Journal: The FASEB Journal

Article Title: Mechanoregulation of p38 activity enhances endoplasmic reticulum stress–mediated inflammation by arterial endothelium

doi: 10.1096/fj.201900236R

Figure Lengend Snippet: VCAM-1 and ICAM-1 expression on inflamed HAECs is regulated by SS. HAEC monolayers were exposed to TNF-α (0.3 ng/ml) and a fluid SS gradient (0–16 dyn/cm2 over 20 mm) for 4 h in a Hele-Shaw microfluidic device. Cells were immunolabeled for VCAM-1 and ICAM-1 protein and images were captured by fluorescence microscopy. A) Representative imaging series for VCAM-1 and ICAM-1 captured at discrete SS levels within a single monolayer. Fold changes for each SS level in this image series expressed relative to 0 dyn/cm2 (static point). Scale bars, 50 µm. B, C) VCAM-1 (B) and ICAM-1 (C) were quantified over multiple experiments and expression was compared at representative low- (2 dyn/cm2) and high- (12 dyn/cm2) SS levels corresponding to the maximum reproducible change in TNF-α–stimulated VCAM-1 expression. Dashed lines represent initial (static, untreated) intensities. A role for PECAM-1–mediated mechanotransduction was assessed using PECAM-1–deficient cells generated by stable lentiviral transfection of shRNA. PI3K was blocked with the small molecule inhibitor LY294002. D) Summary schematic illustrating common PECAM-1–dependent mechanosignaling to both VCAM-1 and ICAM-1. E, F) VCAM-1 (E) and ICAM-1 (F) were quantified as above using a small molecule inhibitor to p38 (SB203580) or ERK1/2 (PD98059). G) Schematic highlighting unique MAPK-signaling dependence of CAM expression. Values are means ± se; n = 3–5 experiments. *P < 0.05.

Article Snippet: Each lot was characterized for a consistent dose response to TNF-α (R&D Systems, Minneapolis, MN, USA) stimulation by flow cytometry.

Techniques: Expressing, Immunolabeling, Fluorescence, Microscopy, Imaging, Generated, Transfection, shRNA

Journal: The FASEB Journal

Article Title: Mechanoregulation of p38 activity enhances endoplasmic reticulum stress–mediated inflammation by arterial endothelium

doi: 10.1096/fj.201900236R

Figure Lengend Snippet: Sustained p38 activation under low SS is accompanied by delayed activation of XBP1 and IRF-1. HAEC monolayers were exposed to TNF-α (0.3 ng/ml) and a fluid SS gradient (0–16 dyn/cm2 over 20 mm) for 5–120 min in a Hele-Shaw microfluidic device. Cells were immunolabeled for p-p38, XBP1s, or IRF-1, and images were captured by confocal microscopy. A) Representative imaging series for each target captured at discrete SS levels within a monolayer at 120 min. Fold changes for each SS level in this image series expressed relative to 0 dyn/cm2 (static point). Scale bars, 50 µm. B–D) Nuclear p-p38 (B), XBP1s (C), and IRF-1 (D) expression over time were quantified over multiple experiments and expression was compared at representative low- (2 dyn/cm2) and high- (12 dyn/cm2) SS levels corresponding to the maximum reproducible change in TNF-α–stimulated VCAM-1 expression. MFI, mean fluorescence intensity. Values are means ± se; n = 3–6 experiments. *P < 0.05.

Article Snippet: Each lot was characterized for a consistent dose response to TNF-α (R&D Systems, Minneapolis, MN, USA) stimulation by flow cytometry.

Techniques: Activation Assay, Immunolabeling, Confocal Microscopy, Imaging, Expressing, Fluorescence

Journal: The FASEB Journal

Article Title: Mechanoregulation of p38 activity enhances endoplasmic reticulum stress–mediated inflammation by arterial endothelium

doi: 10.1096/fj.201900236R

Figure Lengend Snippet: SS sustains phosphorylation of p38 following TNF-α stimulation. HAEC monolayers were exposed to TNF-α (0.3 ng/ml) and a fluid SS gradient in a Hele-Shaw microfluidic device. A) A role for PECAM-1–mediated mechanotransduction was assessed at time points corresponding to peak (30 min) and sustained (120 min) nuclear p38 activity in control and PECAM-1–deficient (shRNA) HAECs. Dashed line represents initial (static, untreated) intensity. Values are means ± se; n = 3–6 experiments. *P < 0.05. B) Representative confocal images captured at discrete SS levels coinciding with the SS-dependent difference in p-p38 expression observed at 120 min. Scale bars, 30 µm. MFI, mean fluorescence intensity.

Article Snippet: Each lot was characterized for a consistent dose response to TNF-α (R&D Systems, Minneapolis, MN, USA) stimulation by flow cytometry.

Techniques: Activity Assay, shRNA, Expressing, Fluorescence

Journal: The FASEB Journal

Article Title: Mechanoregulation of p38 activity enhances endoplasmic reticulum stress–mediated inflammation by arterial endothelium

doi: 10.1096/fj.201900236R

Figure Lengend Snippet: MKP-1 regulates the SS-mediated phosphorylation of p38. HAEC monolayers were exposed to TNF-α (0.3 ng/ml) and a fluid SS gradient in a Hele-Shaw microfluidic device for 2 h. After 30 min of treatment, p38 agonist (anisomycin), p38 inhibitor (SB203580), or MKP-1 inhibitor (NSC 95397) was added to the flow medium. A) Nuclear p-p38 was quantified from confocal images. B) MKP-1 expression was quantified from confocal images in HEACs treated with TNF-α and exposed to the SS gradient. Representative imaging series for MKP-1 captured at discrete SS levels within a monolayer at 45 min. Fold changes for each SS level in this image series expressed relative to 0 dyn/cm2 (static point). Scale bars, 50 µm. C) Cytoplasmic MKP-1 expression was quantified over multiple experiments and expression was compared at discrete SS levels coinciding with the SS-dependent difference in p-p38 expression. Representative images: scale bars, 35 µm. D) A role for PECAM-1–mediated mechanotransduction was assessed in control and PECAM-1–deficient (shRNA) HAECs. E) Schematic illustrating the regulation of p38 by MKP-1 to control VCAM-1 expression. MFI, mean fluorescence intensity. Values are means ± se; n = 3–6 experiments. *P < 0.05.

Article Snippet: Each lot was characterized for a consistent dose response to TNF-α (R&D Systems, Minneapolis, MN, USA) stimulation by flow cytometry.

Techniques: Expressing, Imaging, shRNA, Fluorescence

Journal: The FASEB Journal

Article Title: Mechanoregulation of p38 activity enhances endoplasmic reticulum stress–mediated inflammation by arterial endothelium

doi: 10.1096/fj.201900236R

Figure Lengend Snippet: p-p38 promotes nuclear localization of IRF-1 that promotes VCAM-1 transcription. HAEC monolayers were exposed to TNF-α (0.3 ng/ml) and a fluid SS gradient in the presence or absence of p38 inhibitor (SB203580). A) Expression of the ER resident protein calreticulin was imaged by confocal immunofluorescence microscopy and used to calculate a metric of ER expansion. B) After 30 min of SS and TNF-α exposure, p38 inducer (anisomycin), p38 inhibitor (SB203580), or MKP-1 inhibitor (NSC 95397) was added to the flow medium and nuclear XBP1s was quantified from confocal images at 120 min. C) A role for mechanoregulation of XBP1s activity by p38 was assessed in the presence or absence of p38 inhibition over the entire 120 min of treatment. Representative confocal images captured at discrete SS levels coincide with the SS-dependent difference in p-p38 expression. Scale bars, 30 µm. D) Nuclear XBP1s quantified over multiple experiments. E) IRF-1 activity was assessed in the presence or absence of p38 inhibition over the entire 120 min of treatment. Representative confocal images captured at discrete SS levels coinciding with the SS-dependent difference in p-p38 expression. Scale bars, 30 µm. F) Nuclear IRF-1 quantified over multiple experiments. CV, coefficient of variation; MFI, mean fluorescence intensity. Values are means ± se; n = 3–6 experiments. *P < 0.05, **P < 0.005.

Article Snippet: Each lot was characterized for a consistent dose response to TNF-α (R&D Systems, Minneapolis, MN, USA) stimulation by flow cytometry.

Techniques: Expressing, Immunofluorescence, Microscopy, Activity Assay, Inhibition, Fluorescence

Journal: bioRxiv

Article Title: Proopiomelanocortin (POMC) is a negative regulator of tadpole aggression through opioid receptor signaling

doi: 10.1101/2022.11.28.518266

Figure Lengend Snippet: (A) POMC is enriched in the diencephalon of aggressive tadpoles. Plot shows statistical significance plotted against magnitude of change. (B) There is a significant difference between POMC-pS6 positive cells in the preoptic area of the hypothalamus (POA) of aggressive and control animals. (C) Fluorescent imaging of POMC (magenta), pS6-positive cells (green) and their colocalization (white) in the POA; scale bar is 50 μm. (D) Schematic of experimental injection protocol. Treatment with naltrexone increased the number of attacks compared to MC4R antagonist and vehicle treated animals (E) and total aggressive actions compared to ACTH, MC4R antagonist, and vehicle treated animals (F) .

Article Snippet: A dose-response curve was performed for a selective MC4R antagonist, M4603 (Sigma Aldrich, #SHU9119), which binds aMSH.

Techniques: Imaging, Injection