Journal: Molecular Medicine Reports

Article Title: Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro

doi: 10.3892/mmr.2015.4627

Figure Lengend Snippet: Effects of ZA on the differentiation and maturation of MC3T3-E1 cells. The MC3T3-E1 cells were treated with various concentrations of ZA (0–1 µ M). (A) Alizarin red S staining following 21 days of culture. (B) ALP activity on days 7 and 14. (C) ALP staining on day 7. (D) Gene expression levels of Col I, ALP, OCN and Runx2 on day 7, determined using RT-qPCR analysis normalized to 18S. (E) Protein levels of ALP, OCN and Runx2 on day 7, determined using western blot analysis normalized to GAPDH. (F) Quantitative analysis of the blots for the ALP, OCN and Runx2 proteins. The results are expressed as the mean ± standard error of the mean (n =3 for each group). * P<0.05, ** P<0.01 and *** P<0.001, compared with the 0 µ M group. ZA, zoledronic acid; Col I, collagen type I; ALP, alkaline phosphatase; OCN, osteocalcin; Runx2, runt-related transcription factor 2; GAPDH, glyceraldehyde phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: The PCR reactions were run in a total volume of 20 μ l, containing 10 μ l TransStart ® Top Green qPCR SuperMix (concentration, 2×; Beijing Transgen Biotech Co., Ltd., Beijing, China) and 0.2 μ M of each primer.

Techniques: Staining, Activity Assay, Gene Expression, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro

doi: 10.3892/mmr.2015.4627

Figure Lengend Snippet: Effect of ZA on the expression of BMP-2 and phosphorylation of the ERK 1/2 and p38 pathways in MC3T3-E1 cells. The MC3T3-E1 cells were incubated with various concentrations of ZA (0–1 µ M) for 7 days. (A) Levels of secreted BMP-2, measured using an ELISA. (B) mRNA expression levels of BMP-2, determined using reverse transcription-quantitative polymerase chain reaction analysis. (C) Protein levels of BMP-2, p38, p-p38, ERK 1/2 and p-ERK 1/2, detected using western blot analysis and normalized to GAPDH. (D) Quantitative analysis of the blots for BMP-2. (E) Relative expression of p-ERK, normalized to ERK, and relative expression of p-p38 normalized to p38. The results are expressed as the mean ± standard error of the mean (n =3 for each group). * P<0.05, ** P<0.01 and *** P<0.001, compared with the 0 µ M group. BMP-2, bone morphogenetic protein 2; ERK 1/2, extracellular signal-regulated kinase 1/2; p-, phosphorylated; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: The PCR reactions were run in a total volume of 20 μ l, containing 10 μ l TransStart ® Top Green qPCR SuperMix (concentration, 2×; Beijing Transgen Biotech Co., Ltd., Beijing, China) and 0.2 μ M of each primer.

Techniques: Expressing, Phospho-proteomics, Incubation, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Toxins

Article Title: Environmental Toxin Biliatresone-Induced Biliary Atresia-like Abnormal Cilia and Bile Duct Cell Development of Human Liver Organoids

doi: 10.3390/toxins16030144

Figure Lengend Snippet: Biliatresone-induced aberrant growth of human liver organoids. ( A ) Schematic diagram of the establishment of human liver organoids for biliatresone treatment. ( B ) Representative bright-field images; Hematoxylin & Eosin staining of sections of control and Day-5 biliatresone-treated organoids. ( C ) Growth curve of control and biliatresone-treated human liver organoids. The diameters of organoids (25 randomly chosen organoids were measured in each culture) were measured for the growth curve. *, a statistically significant difference between biliatresone-treated and untreated cultures ( p < 0.05). Error bars indicate standard deviation.

Article Snippet: Therefore, in the current study to investigate the effects of biliatresone, after culturing for 5 days, biliatresone (final concentration: 2 μg/mL; 2867, Axon MEDCHEM; stock solution: 2 mg/mL in DMSO) was added to the medium, and the organoids were cultured for different periods for subsequent analysis.

Techniques: Staining, Control, Standard Deviation

Journal: Toxins

Article Title: Environmental Toxin Biliatresone-Induced Biliary Atresia-like Abnormal Cilia and Bile Duct Cell Development of Human Liver Organoids

doi: 10.3390/toxins16030144

Figure Lengend Snippet: Biliatresone-induced hepatocytic differentiation of human liver organoids. ( A ) Representative images of control and Day-2 biliatresone-treated human liver organoids stained for HNF4A (green). Nuclei were stained with DAPI (blue). The percentage of HNF4A-positive cells (white arrow) in control and Day-2 biliatresone-treated organoids was determined by counting the total number of cells and HNF4A+ cells of the organoids. The number of organoids counted in each group = 3; *, p < 0.05, Student’s t -test; error bars indicated the standard deviation. ( B ) Representative images of control and Day-2 biliatresone-treated human liver organoids stained for MDR1 (red). Nuclei were stained with DAPI (blue). ( C ) MDR1 activity assay of organoids. ( C ) Representative images demonstrating the fluorescent substrate Rhodamine 123 (R123) localization in the lumen of control organoids at 30 min. In contrast, luminal R123 localization of Day-2 biliatresone-treated organoids was minimal during the 30-min incubation. The fluorescent intensity along the white line of images of control and Day-2 biliatresone-treated organoids at 30 min of incubation were plotted.

Article Snippet: Therefore, in the current study to investigate the effects of biliatresone, after culturing for 5 days, biliatresone (final concentration: 2 μg/mL; 2867, Axon MEDCHEM; stock solution: 2 mg/mL in DMSO) was added to the medium, and the organoids were cultured for different periods for subsequent analysis.

Techniques: Control, Staining, Standard Deviation, Activity Assay, Incubation

Journal: Toxins

Article Title: Environmental Toxin Biliatresone-Induced Biliary Atresia-like Abnormal Cilia and Bile Duct Cell Development of Human Liver Organoids

doi: 10.3390/toxins16030144

Figure Lengend Snippet: Biliatresone-induced cytoskeleton defects in human liver organoids. ( A ) Immunostaining of control and Day-2 biliatresone-treated human liver organoids for cytoskeleton protein F-actin (green). Nuclei were stained with DAPI (blue). The number of organoids examined in each group was 20. ( B ) ( Left panel ). Representative images demonstrating the accumulation of FITC-dextran localization in the lumen of Day-2 biliatresone-treated human liver organoids but not of control organoids at 10 and 30 min of incubation. ( Right panel ), the fluorescent intensity along the white line of images of control and Day-2 biliatresone-treated organoids at 10 and 30 min of incubation were plotted. The number of organoids examined in each group was 10.

Article Snippet: Therefore, in the current study to investigate the effects of biliatresone, after culturing for 5 days, biliatresone (final concentration: 2 μg/mL; 2867, Axon MEDCHEM; stock solution: 2 mg/mL in DMSO) was added to the medium, and the organoids were cultured for different periods for subsequent analysis.

Techniques: Immunostaining, Control, Staining, Incubation

Journal: Toxins

Article Title: Environmental Toxin Biliatresone-Induced Biliary Atresia-like Abnormal Cilia and Bile Duct Cell Development of Human Liver Organoids

doi: 10.3390/toxins16030144

Figure Lengend Snippet: Biliatresone decreased the number of ciliated cholangiocytes in human organoids. Representative images of control and Day-1, -2, -3 and -5 biliatresone-treated human liver organoids stained for pericentrin (PCNT; green) and acetylated α-tubulin (Tubulin; red). Nuclei were stained with DAPI (blue). Boxed regions were magnified and shown as insets. The percentages of ciliated cells (white arrow) in control and biliatresone-treated organoids were determined by counting the total number of cells and ciliated cells of the organoids. The number of organoids counted in each group was 20.

Article Snippet: Therefore, in the current study to investigate the effects of biliatresone, after culturing for 5 days, biliatresone (final concentration: 2 μg/mL; 2867, Axon MEDCHEM; stock solution: 2 mg/mL in DMSO) was added to the medium, and the organoids were cultured for different periods for subsequent analysis.

Techniques: Control, Staining

Journal: Toxins

Article Title: Environmental Toxin Biliatresone-Induced Biliary Atresia-like Abnormal Cilia and Bile Duct Cell Development of Human Liver Organoids

doi: 10.3390/toxins16030144

Figure Lengend Snippet: Reduction in cholangiocyte cilia mechanosensory response in biliatresone-treated organoids. ( A ) Representative images of control and biliatresone-treated human cholangiocytes stained with Calbryte 520 after application of shear flow with HBSS at 1 dyne/cm 2 . ( B ) Plot profile of fluorescent intensity changes of selected control and biliatresone-treated human cholangiocytes (highlighted with dotted line and numbered 1, 2 and 3) for 20 s. ( C ) The percentages of evoked cells after perfusion stimulation of control (Ctrl) and biliatresone-treated (BTS) human cholangiocytes were determined by counting the total number of cells and stimulated cells (green) at three randomly chosen fields of the cultures. *, p < 0.05, Student’s t -test; error bars indicated the standard deviation.

Article Snippet: Therefore, in the current study to investigate the effects of biliatresone, after culturing for 5 days, biliatresone (final concentration: 2 μg/mL; 2867, Axon MEDCHEM; stock solution: 2 mg/mL in DMSO) was added to the medium, and the organoids were cultured for different periods for subsequent analysis.

Techniques: Control, Staining, Shear, Standard Deviation