Journal: Journal of Orthopaedic Surgery and Research

Article Title: Circ_0000479 promotes proliferation, invasion, migration and inflammation and inhibits apoptosis of rheumatoid arthritis fibroblast-like synoviocytes via miR-766/FKBP5 axis

doi: 10.1186/s13018-023-03700-0

Figure Lengend Snippet: Circ_0000479 was upregulated in MH7A cells. A Relative expression of circ_0000479 in MH7A cells and normal FLSs. B Relative expression of EPSTI1 in MH7A cells and normal FLSs. C The expression of EPSTI1 protein in MH7A cells and normal FLSs. D Relative RNA levels of circ_0000479 and EPSTI1 after RNase R treatment. E Analysis for RNA abundance of circ_0000479 and EPSTI1 after Actinomycin D treatment at indicated time points. F Detected by qRT-PCR, circ_0000479 was mainly enriched in the cytoplasm. **P < 0.01; ****P < 0.0001

Article Snippet: RA-FLSs (MH7A cells) and normal FLSs (primary cells) were purchased from Wuhan Procell Life Technology Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Circ_0000479 promotes proliferation, invasion, migration and inflammation and inhibits apoptosis of rheumatoid arthritis fibroblast-like synoviocytes via miR-766/FKBP5 axis

doi: 10.1186/s13018-023-03700-0

Figure Lengend Snippet: MiR-766 was the target of circ_0000479. A Detection of miRNA expression levels in MH7A cells by qRT-PCR. B Relative expression of miR-766 in MH7A cells and normal FLSs. C Relative expression of miR-766 in MH7A cells transfected with miR-NC or miR-766. D The binding sites between circ_0000479 and miR-766. E Relative luciferase activity of WT-circ_0000479 and MUT-circ_0000479 after transfection of miR-NC or miR-766. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: RA-FLSs (MH7A cells) and normal FLSs (primary cells) were purchased from Wuhan Procell Life Technology Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Luciferase, Activity Assay

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Patient-Derived Phenotypic High-Throughput Assay to Identify Small Molecules Restoring Lysosomal Function in Tay–Sachs Disease

doi: 10.1177/2472555218814538

Figure Lengend Snippet: Development of a lysosomal calcium release assay in TSD patient-derived fibroblasts. Representative kinetic tracings from WT (A) and TSD (B) fibroblast calcium release in response to GPN measured on the FDSS μCell. Cells were pretreated with 25 μM APB for 15 min prior to GPN addition. Tracings show the baseline recording starting the last 5 min of the APB pretreatment. Ionomycin (5 μM) was added to induce a maximal release from the ER for comparison once the GPN-induced calcium release reached baseline. (C) Peak fluorescence ratios measured across a range of GPN concentrations (6–100 μM) in WT and TSD fibroblasts. The S/B indicated for each GPN concentration was determined comparing the peak fluorescence ratio calcium response in WT (signal) with TSD (background) fibroblasts. A 50 μM concentration of GPN was selected for the screening assay protocol as it produced the best S/B. (D) GPN-induced calcium release from acidic lysosomal calcium stores is significantly reduced in fibroblasts derived from TSD patients. Patient cell lines are WT (GM05659), TSD 1 (GM00221), TSD 2 (GM00502), and TSD 3 (GM11853). ***p < 0.0001 compared with WT cells. The reduced lysosomal calcium response in TSD patient cells was used as the basis to develop the phenotypic HTS assay.

Article Snippet: Human fibroblast cell lines from Tay–Sachs patients (GM00221, GM00502, GM11853) and a healthy normal patient (GM05659) were from Coriell Institute for Medical Research (Camden, NJ); GM00221 donor is homozygous for a 4-base-pair insertion at nucleotide 1278 in exon 11 of the HEXA gene (c.1278insTATC), which leads to a premature termination signal.

Techniques: Release Assay, Derivative Assay, Comparison, Fluorescence, Concentration Assay, Screening Assay, Produced, HTS Assay

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Patient-Derived Phenotypic High-Throughput Assay to Identify Small Molecules Restoring Lysosomal Function in Tay–Sachs Disease

doi: 10.1177/2472555218814538

Figure Lengend Snippet: Autophagy detection in WT and TSD fibroblasts. (A) Immunoblot analyses with anti-LC3 and anti-actin antibodies on WT (GM05659) and TSD (GM00221) patient fibroblast cell lines. Cells were treated with either vehicle (0.2% DMSO), 20 μM pyrimethamine (PYR), 100 nM bafilomycin A1 (Baf. A1), or PYR plus Baf. A1 for 22 h, followed by Western blot analysis. A representative Western blot is shown. (B) Quantification of LC3II intensity normalized to β-actin. Data represent the mean + SEM of at least three independent replicates.

Article Snippet: Human fibroblast cell lines from Tay–Sachs patients (GM00221, GM00502, GM11853) and a healthy normal patient (GM05659) were from Coriell Institute for Medical Research (Camden, NJ); GM00221 donor is homozygous for a 4-base-pair insertion at nucleotide 1278 in exon 11 of the HEXA gene (c.1278insTATC), which leads to a premature termination signal.

Techniques: Western Blot

Journal: FEBS Open Bio

Article Title: Modeling atrial fibrosis in vitro —Generation and characterization of a novel human atrial fibroblast cell line

doi: 10.1002/2211-5463.12896

Figure Lengend Snippet: Morphological and immunocytochemical fibroblast identification. Representative brightfield and immunofluorescence images of the fibroblast markers vimentin, DDR2, collagen 1, and αSMA. The nuclei were stained with DAPI (blue). Upper panel) HVFs. Mid panel) HAFs. Lower panel) PAFs. The scale bars equal 50 µm.

Article Snippet: Using lentiviral transfer of Upcyte ® proliferation genes, a novel human atrial fibroblast cell line (HAF‐SRK01) was generated.

Techniques: Immunofluorescence, Staining

Journal: FEBS Open Bio

Article Title: Modeling atrial fibrosis in vitro —Generation and characterization of a novel human atrial fibroblast cell line

doi: 10.1002/2211-5463.12896

Figure Lengend Snippet: Induction of a cellular fibrosis phenotype in HAFs with TGF‐β. (A) Protein expression of phosphorylated SMAD2/3 after 72 h of TGF‐β stimulation (1, 3, 10 ng·mL −1 ) in HAFs ( n = 3 per concentration) and representative original WB below. (B) Protein expression of αSMA after 72 h of TGF‐β stimulation (1, 3, 10 ng·mL −1 ) in HAFs ( n = 3 per concentration) and representative original WB below. (C) Quantification of fibroblasts positive for fibrillary αSMA microfilaments after stimulation with 10 ng·mL −1 TGF‐β for 72 h and representative immunofluorescence staining of fibrillary αSMA upon TGF‐β stimulation. The scale bars equal 50 µm. (D) Soluble collagen secretion (left) and representative immunofluorescence image for deposited Collagen Iα1 (right) by HAFs upon stimulation with 10 ng·mL −1 TGF‐β ( n = 14 vs. 10). The scale bars equal 50 µm. (E) Proliferation curves of HAFs under control conditions ( n = 4) and upon stimulation with 10 ng·mL −1 TGF‐β ( n = 4). Cells were counted after 7 and 14 days. (F) 24‐h migration capacity of HAFs under control conditions ( n = 6) and upon stimulation with 10 ng·mL −1 TGF‐β ( n = 4). Data are presented as mean ± SEM. Differences between two groups were compared using Student’s t ‐test with Welch’s correction. Differences between multiple groups were compared with one‐way ANOVA with Newman–Keuls post‐test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Using lentiviral transfer of Upcyte ® proliferation genes, a novel human atrial fibroblast cell line (HAF‐SRK01) was generated.

Techniques: Expressing, Concentration Assay, Immunofluorescence, Staining, Control, Migration

Journal: FEBS Open Bio

Article Title: Modeling atrial fibrosis in vitro —Generation and characterization of a novel human atrial fibroblast cell line

doi: 10.1002/2211-5463.12896

Figure Lengend Snippet: Functional analysis of cardiac fibroblast subtypes. (A) Proliferation curves of HVFs ( n = 6), HAFs ( n = 7) and PAFs ( n = 21). Cells were cultured in DMEM (10% FCS, 1% penicillin–streptomycin) at 37 °C, 5% CO 2 , and counted after 7 and 14 days. (B) Quantification of immunostaining experiments for fibrillary αSMA protein abundance ( n HVF = 4, n HAF = 7, n PAF = 6). (C) Basal 24‐h migration capacity of HVFs ( n = 5), HAFs ( n = 5), and PAFs ( n = 6). Data are presented as mean ± SEM. Differences between multiple groups were compared with one‐way ANOVA with Newman–Keuls post‐test. * P < 0.05. ** P < 0.01. *** P < 0.001; n.s., not significant.

Article Snippet: Using lentiviral transfer of Upcyte ® proliferation genes, a novel human atrial fibroblast cell line (HAF‐SRK01) was generated.

Techniques: Functional Assay, Cell Culture, Immunostaining, Quantitative Proteomics, Migration

Journal: FEBS Open Bio

Article Title: Modeling atrial fibrosis in vitro —Generation and characterization of a novel human atrial fibroblast cell line

doi: 10.1002/2211-5463.12896

Figure Lengend Snippet: Adaption of HAF and PAF stiffness in response to different stiffness of the growth matrix. (A) HAFs present a typical fibroblast morphology when grown on CyPhyGels. Nuclei were stained with Hoechst (blue), F‐actin was stained with Phalloidin (red), and ɑSMA was stained in green. The scale bar equals 20 µm. (B) Representative force/ indentation curves used to calculate the stiffness (E eff ) of individual cells cultured on either stiff (black curve) or soft gels (grey curve). The force required to indent a cell on the stiff substrate is higher than on the soft substrate. (C) Measurements of HAF and PAF stiffness on soft (~2.7 kPa) and stiff (~4.6 kPa) CyPhyGels (36 ≤ n ≤ 57). Data are presented as mean ± SEM. Differences between multiple groups were compared with one‐way ANOVA with Newman–Keuls post‐test. *** P < 0.001.

Article Snippet: Using lentiviral transfer of Upcyte ® proliferation genes, a novel human atrial fibroblast cell line (HAF‐SRK01) was generated.

Techniques: Staining, Cell Culture