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: Open Veterinary Journal

Article Title: Generation and characterization of mesenchymal stem cells from the affected femoral heads of dogs with Legg Calvé Perthes disease

doi: 10.5455/OVJ.2024.v14.i5.12

Figure Lengend Snippet: Trilineage differentiation potency. Canine LCPD-affected femoral head-derived adherent cells (A-C) and BMMSCs (D-F) differentiated into adipocytes (A and D), osteoblasts (B and E), and chondrocytes (C and F). (A and D): Sudan III staining, (B and E): Alizarin Red staining, and (C and F): Alcian blue staining. Scale bars = 100 μm or 1 mm.

Article Snippet: To induce differentiation into chondrocytes, 5 × 10 5 cells were placed in 15 ml polypropylene tubes, centrifuged to form pellets, and cultured with canine chondrocyte differentiation medium (Cell Applications, Inc.) for 3 weeks.

Techniques: Derivative Assay, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Ajuga decumbens stimulates mesenchymal stem cell differentiation and regenerates cartilage in a rabbit osteoarthritis model

doi: 10.3892/etm.2018.5981

Figure Lengend Snippet: Chondrocyte differentiation from hMSCs. hMSCs were cultured for 3 weeks with GM or CD and different concentrations of EADE. Cells were subjected to Alcian blue staining and the absorbance was measured at 630 nm. *P<0.05 and **P<0.01. hMSCs, human mesenchymal stem cells; GM, normal growth medium; CD, chondrogenic differentiation medium; EADE, extra Ajuga decumbens extract.

Article Snippet: Human chondrocytes (HC) derived from normal human femoral cartilage were obtained from Cell Applications, Inc. (Merck KGaA).

Techniques: Cell Culture, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Ajuga decumbens stimulates mesenchymal stem cell differentiation and regenerates cartilage in a rabbit osteoarthritis model

doi: 10.3892/etm.2018.5981

Figure Lengend Snippet: Effect of EADE on PGE2 production in chondrocytes. Human chondrocytes were stimulated with IL-1β (1 ng/ml) and treated with 10 or 100 µg/ml EADE. The concentration of PGE2 was significantly increased by IL-1β stimulation, and EADE significantly suppressed this increase. *P<0.05. IL, interleukin; EADE, extra Ajuga decumbens extract; PGE2, prostaglandin E2.

Article Snippet: Human chondrocytes (HC) derived from normal human femoral cartilage were obtained from Cell Applications, Inc. (Merck KGaA).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 chondrocytes were treated with either PGD 2 (A) or 15d-PGJ 2 (B) for 48 h, or PGE 2 (C) for 2 h. TLR4, caveolin-1 and IL-6 protein (upper) and mRNA (lower) expression was determined by Western blotting or qRT-PCR, respectively. ß-actin and GAPDH served as internal controls in immunoblotting and qRT-PCR, respectively. The Western blots are representative of three independent experiments, all revealing similar results. Data represent the mean ± S.E. of 3 independent qRT-PCR experiments. * and ▴, p <0.05 with respect to the corresponding vehicle control.

Article Snippet: Human primary articular chondrocytes (Cell Applications, Inc) or T/C-28a2 chondrocytic cells (T/C-28a2 chondrocytic cells were kindly provided by Dr. Goldring at Harvard Medical School, Boston, MA, USA) were seeded on 6-cm tissue culture dishes (10 6 cells per dish) in human chondrocyte growth medium (Cell Applications, Inc) or in DMEM/F12 medium supplemented with 10% FBS, respectively , , , , , .

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: PGE 2 stimulates TLR4 synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human chondrocytes. PGD 2 or 15d-PGJ 2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE 2 -dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in chondrocytes.

Article Snippet: Human primary articular chondrocytes (Cell Applications, Inc) or T/C-28a2 chondrocytic cells (T/C-28a2 chondrocytic cells were kindly provided by Dr. Goldring at Harvard Medical School, Boston, MA, USA) were seeded on 6-cm tissue culture dishes (10 6 cells per dish) in human chondrocyte growth medium (Cell Applications, Inc) or in DMEM/F12 medium supplemented with 10% FBS, respectively , , , , , .

Techniques: Activation Assay, Binding Assay

Journal: Aging Cell

Article Title: Urolithin A improves mitochondrial health, reduces cartilage degeneration, and alleviates pain in osteoarthritis

doi: 10.1111/acel.13662

Figure Lengend Snippet: (a–c) Oxygen consumption rates (OCR) showing basal (a) maximal (b) and ATP‐linked (c) mitochondrial respiration in primary healthy chondrocytes (HC), treated with DMSO or Urolithin A (UA) at 6.25 μM and 12 μM for 24 h. (technical replicates for a, 15–33; for b, 5–9; for c, 5–9). * p < 0.05; **** p < 0.0001 after one‐way ANOVA. Error bars represent mean ± SEM. (d) mRNA levels of mitophagy/autophagy genes PARK2 , SQSTM1 , MAP1LC3B , and BNIP3 in HC treated with DMSO or the indicated doses of UA for 24 h ( N = 5). * p < 0.05; *** p < 0.001 after one‐way ANOVA. Error bars represent mean ± SEM. (e) Mitotracker green fluorescent signal, representing the mitochondria quantity in cells treated with DMSO, 6.25 μM UA, 100 nM bafilomycin A1 (BafA1) or cotreated with both 6.25 μM UA and 100 nM BafA1 for 24 h. ( N = 12–18). *** p < 0.001; **** p < 0.0001 after one‐way ANOVA. Error bars represent mean ± SEM. (f, g) Representative images of HC treated as in (d) for 24 h and stained for TOM20 (green) and phospho‐ubiquitin (ph‐Ub, red). White squares indicate regions corresponding to insets. Nuclei were stained in blue with DAPI (f). Corresponding quantification of the intensity of ph‐Ub over TOM20 (g). ( N = 27–43) * p < 0.05; *** p < 0.001 after one‐way ANOVA. Error bars represent mean ± SEM. (h) Western blot of HC treated DMSO or UA at 6.25 μM for 24 h and probed with antibodies against phospho‐ubiquitin (ph‐Ub), TOM20 and HSP90 mitochondrial and cellular loading controls

Article Snippet: Human chondrocytes from healthy and OA donors were seeded in 96‐well seahorse plates (Agilent Seahorse XF96/XFe96 FluxPak, 102,416–100) and treated the day after with the indicated doses of UA in chondrocyte growth medium (Cell applications, 411–500).

Techniques: Staining, Ubiquitin Proteomics, Western Blot

Journal: Aging Cell

Article Title: Urolithin A improves mitochondrial health, reduces cartilage degeneration, and alleviates pain in osteoarthritis

doi: 10.1111/acel.13662

Figure Lengend Snippet: (a, b) Oxygen consumption rates (OCR) showing basal (a) and FCCP‐induced maximal respiration (b) in human primary chondrocytes (HC) from an OA patient, treated with DMSO or UA at 6.25 μM and 12 μM for 24 h (a). ( N = 7–10). * p < 0.05; ** p < 0.01; after one‐way ANOVA. Error bars represent mean ± SEM. (c) Mitotracker green fluorescent signal, in cells treated with DMSO, 12 μM UA, 100 nM bafilomycin A1 (BafA1) or cotreated with both 12 μM UA and 100 nM BafA1 for 24 h. ( N = 22–24). * p < 0.05, after one‐way ANOVA. Error bars represent mean ± SEM. (d, e) Representative images of HC treated as above for 24 h and stained for TOM20 (green) and phospho‐ubiquitin (ph‐Ub, red). White squares indicate the regions corresponding to insets. Nuclei were stained in blue with DAPI (D). Corresponding quantification of the intensity of ph‐Ub over TOM20 (E). ( N = 35–40) * p < 0.05; *** p < 0.001 after one‐way ANOVA. Error bars represent mean ± SEM

Article Snippet: Human chondrocytes from healthy and OA donors were seeded in 96‐well seahorse plates (Agilent Seahorse XF96/XFe96 FluxPak, 102,416–100) and treated the day after with the indicated doses of UA in chondrocyte growth medium (Cell applications, 411–500).

Techniques: Staining, Ubiquitin Proteomics