Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Advanced healthcare materials

Article Title: Designing Inherently Photodegradable Cell-Adhesive Hydrogels for 3D Cell Culture.

doi: 10.1002/adhm.202100632

Figure Lengend Snippet: Figure 2. A) Live/dead staining of normal human dermal fibroblasts (NHDF) with calcein-AM (green, live cells) and propidium iodide (red, dead cells) embedded in GelMA-free PEGMA/PEGDMA (Gel-N0) and GelMA-containing (Gel-N10) hydrogels on day 1, 7, 14 postencapsulation, followed by imaging with confocal microscopy (Leica TCS SPE, scale bar: 200 µm). B-i) NHDF embedded in Gel-N10 hydrogels on day 1 postencapsulation. Comparison of HepG2 cells encapsulated in (ii) Gel-N10 and (iii) Gel-N0 hydrogels (scale bar: 100 µm). C) Proliferation behavior of encapsulated HepG2 cells in Gel-N10 and Gel-N0 hydrogels over a period of 7 d assessed by PrestoBlue assay (n = 3). D) Toxicity evaluation of Gel-N10 photodegradation products by MTT proliferation assay with HeLa cells after 72 h exposure (n = 3). The concentration of nondiluted degradation products was 53 mg mL−1. Data are presented as mean ± SD and statistically evaluated by (C) Student’s t-test and (D) one-way ANOVA. *, **, *** represent p < 0.05, 0.01, and 0.001, respectively.

Article Snippet: Normal human dermal fibroblasts (NHDF), human liver cancer cells (HepG2), and HeLa cells were purchased from PromoCell GmbH (Heidelberg, Germany).

Techniques: Staining, Imaging, Confocal Microscopy, Comparison, Prestoblue Assay, Proliferation Assay, Concentration Assay

Journal: PLoS ONE

Article Title: C/EBPβ-Thr217 Phosphorylation Signaling Contributes to the Development of Lung Injury and Fibrosis in Mice

doi: 10.1371/journal.pone.0025497

Figure Lengend Snippet: A. Immunoblots for RSK, C/EBPβ-phospho-Thr217, procaspase 8 and C/EBPβ were performed on C/EBPβ immunoprecipitates from activated primary human LMF lysates as described in . RSK and phosphorylated C/EBPβ were induced in activated LMF but decreased in activated LMF treated with the ERK1/2 inhibitor (10 µg for 24 hr) or with the C/EBPβ peptide (200 µg for 24 hr). Inactive procaspase 8 was associated with phosphorylated C/EBPβ in untreated, activated LMF, while active caspase 8 was associated with unphosphorylated C/EBPβ in activated LMF treated with the ERK1/2 inhibitor or with the C/EBPβ peptide. Human LMF expressed full-length C/EBPβ from the second AUG . β-Actin was used to correct for lung lysate input. We performed single analysis of the samples. Results from triplicate samples of two independent experiments are shown. B. The ERK1/2 inhibitor decreased the fold association of RSK with C/EBPβ (n: 6 per group; 0.07+/−0.01, P <0.0001), and the fold expression of C/EBPβ (n: 6 per group; 0.05+/−0.006, P <0.0001) and C/EBPβ-PhosphoThr266 (n: 6 per group; 0.33+/−0.078, P <0.0001) in activated, human lung fibroblasts. There was also an increased fold association between unphosphorylated human C/EBPβ and active caspase 8 (n: 6 per group; active caspase 8; 5.70+/−0.59, P <0.0001). The cell permeant Ac-KAla217VD-CHO peptide also inhibited C/EBPβ expression (n: 6 per group; 0.12+/−0.01, P <0.0001), the phosphorylation of C/EBPβ-Thr266 (n: 6 per group; 0.17+/−0.03, P <0.0001), the association of RSK with C/EBPβ (n: 6 per group; 0.24+/−0.08, P <0.0001) and increased the association of RSK with C/EBPβ (n: 6 per group; 0.24+/−0.08, P <0.0001). We performed single analysis of the samples. C. α-SMA and TGF-β were induced in activated LMF but the expression of these fibrogenic genes was inhibited by treatment with the ERK1/2 inhibitor (10 µg for 24 hr) or with the peptide (200 µg for 24 hr). β-Actin was used to correct for lung lysate input. Representative results from two independent studies. D. The ERK1/2 inhibitor decreased the fold expression of α-SMA (n: 6 per group; 0.24+/−0.11, P <0.0001) and TGF-β1 (n: 6 per group; 0.14+/−0.02, P <0.0001). The cell permeant Ac-KAla217VD-CHO peptide also inhibited the fold expression of α-SMA (n: 6 per group; 0.24+/−0.11, P <0.0001) and TGF-β1 (n: 6 per group; 0.14+/−0.02, P <0.0001). We performed single analysis of the samples. E. Annexin-V-PE binding in vivo in activated LMF was increased after treatment with the ERK1/2 inhibitor (20 µg for 8 hr) or with the peptide (200 µg for 24 hr). Values are the percentage of cells expressing annexin-V-PE binding as described in . Activated, human lung fibroblasts treated with the ERK1/2 inhibitor (n: 6; 66.33+/−5.68%, P <0.0001) or with the Ac-KAla217VD-CHO peptide (n: 6; 61.00+/−9.27%, P <0.0001) displayed increased percent annexin-V binding compared to control (n: 6; 4.15+/−0.94%). We performed single analysis of the samples. Results from triplicate samples of three independent experiments are shown.

Article Snippet: Primary human lung fibroblasts isolated from normal human lung parenchyma (Cell Applications; San Diego, CA) were cryopreserved at first passage and can be cultured and propagated at least 12 population doublings.

Techniques: Western Blot, Expressing, Phospho-proteomics, Binding Assay, In Vivo, Control

Journal: Matrix Biology Plus

Article Title: Temporal expression and spatial distribution of the proteoglycan versican during cardiac fibrosis development

doi: 10.1016/j.mbplus.2023.100135

Figure Lengend Snippet: Versican ( VCAN ) expression is regulated by cytokines and mechanical stretch in cardiac cells. (A) mRNA expression of versican was upregulated by TGF-β1, and downregulated by TNF-α, and IL-1β in cardiac fibroblasts, and (B) in addition by IL-4 in cardiomyocytes compared to control (Control, n = 3). (C, D) Versican and α-SMA ( ACTA2 ) mRNA expression were increased after biaxial strain of 10% (stretch) for 24 h at 1 Hz frequency in fibroblasts compared to non-stretched cells (Control, n = 9). Gene expression is presented as values relative to Control. Bar graphs represent mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test (A, B) and Student’s t -test (C, D) were used for statistical analysis. P values < 0.05 were considered statistically significant. *P < 0.05 Control vs. cytokine treatment or stretch.

Article Snippet: Fetal human cardiac fibroblasts (CFs) (Cell Applications, CA, #306-05F) and primary human cardiomyocytes (CMs) (PromoCell, Germany, #C-12810) were used for in vitro experiments.

Techniques: Expressing, Control, Gene Expression