Hsp90ab1 served as reference; comparable results were obtained if Gapdh was chosen as reference). The functional effect of the Nfat5 knockout was analyzed by comparing the cholesterol accumulation in cholesterol-exposed murine Nfat5fl/fl and Nfat5−/− VSMCs (C, Oil Red O (ORO)-stained area, **P < .01 vs. control, one out of two independent experiments with comparable results analyzing 37-62 cells per group is shown, scale bar: 50 µm). Additionally, the cellular cholesterol content was assessed by utilizing a direct colorimetric cholesterol assay (D, *P < .05 vs control, n = 4). Lipids were also visualized in murine aortic VSMCs overexpressing NFAT5 (AdN5; control: AdPl) after exposure to cholesterol (E, ***P < .001 vs. control, one out of three independent experiments with comparable results determining the ORO-positive area of 37-39 cells, scale bars: 50 µm)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Control, Functional Assay, Knock-Out, Staining, Cholesterol Assay

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 6 Analyses of blood cholesterol levels and lipid accumulation in the aorta of mice fed an atherogenic diet upon SMC-specific genetic ablation of Nfat5. Nfat5fl/fl (N5fl/fl) and Nfat5(SMC)−/− (N5(SMC)−/−) mice were fed an atherogenic diet (AD) for either 14 or 25 weeks or a normal chow diet (ND). Blood cholesterol levels were significantly elevated in mice fed an atherogenic diet (A, *P < .05 vs N5fl/fl ND, n = 4-8; **P < .01 vs N5(SMC)−/− ND, n = 6-9). Aortae were excised and stained with Oil Red O (ORO) to detect lipid-containing areas (B, scale bars: 1 mm). The cumulative ORO-stained vessel area was recorded via light microscopy and quantified (C, ***P < .001, **P < .01, *P < .05 vs control, n = 5-9, representative images are shown in D) and the location of ORO-positive areas in Nfat5(SMC)−/− mice was mapped (E). Confocal microscopy-based analyses of whole mount preparations of these aortae revealed ORO-positive droplets in the subintimal layers (F, scale bars: 100 and 40 µm)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Staining, Light Microscopy, Control, Confocal Microscopy